Updated on 2021/10/21

写真a

 
TAMA Florence
 
Organization
Graduate School of Science Division of Material Science Molecular Biophysics Professor
Institute of Transformative Bio-Molecules Professor
Graduate School
Graduate School of Science
Undergraduate School
School of Science Department of Physics
Title
Professor

Degree 2

  1. PhD, Computational Biophysics ( 2000.10 ) 

  2. BS, Physics & Chemistry ( 1996.6   University Paul Sabatier, France ) 

Research Interests 3

  1. structure

  2. simulations

  3. biophysics

Research Areas 1

  1. Life Science / Biophysics

Research History 4

  1. Nagoya University   Institute of Transformative Bio-Molecules   Researcher

    2016.4

  2. Nagoya University   Professor

    2015.3

  3. RIKEN - Center for Computational Sciences   Team Leader

    2013.1

  4. The Scripps Research Institute   Post-Doctoral researcher

    2001.1 - 2005.10

 

Papers 82

  1. Protocol for Retrieving Three-Dimensional Biological Shapes for a Few XFEL Single-Particle Diffraction Patterns

    Tiwari Sandhya P., Tama Florence, Miyashita Osamu

    JOURNAL OF CHEMICAL INFORMATION AND MODELING   Vol. 61 ( 8 ) page: 4108 - 4119   2021.8

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    Language:Japanese  

    DOI: 10.1021/acs.jcim.1c00602

    Web of Science

  2. Reconstruction of Three-Dimensional Conformations of Bacterial ClpB from High-Speed Atomic-Force-Microscopy Images

    Dasgupta Bhaskar, Miyashita Osamu, Uchihashi Takayuki, Tama Florence

    FRONTIERS IN MOLECULAR BIOSCIENCES   Vol. 8   2021.8

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    Language:Japanese  

    DOI: 10.3389/fmolb.2021.704274

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  3. Structural differences in the FAD-binding pockets and lid loops of mammalian CRY1 and CRY2 for isoform-selective regulation

    Miller Simon, Srivastava Ashutosh, Nagai Yoshiko, Aikawa Yoshiki, Tama Florence, Hirota Tsuyoshi

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 118 ( 26 )   2021.6

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    Language:Japanese  

    DOI: 10.1073/pnas.2026191118

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  4. Reversible modulation of circadian time with chronophotopharmacology

    Kolarski Dusan, Miro-Vinyals Carla, Sugiyama Akiko, Srivastava Ashutosh, Ono Daisuke, Nagai Yoshiko, Iida Mui, Itami Kenichiro, Tama Florence, Szymanski Wiktor, Hirota Tsuyoshi, Feringa Ben L.

    NATURE COMMUNICATIONS   Vol. 12 ( 1 )   2021.5

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  5. How CRY Regulates the Clock: Structural Studies of a Dynamic Mammalian Circadian Complex

    Sandate C., Fribourgh J., Michael A., Srivastava A., Hura G., Schneidman-Duhovny D., Tripathi S., Takahashi J., Lander G., Hirota T., Tama F., Partch C.

    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES   Vol. 76   page: A122 - A122   2020.8

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    Language:Japanese  

    DOI: 10.1107/S0108767320098785

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  6. Photopharmacological Manipulation of Mammalian CRY1 for Regulation of the Circadian Clock. Reviewed International coauthorship International journal

    Dušan Kolarski, Simon Miller, Tsuyoshi Oshima, Yoshiko Nagai, Yugo Aoki, Piermichele Kobauri, Ashutosh Srivastava, Akiko Sugiyama, Kazuma Amaike, Ayato Sato, Florence Tama, Wiktor Szymanski, Ben L Feringa, Kenichiro Itami, Tsuyoshi Hirota

    Journal of the American Chemical Society   Vol. 143 ( 4 ) page: 2078 - 2087   2021.2

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    Language:Japanese   Publishing type:Research paper (scientific journal)  

    CRY1 and CRY2 proteins are highly conserved components of the circadian clock that controls daily physiological rhythms. Disruption of CRY functions are related to many diseases, including circadian sleep phase disorder. Development of isoform-selective and spatiotemporally controllable tools will facilitate the understanding of shared and distinct functions of CRY1 and CRY2. Here, we developed CRY1-selective compounds that enable light-dependent manipulation of the circadian clock. From phenotypic chemical screening in human cells, we identified benzophenone derivatives that lengthened the circadian period. These compounds selectively interacted with the CRY1 photolyase homology region, resulting in activation of CRY1 but not CRY2. The benzophenone moiety rearranged a CRY1 region called the "lid loop" located outside of the compound-binding pocket and formed a unique interaction with Phe409 in the lid loop. Manipulation of this key interaction was achieved by rationally designed replacement of the benzophenone with a switchable azobenzene moiety whose cis-trans isomerization can be controlled by light. The metastable cis form exhibited sufficiently high half-life in aqueous solutions and structurally mimicked the benzophenone unit, enabling reversible period regulation over days by cellular irradiation with visible light. This study revealed an unprecedented role of the lid loop in CRY-compound interaction and paves the way for spatiotemporal regulation of CRY1 activity by photopharmacology for molecular understanding of CRY1-dependent functions in health and disease.

    DOI: 10.1021/jacs.0c12280

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  7. Isoform-selective regulation of mammalian cryptochromes. Reviewed International journal

    Simon Miller, You Lee Son, Yoshiki Aikawa, Eri Makino, Yoshiko Nagai, Ashutosh Srivastava, Tsuyoshi Oshima, Akiko Sugiyama, Aya Hara, Kazuhiro Abe, Kunio Hirata, Shinya Oishi, Shinya Hagihara, Ayato Sato, Florence Tama, Kenichiro Itami, Steve A Kay, Megumi Hatori, Tsuyoshi Hirota

    Nature chemical biology   Vol. 16 ( 6 ) page: 676 - +   2020.6

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    Language:Japanese   Publishing type:Research paper (scientific journal)  

    CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly homologous proteins, in addition to redundant functions. Therefore, the development of isoform-selective compounds represents an effective approach towards understanding the similarities and differences of CRY1 and CRY2 by controlling each isoform individually. We conducted phenotypic screenings of circadian clock modulators, and identified KL101 and TH301 that selectively stabilize CRY1 and CRY2, respectively. Crystal structures of CRY-compound complexes revealed conservation of compound-binding sites between CRY1 and CRY2. We further discovered a unique mechanism underlying compound selectivity in which the disordered C-terminal region outside the pocket was required for the differential effects of KL101 and TH301 against CRY isoforms. By using these compounds, we found a new role of CRY1 and CRY2 as enhancers of brown adipocyte differentiation, providing the basis of CRY-mediated regulation of energy expenditure.

    DOI: 10.1038/s41589-020-0505-1

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  8. Computational Protocol for Assessing the Optimal Pixel Size to Improve the Accuracy of Single-particle Cryo-electron Microscopy Maps. Reviewed International coauthorship International journal

    Sandhya P Tiwari, Sahil Chhabra, Florence Tama, Osamu Miyashita

    Journal of chemical information and modeling   Vol. 60 ( 5 ) page: 2570 - 2580   2020.5

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    Cryo-electron microscopy (cryo-EM) single-particle analysis has come a long way in achieving atomic-level resolution when imaging biomolecules. To obtain the best possible three-dimensional (3D) structure in cryo-EM, many parameters have to be carefully considered. Here we address the often-overlooked parameter of the pixel size, which describes the magnification of the image produced by the experiment. While efforts are made to refine and validate this parameter in the analysis of cryo-EM experimental data, there is no systematic protocol in place. Since the pixel size parameter can have an impact on the resolution and accuracy of a cryo-EM map, and the atomic resolution 3D structure models derived from it, we propose a computational protocol to estimate the appropriate pixel size parameter. In our protocol, we fit and refine atomic structures against cryo-EM maps at multiple pixel sizes. The resulting fitted and refined structures are evaluated using the GOAP (generalized orientation-dependent, all-atom statistical potential) score, which we found to perform better than other commonly used functions, such as Molprobity and the correlation coefficient from refinement. Finally, we describe the efficacy of this protocol in retrieving appropriate pixel sizes for several examples; simulated data based on yeast elongation factor 2 and experimental data from Gro-EL chaperone, beta-galactosidase, and the TRPV1 ion channel.

    DOI: 10.1021/acs.jcim.9b01107

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  9. Integrative/Hybrid Modeling Approaches for Studying Biomolecules. Reviewed International journal

    Ashutosh Srivastava, Sandhya Premnath Tiwari, Osamu Miyashita, Florence Tama

    Journal of molecular biology   Vol. 432 ( 9 ) page: 2846 - 2860   2020.4

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    The structural and dynamical characterization of biomolecules holds central importance in the endeavor to understand the molecular mechanisms regulating living systems. However, owing to the inherent heterogeneity of biomolecular interactions within cells, it is often difficult to understand the overall structure and dynamics of biomolecules using any experimental method in isolation. In this regard, hybrid methods that combine data from multiple experiments to generate a comprehensive model of biomolecular complexes have gained prominence in the last few years. In this article, we discuss the advancements in hybrid methods, with a particular focus on the role of computation in their development and application. We further outline the future directions that hybrid methods are likely to take, regarding the advancements in techniques such as X-ray free-electron laser single- particle imaging, and electron cryo-tomography. Finally, we conclude the review by highlighting the future goals of broader consensus and collaboration within the integrative/hybrid structural biology community and for disseminating the data generated by hybrid modeling efforts.

    DOI: 10.1016/j.jmb.2020.01.039

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  10. Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing. Reviewed International journal

    Jennifer L Fribourgh, Ashutosh Srivastava, Colby R Sandate, Alicia K Michael, Peter L Hsu, Christin Rakers, Leslee T Nguyen, Megan R Torgrimson, Gian Carlo G Parico, Sarvind Tripathi, Ning Zheng, Gabriel C Lander, Tsuyoshi Hirota, Florence Tama, Carrie L Partch

    eLife   Vol. 9   2020.2

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    Language:Japanese   Publishing type:Research paper (scientific journal)  

    Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.

    DOI: 10.7554/eLife.55275

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  11. Conformational ensemble of an intrinsically flexible loop in mitochondrial import protein Tim21 studied by modeling and molecular dynamics simulations. Reviewed International journal

    Arpita Srivastava, Siqin Bala, Hajime Motomura, Daisuke Kohda, Florence Tama, Osamu Miyashita

    Biochimica et biophysica acta. General subjects   Vol. 1864 ( 2 ) page: 129417 - 129417   2020.2

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    BACKGROUND: Tim21, a subunit of a highly dynamic translocase of the inner mitochondrial membrane (TIM23) complex, translocates proteins by interacting with subunits in the translocase of the outer membrane (TOM) complex and Tim23 channel in the TIM23 complex. A loop segment in Tim21, which is in close proximity of the binding site of Tim23, has different conformations in X-ray, NMR and new crystal contact-free space (CCFS) structures. MD simulations can provide information on the structure and dynamics of the loop in solution. METHODS: The conformational ensemble of the loop was characterized using loop modeling and molecular dynamics (MD) simulations. RESULTS: MD simulations confirmed mobility of the loop. Multidimensional scaling and clustering were used to characterize the dynamic conformational ensemble of the loop. Free energy landscape showed that the CCFS crystal structure occupied a low energy region as compared to the conventional X-ray crystal structure. Analysis of crystal packing indicates that the CCFS provides larger conformational space for the motions of the loop. CONCLUSIONS: Our work reported the conformational ensemble of the loop in solution, which is in agreement with the structure obtained from CCFS approach. The combination of the experimental techniques and computational methods is beneficial for studying highly flexible regions of proteins. GENERAL SIGNIFICANCE: Computational methods, such as loop modeling and MD simulations, have proved to be useful for studying conformational flexibility of proteins. These methods in integration with experimental techniques such as CCFS has the potential to transform the studies on flexible regions of proteins.

    DOI: 10.1016/j.bbagen.2019.129417

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  12. Crystal contact-free conformation of an intrinsically flexible loop in protein crystal: Tim21 as the case study. Reviewed International journal

    Siqin Bala, Shoko Shinya, Arpita Srivastava, Marie Ishikawa, Atsushi Shimada, Naohiro Kobayashi, Chojiro Kojima, Florence Tama, Osamu Miyashita, Daisuke Kohda

    Biochimica et biophysica acta. General subjects   Vol. 1864 ( 2 ) page: 129418 - 129418   2020.2

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    BACKGROUND: In protein crystals, flexible loops are frequently deformed by crystal contacts, whereas in solution, the large motions result in the poor convergence of such flexible loops in NMR structure determinations. We need an experimental technique to characterize the structural and dynamic properties of intrinsically flexible loops of protein molecules. METHODS: We designed an intended crystal contact-free space (CCFS) in protein crystals, and arranged the flexible loop of interest in the CCFS. The yeast Tim 21 protein was chosen as the model protein, because one of the loops (loop 2) is distorted by crystal contacts in the conventional crystal. RESULTS: Yeast Tim21 was fused to the MBP protein by a rigid α-helical linker. The space created between the two proteins was used as the CCFS. The linker length provides adjustable freedom to arrange loop 2 in the CCFS. We re-determined the NMR structure of yeast Tim21, and conducted MD simulations for comparison. Multidimensional scaling was used to visualize the conformational similarity of loop 2. We found that the crystal contact-free conformation of loop 2 is located close to the center of the ensembles of the loop 2 conformations in the NMR and MD structures. CONCLUSIONS: Loop 2 of yeast Tim21 in the CCFS adopts a representative, dominant conformation in solution. GENERAL SIGNIFICANCE: No single powerful technique is available for the characterization of flexible structures in protein molecules. NMR analyses and MD simulations provide useful, but incomplete information. CCFS crystallography offers a third route to this goal.

    DOI: 10.1016/j.bbagen.2019.129418

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  13. Reconstruction of low-resolution molecular structures from simulated atomic force microscopy images. Reviewed International journal

    Bhaskar Dasgupta, Osamu Miyashita, Florence Tama

    Biochimica et biophysica acta. General subjects   Vol. 1864 ( 2 ) page: 129420 - 129420   2020.2

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    BACKGROUND: Atomic Force Microscopy (AFM) is an experimental technique to study structure-function relationship of biomolecules. AFM provides images of biomolecules at nanometer resolution. High-speed AFM experiments produce a series of images following dynamics of biomolecules. To further understand biomolecular functions, information on three-dimensional (3D) structures is beneficial. METHOD: We aim to recover 3D information from an AFM image by computational modeling. The AFM image includes only low-resolution representation of a molecule; therefore we represent the structures by a coarse grained model (Gaussian mixture model). Using Monte-Carlo sampling, candidate models are generated to increase similarity between AFM images simulated from the models and target AFM image. RESULTS: The algorithm was tested on two proteins to model their conformational transitions. Using a simulated AFM image as reference, the algorithm can produce a low-resolution 3D model of the target molecule. Effect of molecular orientations captured in AFM images on the 3D modeling performance was also examined and it is shown that similar accuracy can be obtained for many orientations. CONCLUSIONS: The proposed algorithm can generate 3D low-resolution protein models, from which conformational transitions observed in AFM images can be interpreted in more detail. GENERAL SIGNIFICANCE: High-speed AFM experiments allow us to directly observe biomolecules in action, which provides insights on biomolecular function through dynamics. However, as only partial structural information can be obtained from AFM data, this new AFM based hybrid modeling method would be useful to retrieve 3D information of the entire biomolecule.

    DOI: 10.1016/j.bbagen.2019.129420

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  14. Controlling the Circadian Clock with High Temporal Resolution through Photodosing. Reviewed

    Kolarski D, Sugiyama A, Breton G, Rakers C, Ono D, Schulte A, Tama F, Itami K, Szymanski W, Hirota T, Feringa BL

    Journal of the American Chemical Society   Vol. 141 ( 40 ) page: 15784 - 15791   2019.10

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  15. Bipartite anchoring of SCREAM enforces stomatal initiation by coupling MAP kinases to SPEECHLESS. Reviewed

    Putarjunan A, Ruble J, Srivastava A, Zhao C, Rychel AL, Hofstetter AK, Tang X, Zhu JK, Tama F, Zheng N, Torii KU

    Nature plants   Vol. 5 ( 7 ) page: 742 - 754   2019.7

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  16. Hybrid Modeling Approaches to Study Structures and Dynamics of Biological Systems

    Tama Florence

    BIOPHYSICAL JOURNAL   Vol. 116 ( 3 ) page: 331A - 331A   2019.2

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  17. Cryo-Cooling Effect on DHFR Crystal Studied by Replica-Exchange Molecular Dynamics Simulations. Reviewed

    Nagai T, Tama F, Miyashita O

    Biophysical journal   Vol. 116 ( 3 ) page: 395 - 405   2019.2

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  18. Acceleration of cryo-EM Flexible Fitting for Large Biomolecular Systems by Efficient Space Partitioning Reviewed

    Mori Takaharu, Kulik Marta, Miyashita Osamu, Jung Jaewoon, Tama Florence, Sugita Yuji

    STRUCTURE   Vol. 27 ( 1 ) page: 161 - +   2019.1

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    DOI: 10.1016/j.str.2018.09.004

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  19. Cell-based screen identifies a new potent and highly selective CK2 inhibitor for modulation of circadian rhythms and cancer cell growth. Reviewed

    Oshima T, Niwa Y, Kuwata K, Srivastava A, Hyoda T, Tsuchiya Y, Kumagai M, Tsuyuguchi M, Tamaru T, Sugiyama A, Ono N, Zolboot N, Aikawa Y, Oishi S, Nonami A, Arai F, Hagihara S, Yamaguchi J, Tama F, Kunisaki Y, Yagita K, Ikeda M, Kinoshita T, Kay SA, Itami K, Hirota T

    Science advances   Vol. 5 ( 1 ) page: eaau9060   2019.1

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  20. Computational investigation of the conformational dynamics in Tom20-mitochondrial presequence tethered complexes Reviewed

    Srivastava Arpita, Tama Florence, Kohda Daisuke, Miyashita Osamu

    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS   Vol. 87 ( 1 ) page: 81 - 90   2019.1

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    DOI: 10.1002/prot.25625

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  21. Parameter optimization for 3D-reconstruction from XFEL diffraction patterns based on Fourier slice matching. Reviewed

    Miki Nakano, Osamu Miyashita, Florence Tama

    Biophysics and physicobiology   Vol. 16   page: 367 - 376   2019

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    Single-particle analysis (SPA) by X-ray free electron laser (XFEL) is a novel method that can observe biomolecules and living tissue that are difficult to crystallize in a state close to nature. To reconstruct three-dimensional (3D) molecular structure from two-dimensional (2D) XFEL diffraction patterns, we have to estimate the incident beam angle to the molecule for each pattern to assemble the 3D-diffraction intensity distribution using interpolation, and retrieve the phase information. In this study, we investigated the optimal parameter sets to assemble the 3D-diffraction intensity distribution from simulated 2D-diffraction patterns of ribosome. In particular, we examined how the parameters need to be adjusted for diffraction patterns with different binning sizes and beam intensities to obtain the highest resolution of molecular structure phase retrieved from the 3D-diffraction intensity. We found that resolution of restored molecular structure is sensitive to the interpolation parameters. Using the optimal parameter set, a linear oversampling ratio of around four is found to be sufficient for correct angle estimation and phase retrieval from the diffraction patterns of SPA by XFEL.

    DOI: 10.2142/biophysico.16.0_367

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  22. Poisson image denoising by piecewise principal component analysis and its application in single-particle X-ray diffraction imaging Reviewed

    Jin Qiyu, Miyashita Osamu, Tama Florence, Yang Jie, Jonic Slavica

    IET IMAGE PROCESSING   Vol. 12 ( 12 ) page: 2264 - 2274   2018.12

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    DOI: 10.1049/iet-ipr.2018.5145

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  23. Role of Computational Methods in Going beyond X-ray Crystallography to Explore Protein Structure and Dynamics. Reviewed

    Srivastava A, Nagai T, Srivastava A, Miyashita O, Tama F

    International journal of molecular sciences   Vol. 19 ( 11 )   2018.11

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  24. Gaussian mixture model for coarse-grained modeling from XFEL. Reviewed

    Nagai T, Mochizuki Y, Joti Y, Tama F, Miyashita O

    Optics express   Vol. 26 ( 20 ) page: 26734 - 26749   2018.10

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  25. Searching for 3D structural models from a library of biological shapes using a few 2D experimental images Reviewed

    Tiwari Sandhya P, Tama Florence, Miyashita Osamu

    BMC BIOINFORMATICS   Vol. 19   2018.9

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    DOI: 10.1186/s12859-018-2358-0

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  26. Single-particle XFEL 3D reconstruction of ribosome-size particles based on Fourier slice matching: requirements to reach subnanometer resolution Reviewed

    Nakano Miki, Miyashita Osamu, Jonic Slavica, Tokuhisa Atsushi, Tama Florence

    JOURNAL OF SYNCHROTRON RADIATION   Vol. 25   page: 1010 - 1021   2018.7

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    DOI: 10.1107/S1600577518005568

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  27. Conformational dynamics of human protein kinase CK2α and its effect on function and inhibition Reviewed

    Ashutosh Srivastava, Tsuyoshi Hirota, Stephan Irle, Florence Tama

    Proteins: Structure, Function and Bioinformatics   Vol. 86 ( 3 ) page: 344 - 353   2018.3

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:John Wiley and Sons Inc.  

    Protein kinase, casein kinase II (CK2), is ubiquitously expressed and highly conserved protein kinase that shows constitutive activity. It phosphorylates a diverse set of proteins and plays crucial role in several cellular processes. The catalytic subunit of this enzyme (CK2α) shows remarkable flexibility as evidenced in numerous crystal structures determined till now. Here, using analysis of multiple crystal structures and long timescale molecular dynamics simulations, we explore the conformational flexibility of CK2α. The enzyme shows considerably higher flexibility in the solution as compared to that observed in crystal structure ensemble. Multiple conformations of hinge region, located near the active site, were observed during the dynamics. We further observed that among these multiple conformations, the most populated conformational state was inadequately represented in the crystal structure ensemble. The catalytic spine, was found to be less dismantled in this state as compared to the “open” hinge/αD state crystal structures. The comparison of dynamics in unbound (Apo) state and inhibitor (CX4945) bound state exhibits inhibitor induced suppression in the overall dynamics of the enzyme. This is especially true for functionally important glycine-rich loop above the active site. Together, this work gives novel insights into the dynamics of CK2α in solution and relates it to the function. This work also explains the effect of inhibitor on the dynamics of CK2α and paves way for development of better inhibitors.

    DOI: 10.1002/prot.25444

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  28. Hybrid Methods for Macromolecular Modeling by Molecular Mechanics Simulations with Experimental Data. Reviewed

    Miyashita O, Tama F

    Advances in experimental medicine and biology   Vol. 1105   page: 199 - 217   2018

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  29. Three-dimensional reconstruction for coherent diffraction patterns obtained by XFEL. Reviewed

    Nakano M, Miyashita O, Jonic S, Song C, Nam D, Joti Y, Tama F

    Journal of synchrotron radiation   Vol. 24 ( Pt 4 ) page: 727 - 737   2017.7

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:International Union of Crystallography ({IUCr})  

    The three-dimensional (3D) structural analysis of single particles using an X-ray free-electron laser (XFEL) is a new structural biology technique that enables observations of molecules that are difficult to crystallize, such as flexible biomolecular complexes and living tissue in the state close to physiological conditions. In order to restore the 3D structure from the diffraction patterns obtained by the XFEL, computational algorithms are necessary as the orientation of the incident beam with respect to the sample needs to be estimated. A program package for XFEL single-particle analysis based on the Xmipp software package, that is commonly used for image processing in 3D cryo-electron microscopy, has been developed. The reconstruction program has been tested using diffraction patterns of an aerosol nanoparticle obtained by tomographic coherent X-ray diffraction microscopy.

    DOI: 10.1107/S1600577517007767

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  30. Computational investigation of binding and dynamics in Tom20-mitochondrial targeting signal complex

    Srivastava A., Miyashita O., Tama F.

    EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS   Vol. 46   page: S232 - S232   2017.7

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  31. Flexible fitting to cryo-EM density map using ensemble molecular dynamics simulations Reviewed

    Osamu Miyashita, Chigusa Kobayashi, Takaharu Mori, Yuji Sugita, Florence Tama

    JOURNAL OF COMPUTATIONAL CHEMISTRY   Vol. 38 ( 16 ) page: 1447 - 1461   2017.6

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:WILEY  

    Flexible fitting is a computational algorithm to derive a new conformational model that conforms to low-resolution experimental data by transforming a known structure. A common application is against data from cryo-electron microscopy to obtain conformational models in new functional states. The conventional flexible fitting algorithms cannot derive correct structures in some cases due to the complexity of conformational transitions. In this study, we show the importance of conformational ensemble in the refinement process by performing multiple fittings trials using a variety of different force constants. Application to simulated maps of Ca2+ ATPase and diphtheria toxin as well as experimental data of release factor 2 revealed that for these systems, multiple conformations with similar agreement with the density map exist and a large number of fitting trials are necessary to generate good models. Clustering analysis can be an effective approach to avoid over-fitting models. In addition, we show that an automatic adjustment of the biasing force constants during the fitting process, implemented as replica-exchange scheme, can improve the success rate. (c) 2017 Wiley Periodicals, Inc.

    DOI: 10.1002/jcc.24785

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  32. Editorial overview: Macromolecular assemblies Reviewed

    Toshiyuki Shimizu, Florence Tama

    CURRENT OPINION IN STRUCTURAL BIOLOGY   Vol. 43   page: VII - ix   2017.4

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:CURRENT BIOLOGY LTD  

    DOI: 10.1016/j.sbi.2017.04.009

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  33. Local thermodynamics of the water molecules around single- and double-stranded DNA studied by grid inhomogeneous solvation theory Reviewed

    Miki Nakano, Hisae Tateishi-Karimata, Shigenori Tanaka, Florence Tama, Osamu Miyashita, Shu-ichi Nakano, Naoki Sugimoto

    CHEMICAL PHYSICS LETTERS   Vol. 660   page: 250 - 255   2016.9

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:ELSEVIER SCIENCE BV  

    Thermodynamic properties of water molecules around single- and double-stranded DNAs (ssDNAs and dsDNAs) with different sequences were investigated using grid inhomogeneous solvation theory. Free energies of water molecules solvating the minor groove of dsDNAs are lower than those near ssDNAs, while water molecules should be released during the formation of dsDNA. Free energies of water molecules around dsDNA are lower than those around ssDNA even in the second and third hydration shells. Our findings will help to clarify the role of water molecules in the formation of dsDNA from ssDNAs, thus facilitating the designs of drugs or nanomaterials using DNA. (C) 2016 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.cplett.2016.08.032

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  34. Hybrid approach for structural modeling of biological systems from X-ray free electron laser diffraction patterns Reviewed

    Atsushi Tokuhisa, Slavica Jonic, Florence Tama, Osamu Miyashita

    JOURNAL OF STRUCTURAL BIOLOGY   Vol. 194 ( 3 ) page: 325 - 336   2016.6

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    We present a new hybrid approach for structural modeling using X-ray free electron laser (XFEL) diffraction patterns from non-crystalline biological samples. Reconstruction of a 3D structure requires a large number of diffraction patterns; however, in the current XFEL experiments with biological systems, the analysis often relies on a small number of 2D diffraction patterns. In this study, we explore the strategies to identify plausible 3D structural models by combining the 2D analysis of such diffraction patterns with computational modeling (normal mode analysis or molecular dynamics simulations). As the first step toward such hybrid modeling, we established a protocol to assess the agreement between the model structure and the target XFEL diffraction pattern and showed that XFEL data can be used to study the conformational transitions of biological molecules. We tested the proposed algorithms using data of three biomolecular complexes of different sizes (elongation factor 2, CCM virus, and ribosome) and examined the experimental conditions that are required to perform such studies, in particular the XFEL beam intensity requirements. The results indicate that the current beam intensity is close to a strength that enables us to study conformational transitions of macromolecules, such as ribosomes. The proposed algorithm can be combined with molecular mechanics approaches, such as molecular dynamics simulations and normal mode analysis, to generate a large number of candidate structures to perform hybrid structural modeling. (C) 2016 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.jsb.2016.03.009

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  35. Thermodynamic properties of water molecules in the presence of cosolute depend on DNA structure: a study using grid inhomogeneous solvation theory Reviewed

    Nakano Miki, Tateishi-Karimata Hisae, Tanaka Shigenori, Tama Florence, Miyashita Osamu, Nakano Shu-ichi, Sugimoto Naoki

    NUCLEIC ACIDS RESEARCH   Vol. 43 ( 21 ) page: 10114-10125   2015.12

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    DOI: 10.1093/nar/gkv1133

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  36. Hybrid Electron Microscopy Normal Mode Analysis graphical interface and protocol Reviewed

    Carlos Oscar S. Sorzano, Jose Miguel de la Rosa-Trevin, Florence Tama, Slavica Jonic

    JOURNAL OF STRUCTURAL BIOLOGY   Vol. 188 ( 2 ) page: 134 - 141   2014.11

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    This article presents an integral graphical interface to the Hybrid Electron Microscopy Normal Mode Analysis (HEMNMA) approach that was developed for capturing continuous motions of large macromolecular complexes from single-particle EM images. HEMNMA was shown to be a good approach to analyze multiple conformations of a macromolecular complex but it could not be widely used in the EM field due to a lack of an integral interface. In particular, its use required switching among different software sources as well as selecting modes for image analysis was difficult without the graphical interface. The graphical interface was thus developed to simplify the practical use of HEMNMA. It is implemented in the open-source software package Xmipp 3.1 (http://xmipp.cnb.csic.es) and only a small part of it relies on MATLAB that is accessible through the main interface. Such integration provides the user with an easy way to perform the analysis of macromolecular dynamics and forms a direct connection to the single-particle reconstruction process. A step-by-step HEMNMA protocol with the graphical interface is given in full details in Supplementary material. The graphical interface will be useful to experimentalists who are interested in studies of continuous conformational changes of macromolecular complexes beyond the modeling of continuous heterogeneity in single particle reconstruction. (C) 2014 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.jsb.2014.09.005

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  37. Replica Exchange Molecular Dynamics Simulations Provide Insight into Substrate Recognition by Small Heat Shock Proteins Reviewed

    Sunita Patel, Elizabeth Vierling, Florence Tama

    BIOPHYSICAL JOURNAL   Vol. 106 ( 12 ) page: 2644 - 2655   2014.6

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    The small heat shock proteins (sHSPs) are a virtually ubiquitous and diverse group of molecular chaperones that can bind and protect unfolding proteins from irreversible aggregation. It has been suggested that intrinsic disorder of the N-terminal arm (NTA) of sHSPs is important for substrate recognition. To investigate conformations of the NTA that could recognize substrates we performed replica exchange molecular dynamics simulations. Behavior at normal and stress temperatures of the dimeric building blocks of dodecameric HSPs from wheat (Ta16.9) and pea (Ps18.1) were compared because they display high sequence similarity, but Ps18.1 is more efficient in binding specific substrates. In our simulations, the NTAs of the dimer are flexible and dynamic; however, rather than exhibiting highly extended conformations they retain considerable alpha-helical character and contacts with the conserved alpha-crystallin domain (ACD). Network analysis and clustering methods reveal that there are two major conformational forms designated either "open" or "closed" based on the relative position of the two NTAs and their hydrophobic solvent accessible surface area. The equilibrium constant for the closed to open transition is significantly different for Ta16.9 and Ps18.1, with the latter showing more open conformations at elevated temperature correlated with its more effective chaperone activity. In addition, the Ps18.1 NTAs have more hydrophobic solvent accessible surface than those of Ta16.9. NTA hydrophobic patches are comparable in size to the area buried in many protein-protein interactions, which would enable sHSPs to bind early unfolding intermediates. Reduced interactions of the Ps18.1 NTAs with each other and with the ACD contribute to the differences in dynamics and hydrophobic surface area of the two sHSPs. These data support a major role for the conformational equilibrium of the NTA in substrate binding and indicate features of the NTA that contribute to sHSP chaperone efficiency.

    DOI: 10.1016/j.bpj.2014.04.048

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  38. Macromolecular structures probed by combining single-shot free-electron laser diffraction with synchrotron coherent X-ray imaging Reviewed

    Marcus Gallagher-Jones, Yoshitaka Bessho, Sunam Kim, Jaehyun Park, Sangsoo Kim, Daewoong Nam, Chan Kim, Yoonhee Kim, Do Young Noh, Osamu Miyashita, Florence Tama, Yasumasa Joti, Takashi Kameshima, Takaki Hatsui, Kensuke Tono, Yoshiki Kohmura, Makina Yabashi, S. Samar Hasnain, Tetsuya Ishikawa, Changyong Song

    NATURE COMMUNICATIONS   Vol. 5   2014.5

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    Nanostructures formed from biological macromolecular complexes utilizing the self-assembly properties of smaller building blocks such as DNA and RNA hold promise for many applications, including sensing and drug delivery. New tools are required for their structural characterization. Intense, femtosecond X-ray pulses from X-ray free-electron lasers enable single-shot imaging allowing for instantaneous views of nanostructures at ambient temperatures. When combined judiciously with synchrotron X-rays of a complimentary nature, suitable for observing steady-state features, it is possible to perform ab initio structural investigation. Here we demonstrate a successful combination of femtosecond X-ray single-shot diffraction with an X-ray free-electron laser and coherent diffraction imaging with synchrotron X-rays to provide an insight into the nanostructure formation of a biological macromolecular complex: RNA interference microsponges. This newly introduced multimodal analysis with coherent X-rays can be applied to unveil nano-scale structural motifs from functional nanomaterials or biological nanocomplexes, without requiring a priori knowledge.

    DOI: 10.1038/ncomms4798

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  39. Iterative Elastic 3D-to-2D Alignment Method Using Normal Modes for Studying Structural Dynamics of Large Macromolecular Complexes Reviewed

    Qiyu Jin, Carlos Oscar S. Sorzano, Jose Miguel de la Rosa-Trevin, Jose Roman Bilbao-Castro, Rafael Nunez-Ramirez, Oscar Llorca, Florence Tama, Slavica Jonic

    STRUCTURE   Vol. 22 ( 3 ) page: 496 - 506   2014.3

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    This article presents a method to study large-scale conformational changes by combining electron microscopy (EM) single-particle image analysis and normal mode analysis (NMA). It is referred to as HEMNMA, which stands for hybrid electron microscopy normal mode analysis. NMA of a reference structure (atomic-resolution structure or EM volume) is used to predict possible motions that are then confronted with EM images within an automatic iterative elastic 3D-to-2D alignment procedure to identify actual motions in the imaged samples. HEMNMA can be used to extensively analyze the conformational changes and may be used in combination with classic discrete procedures. The identified conformations allow modeling of deformation pathways compatible with the experimental data. HEMNMA was tested with synthetic and experimental data sets of E. coli 70S ribosome, DNA polymerase Pol alpha and B subunit complex of the eukaryotic primosome, and tomato bushy stunt virus.

    DOI: 10.1016/j.str.2014.01.004

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  40. ELASTIC IMAGE REGISTRATION TO FULLY EXPLORE MACROMOLECULAR DYNAMICS BY ELECTRON MICROSCOPY Reviewed

    Jin Qiyu, Sanchez Sorzano Carlos Oscar, Callebaut Isabelle, Tama Florence, Jonic Slavica

    2014 IEEE INTERNATIONAL CONFERENCE ON IMAGE PROCESSING (ICIP)     page: 2075-2079   2014

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  41. ELASTIC IMAGE REGISTRATION TO FULLY EXPLORE MACROMOLECULAR DYNAMICS BY ELECTRON MICROSCOPY Reviewed

    Qiyu Jin, Carlos Oscar Sanchez Sorzano, Isabelle Callebaut, Florence Tama, Slavica Jonic

    2014 IEEE INTERNATIONAL CONFERENCE ON IMAGE PROCESSING (ICIP)     page: 2075 - 2079   2014

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    Structural changes are critical for biological functions of proteins and describing conformational changes in large macromolecular complexes is a major challenge. We have recently developed a hybrid method (HEMNMA) combining transmission electron microscopy (EM), normal mode analysis (NMA), and image analysis to study macromolecular dynamics. NMA is traditionally used to study macromolecular motions while HEMNMA provides insight into actual conformational changes seen by EM. HEMNMA uses normal modes to elastically align EM images with a reference structure in order to determine the conformations present in images and evaluate their pertinence. In this paper, we show how HEMNMA can be used with an atomic-resolution reference structure, using as an example the study of the conformational dynamics of Tomato Bushy Stunt Virus.

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  42. Allosteric Regulation of DNA Cleavage and Sequence-Specificity through Run-On Oligomerization Reviewed

    Dmitry Lyumkis, Heather Talley, Andrew Stewart, Santosh Shah, Chad K. Park, Florence Tama, Clinton S. Potter, Bridget Carragher, Nancy C. Horton

    STRUCTURE   Vol. 21 ( 10 ) page: 1848 - 1858   2013.10

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    SgrAl is a sequence specific DNA endonuclease that functions through an unusual enzymatic mechanism that is allosterically activated 200- to 500-fold by effector DNA, with a concomitant expansion of its DNA sequence specificity. Using single-particle transmission electron microscopy to reconstruct distinct populations of SgrAl oligomers, we show that in the presence of allosteric, activating DNA, the enzyme forms regular, repeating helical structures characterized by the addition of DNA-binding dimeric SgrAl subunits in a run-on manner. We also present the structure of oligomeric SgrAl at 8.6 angstrom resolution, demonstrating the conformational state of SgrAl in its activated form. Activated and oligomeric SgrAl displays key protein-protein interactions near the helix axis between its N termini, as well as allosteric protein-DNA interactions that are required for enzymatic activation. The hybrid approach reveals an unusual mechanism of enzyme activation that explains SgrAl's oligomerization and allosteric behavior.

    DOI: 10.1016/j.str.2013.08.012

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  43. 3DEM Loupe: analysis of macromolecular dynamics using structures from electron microscopy Reviewed

    R. Nogales-Cadenas, S. Jonic, F. Tama, A. A. Arteni, D. Tabas-Madrid, M. Vazquez, A. Pascual-Montano, C. O. S. Sorzano

    NUCLEIC ACIDS RESEARCH   Vol. 41 ( W1 ) page: W363 - W367   2013.7

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    Electron microscopy (EM) provides access to structural information of macromolecular complexes in the 3-20 angstrom resolution range. Normal mode analysis has been extensively used with atomic resolution structures and successfully applied to EM structures. The major application of normal modes is the identification of possible conformational changes in proteins. The analysis can throw light on the mechanism following ligand binding, protein-protein interactions, channel opening and other functional macromolecular movements. In this article, we present a new web server, 3DEM Loupe, which allows normal mode analysis of any uploaded EM volume using a user-friendly interface and an intuitive workflow. Results can be fully explored in 3D through animations and movies generated by the server. The application is freely available at http://3demloupe.cnb.csic.es.

    DOI: 10.1093/nar/gkt385

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  44. Consensus among multiple approaches as a reliability measure for flexible fitting into cryo-EM data Reviewed

    Aqeel Ahmed, Florence Tama

    JOURNAL OF STRUCTURAL BIOLOGY   Vol. 182 ( 2 ) page: 67 - 77   2013.5

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    Cryo-electron microscopy (cryo-EM) can provide low-resolution density maps of large macromolecular assemblies. As the number of structures deposited in the Protein Data Bank by fitting a high-resolution structure into a low-resolution cryo-EM map is increasing, there is a need to revise the protocols and improve the measures for fitting. A recent study suggested using a combination of multiple automated flexible fitting approaches to improve the interpretation of cryo-EM data. The current work further explores the use of multiple approaches by validating this "consensus" fitting approach and deriving a local reliability measure. Here four different flexible fitting approaches are applied for fitting an initial structure into a simulated density map of known target structure from a dataset of proteins. It is found that the models produced from different approaches often have a consensus in conformation and are also near to the target structure, whereas cases not showing consensus are away from the target. A high correlation is also observed between the RMSF profiles calculated with respect to the average and the target structures, which indicates that the relation between consensus and accuracy can also be extended to a per-residue level. Therefore, the RMSF among the fitted models is proposed as a local reliability measure, which can be used to assess the reliability of the fit at specific regions. Hence, we encourage the community to use consensus flexible fitting with different methods to report on local reliability of the resulting models and improve the interpretation of cryo-EM data. (c) 2013 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.jsb.2013.02.002

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  45. Steered Molecular Dynamics Simulations of a Type IV Pilus Probe Initial Stages of a Force-Induced Conformational Transition Reviewed

    Joseph L. Baker, Nicolas Biais, Florence Tama

    PLOS COMPUTATIONAL BIOLOGY   Vol. 9 ( 4 )   2013.4

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    Type IV pili are long, protein filaments built from a repeating subunit that protrudes from the surface of a wide variety of infectious bacteria. They are implicated in a vast array of functions, ranging from bacterial motility to microcolony formation to infection. One of the most well-studied type IV filaments is the gonococcal type IV pilus (GC-T4P) from Neisseria gonorrhoeae, the causative agent of gonorrhea. Cryo-electron microscopy has been used to construct a model of this filament, offering insights into the structure of type IV pili. In addition, experiments have demonstrated that GC-T4P can withstand very large tension forces, and transition to a force-induced conformation. However, the details of force-generation, and the atomic-level characteristics of the force-induced conformation, are unknown. Here, steered molecular dynamics (SMD) simulation was used to exert a force in silico on an 18 subunit segment of GC-T4P to address questions regarding the nature of the interactions that lead to the extraordinary strength of bacterial pili. SMD simulations revealed that the buried pilin alpha 1 domains maintain hydrophobic contacts with one another within the core of the filament, leading to GC-T4P's structural stability. At the filament surface, gaps between pilin globular head domains in both the native and pulled states provide water accessible routes between the external environment and the interior of the filament, allowing water to access the pilin alpha 1 domains as reported for VC-T4P in deuterium exchange experiments. Results were also compared to the experimentally observed force-induced conformation. In particular, an exposed amino acid sequence in the experimentally stretched filament was also found to become exposed during the SMD simulations, suggesting that initial stages of the force induced transition are well captured. Furthermore, a second sequence was shown to be initially hidden in the native filament and became exposed upon stretching.

    DOI: 10.1371/journal.pcbi.1003032

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  46. Molecular Model of a Soluble Guanylyl Cyclase Fragment Determined by Small-Angle X-ray Scattering and Chemical Cross-Linking Reviewed

    Bradley G. Fritz, Sue A. Roberts, Aqeel Ahmed, Linda Breci, Wenzhou Li, Andrzej Weichsel, Jacqueline L. Brailey, Vicki H. Wysocki, Florence Tama, William R. Montfort

    BIOCHEMISTRY   Vol. 52 ( 9 ) page: 1568 - 1582   2013.3

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    Soluble guanylyl/guanylate cyclase (sGC) converts GTP to cGMP after binding nitric oxide, leading to smooth muscle relaxation and vasodilation. Impaired sGC activity is common in cardiovascular disease, and sGC stimulatory compounds are vigorously sought. sGC is a 150 kDa heterodimeric protein with two H-NOX domains (one with heme, one without), two PAS domains, a coiled-coil domain, and two cyclase domains. Binding of NO to the sGC heme leads to proximal histidine release and stimulation of catalytic activity. To begin to understand how binding leads to activation, we examined truncated sGC proteins from Manduca sexta (tobacco hornworm) that bind NO, CO, and stimulatory compound YC-1 but lack the cyclase domains. We determined the overall shape of truncated M. sexta sGC using analytical ultracentrifugation and small-angle X-ray scattering (SAXS), revealing an elongated molecule with dimensions of 115 angstrom x 90 angstrom x 75 angstrom. Binding of NO, CO, or YC-1 had little effect on shape. Using chemical cross-linking and tandem mass spectrometry, we identified 20 intermolecular contacts, allowing us to fit homology models of the individual domains into the SAXS-derived molecular envelope. The resulting model displays a central parallel coiled-coil platform upon which the H-NOX and PAS domains are assembled. The beta(1) H-NOX and alpha(1) PAS domains are in contact and form the core signaling complex, while the alpha(1) H-NOX domain can be removed without a significant effect on ligand binding or overall shape. Removal of 21 residues from the C-terminus yields a protein with dramatically increased proximal histidine release rates upon NO binding.

    DOI: 10.1021/bi301570m

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  47. Network visualization of conformational sampling during molecular dynamics simulation Reviewed

    Logan S. Ahlstrom, Joseph Lee Baker, Kent Ehrlich, Zachary T. Campbell, Sunita Patel, Ivan I. Vorontsov, Florence Tama, Osamu Miyashita

    Journal of Molecular Graphics and Modelling   Vol. 46   page: 140 - 149   2013

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    Effective data reduction methods are necessary for uncovering the inherent conformational relationships present in large molecular dynamics (MD) trajectories. Clustering algorithms provide a means to interpret the conformational sampling of molecules during simulation by grouping trajectory snapshots into a few subgroups, or clusters, but the relationships between the individual clusters may not be readily understood. Here we show that network analysis can be used to visualize the dominant conformational states explored during simulation as well as the connectivity between them, providing a more coherent description of conformational space than traditional clustering techniques alone. We compare the results of network visualization against 11 clustering algorithms and principal component conformer plots. Several MD simulations of proteins undergoing different conformational changes demonstrate the effectiveness of networks in reaching functional conclusions. © 2013 Elsevier Inc.

    DOI: 10.1016/j.jmgm.2013.10.003

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    PubMed

  48. Twelve Transmembrane Helices Form the Functional Core of Mammalian MATE1 (Multidrug and Toxin Extruder 1) Protein Reviewed

    Xiaohong Zhang, Xiao He, Joseph Baker, Florence Tama, Geoffrey Chang, Stephen H. Wright

    JOURNAL OF BIOLOGICAL CHEMISTRY   Vol. 287 ( 33 ) page: 27971 - 27982   2012.8

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    The x-ray structure of the prototypic MATE family member, NorM from Vibrio cholerae, reveals a protein fold composed of 12 transmembrane helices (TMHs), confirming hydropathy analyses of the majority of (prokaryotic and plant) MATE transporters. However, the mammalian MATEs are generally predicted to have a 13th TMH and an extracellular C terminus. Here we affirm this prediction, showing that the C termini of epitope-tagged, full-length human, rabbit, and mouse MATE1 were accessible to antibodies from the extracellular face of the membrane. Truncation of these proteins at or near the predicted junction between the 13th TMH and the long cytoplasmic loop that precedes it resulted in proteins that (i) trafficked to the membrane and (ii) interacted with antibodies only after permeabilization of the plasma membrane. CHO cells expressing rbMate1 truncated at residue Gly-545 supported levels of pH-sensitive transport similar to that of cells expressing the full-length protein. Although the high transport rate of the Gly-545 truncation mutant was associated with higher levels of membrane expression (than full-length MATE1), suggesting the 13th TMH may influence substrate translocation, the selectivity profile of the mutant indicated that TMH13 has little impact on ligand binding. We conclude that the functional core of MATE1 consists of 12 (not 13) TMHs. Therefore, we used the x-ray structure of NorM to develop a homology model of the first 12 TMHs of MATE1. The model proved to be stable in molecular dynamic simulations and agreed with topology evident from preliminary cysteine scanning of intracellular versus extracellular loops.

    DOI: 10.1074/jbc.M112.386979

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  49. Simulations of substrate transport in the multidrug transporter EmrD Reviewed

    Joseph Baker, Stephen H. Wright, Florence Tama

    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS   Vol. 80 ( 6 ) page: 1620 - 1632   2012.6

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    EmrD is a multidrug resistance (MDR) transporter from Escherichia coli, which is involved in the efflux of amphipathic compounds from the cytoplasm, and the first MDR member of the major facilitator superfamily to be crystallized. Molecular dynamics simulation of EmrD in a phospholipid bilayer was used to characterize the conformational dynamics of the protein. Motions that support a previously proposed lateral diffusion pathway for substrate from the cytoplasmic membrane leaflet into the EmrD central cavity were observed. In addition, the translocation pathway of meta-chloro carbonylcyanide phenylhydrazone (CCCP) was probed using both standard and steered molecular dynamics simulation. In particular, interactions of a few specific residues with CCCP have been identified. Finally, a large motion of two residues, Val 45 and Leu 233, was observed with the passage of CCCP into the periplasmic space, placing a lower bound on the extent of opening required at this end of the protein for substrate transport. Overall, our simulations probe details of the transport pathway, motions of EmrD at an atomic level of detail, and offer new insights into the functioning of MDR transporters. Proteins 2012; (c) 2012 Wiley Periodicals, Inc.

    DOI: 10.1002/prot.24056

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  50. Twelve transmembrane helices form the functional core of mammalian MATE1 Reviewed

    Zhang Xiaohong, He Xiao, Baker Joseph, Tama Florence, Chang Geoffrey, Wright Stephen

    FASEB JOURNAL   Vol. 26   page: .   2012.4

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  51. Twelve transmembrane helices form the functional core of mammalian MATE1 Reviewed

    Xiaohong Zhang, Xiao He, Joseph Baker, Florence Tama, Geoffrey Chang, Stephen Wright

    FASEB JOURNAL   Vol. 26   2012.4

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  52. Consensus among flexible fitting approaches improves the interpretation of cryo-EM data Reviewed

    Aqeel Ahmed, Paul C. Whitford, Karissa Y. Sanbonmatsu, Florence Tama

    JOURNAL OF STRUCTURAL BIOLOGY   Vol. 177 ( 2 ) page: 561 - 570   2012.2

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    Cryo-elecron microscopy (cryo-EM) can provide important structural information of large macromolecular assemblies in different conformational states. Recent years have seen an increase in structures deposited in the Protein Data Bank (PDB) by fitting a high-resolution structure into its low-resolution cryo-EM map. A commonly used protocol for accommodating the conformational changes between the X-ray structure and the cryo-EM map is rigid body fitting of individual domains. With the emergence of different flexible fitting approaches, there is a need to compare and revise these different protocols for the fitting. We have applied three diverse automated flexible fitting approaches on a protein dataset for which rigid domain fitting (RDF) models have been deposited in the PDB. In general, a consensus is observed in the conformations, which indicates a convergence from these theoretically different approaches to the most probable solution corresponding to the cryo-EM map. However, the result shows that the convergence might not be observed for proteins with complex conformational changes or with missing densities in cryo-EM map. In contrast, RDF structures deposited in the PDB can represent conformations that not only differ from the consensus obtained by flexible fitting but also from X-ray crystallography. Thus, this study emphasizes that a "consensus" achieved by the use of several automated flexible fitting approaches can provide a higher level of confidence in the modeled configurations. Following this protocol not only increases the confidence level of fitting, but also highlights protein regions with uncertain fitting. Hence, this protocol can lead to better interpretation of cryo-EM data. (C) 2011 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.jsb.2011.10.002

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  53. Phosphorylated Smooth Muscle Heavy Meromyosin Shows an Open Conformation: Implications for the Structure of Myosin with One Head Phosphorylated Reviewed

    Taylor Kenneth A., Baumann Bruce A. J., Taylor Dianne W., Huang Zhong, Tama Florence, Fagnant Patricia M., Trybus Kathleen M.

    BIOPHYSICAL JOURNAL   Vol. 102 ( 3 ) page: 159A-159A   2012.1

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  54. Phosphorylated Smooth Muscle Heavy Meromyosin Shows an Open Conformation Linked to Activation Reviewed

    Bruce A. J. Baumann, Dianne W. Taylor, Zhong Huang, Florence Tama, Patricia M. Fagnant, Kathleen M. Trybus, Kenneth A. Taylor

    JOURNAL OF MOLECULAR BIOLOGY   Vol. 415 ( 2 ) page: 274 - 287   2012.1

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    Smooth muscle myosin and smooth muscle heavy meromyosin (smHMM) are activated by regulatory light chain phosphorylation, but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head head interactions between motor domains. The "free head" can bind to actin, but the actin binding interface of the "blocked head" is involved in interactions with the free head. We report here a three-dimensional structure for phosphorylated, active smHMM obtained using electron crystallography of two-dimensional arrays. Head head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state but occur between different molecules, not within the same molecule. The light chain binding domain structure of phosphorylated smHMM differs markedly from that of the "blocked" head of dephosphorylated smHMM. We hypothesize that regulatory light chain phosphorylation opens the inhibited conformation primarily by its effect on the blocked head. Singly phosphorylated smHMM is not compatible with the closed conformation if the blocked head is phosphorylated. This concept has implications for the extent of myosin activation at low levels of phosphorylation in smooth muscle. (C) 2011 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.jmb.2011.10.047

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  55. Excited states of ribosome translocation revealed through integrative molecular modeling Reviewed

    Paul C. Whitford, Aqeel Ahmed, Yanan Yu, Scott P. Hennelly, Florence Tama, Christian M. T. Spahn, Jose N. Onuchic, Karissa Y. Sanbonmatsu

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 108 ( 47 ) page: 18943 - 18948   2011.11

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    The dynamic nature of biomolecules leads to significant challenges when characterizing the structural properties associated with function. While X-ray crystallography and imaging techniques (such as cryo-electron microscopy) can reveal the structural details of stable molecular complexes, strategies must be developed to characterize configurations that exhibit only marginal stability (such as intermediates) or configurations that do not correspond to minima on the energy landscape (such as transition-state ensembles). Here, we present a methodology (MDfit) that utilizes molecular dynamics simulations to generate configurations of excited states that are consistent with available biophysical and biochemical measurements. To demonstrate the approach, we present a sequence of configurations that are suggested to be associated with transfer RNA (tRNA) movement through the ribosome (translocation). The models were constructed by combining information from X-ray crystallography, cryo-electron microscopy, and biochemical data. These models provide a structural framework for translocation that may be further investigated experimentally and theoretically to determine the precise energetic character of each configuration and the transition dynamics between them.

    DOI: 10.1073/pnas.1108363108

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  56. Structure modeling from small angle X-ray scattering data with elastic network normal mode analysis Reviewed

    Osamu Miyashita, Christian Gorba, Florence Tama

    JOURNAL OF STRUCTURAL BIOLOGY   Vol. 173 ( 3 ) page: 451 - 460   2011.3

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    Computational algorithms to construct structural models from SAXS experimental data are reviewed. SAXS data provides a wealth of information to study the structure and dynamics of biological molecules, however it does not provide atomic details of structures. Thus combining the low-resolution data with already known X-ray structure is a common approach to study conformational transitions of biological molecules. This review provides a survey of SAXS modeling approaches. In addition, we will discuss theoretical backgrounds and performance of our approach, in which elastic network normal mode analysis is used to predict reasonable conformational transitions from known X-ray structures, and find alternative conformations that are consistent with SAXS data. (C) 2010 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.jsb.2010.09.008

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  57. Three-dimensional structure of the anthrax toxin pore inserted into lipid nanodiscs and lipid vesicles Reviewed

    H. Katayama, J. Wang, F. Tama, L. Chollet, E. P. Gogol, R. J. Collier, M. T. Fisher

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 107 ( 8 ) page: 3453 - 3457   2010.2

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    A major goal in understanding the pathogenesis of the anthrax bacillus is to determine how the protective antigen (PA) pore mediates translocation of the enzymatic components of anthrax toxin across membranes. To obtain structural insights into this mechanism, we constructed PA-pore membrane complexes and visualized them by using negative-stain electron microscopy. Two populations of PA pores were visualized in membranes, vesicle-inserted and nanodisc-inserted, allowing us to reconstruct two virtually identical PA-pore structures at 22-angstrom resolution. Reconstruction of a domain 4-truncated PA pore inserted into nanodiscs showed that this domain does not significantly influence pore structure. Normal mode flexible fitting of the x-ray crystallographic coordinates of the PA prepore indicated that a prominent flange observed within the pore lumen is formed by the convergence of mobile loops carrying Phe427, a residue known to catalyze protein translocation. Our results have identified the location of a crucial functional element of the PA pore and documented the value of combining nanodisc technology with electron microscopy to examine the structures of membrane-interactive proteins.

    DOI: 10.1073/pnas.1000100107

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  58. Biased coarse-grained molecular dynamics simulation approach for flexible fitting of X-ray structure into cryo electron microscopy maps Reviewed

    Ivan Grubisic, Maxim N. Shokhirev, Marek Orzechowski, Osamu Miyashita, Florence Tama

    JOURNAL OF STRUCTURAL BIOLOGY   Vol. 169 ( 1 ) page: 95 - 105   2010.1

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    Several approaches have been introduced to interpret, in terms of high-resolution structure, low-resolution structural data as obtained from cryo-EM. As conformational changes are often observed in biological molecules, these techniques need to take into account the flexibility of proteins. Flexibility has been described in terms of movement between rigid domains and between rigid secondary structure elements, which present some limitations for studying dynamical properties. Normal mode analysis has also been used, but is limited to medium resolution data. All-atom molecular dynamics fitting techniques are more appropriate to fit structures into higher-resol uti on data as full protein flexibility is considered, but are cumbersome in terms of computational time. Here, we introduce a coarse-grained approach; a Go-model was used to represent biological molecules, combined with biased molecular dynamics to reproduce accurately conformational transitions. Illustrative examples on simulated data are shown. Accurate fittings can be obtained for resolution ranging from 5 to 20 angstrom. The approach was also tested on experimental data of Elongation Factor G and Escherichia coli RNA polymerase, where its validity is compared to previous models obtained from different techniques. This comparison demonstrates that quantitative flexible techniques, as opposed to manual docking, need to be considered to interpret low-resolution data. (C) 2009 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.jsb.2009.09.010

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  59. Flexible Fitting of High-Resolution X-Ray Structures into Cryoelectron Microscopy Maps Using Biased Molecular Dynamics Simulations Reviewed

    Marek Orzechowski, Florence Tama

    BIOPHYSICAL JOURNAL   Vol. 95 ( 12 ) page: 5692 - 5705   2008.12

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    A methodology for flexible fitting of all-atom high-resolution structures into low-resolution cryoelectron microscopy (cryo-EM) maps is presented. Flexibility of the modeled structure is simulated by classical molecular dynamics and an additional effective potential is introduced to enhance the fitting process. The additional potential is proportional to the correlation coefficient between the experimental cryo-EM map and a synthetic map generated for an all-atom structure being fitted to the map. The additional forces are calculated as a gradient of the correlation coefficient. During the molecular dynamics simulations under the additional forces, the molecule undergoes a conformational transition that maximizes the correlation coefficient, which results in a high-accuracy fit of all-atom structure into a cryo-EM map. Using five test proteins that exhibit structural rearrangement during their biological activity, we demonstrate performance of our method. We also test our method on the experimental cryo-EM of elongation factor G and show that the model obtained is comparable to previous studies. In addition, we show that over fitting can be avoided by assessing the quality of the fitted model in terms of correlation coefficient and secondary structure preservation.

    DOI: 10.1529/biophysj.108.139451

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  60. Normal-mode flexible fitting of high-resolution structure of biological molecules toward one-dimensional low-resolution data Reviewed

    Christian Gorba, Osamu Miyashita, Florence Tama

    BIOPHYSICAL JOURNAL   Vol. 94 ( 5 ) page: 1589 - 1599   2008.3

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    We present a method for reconstructing a 3D structure from a pair distribution function by flexibly fitting known x-ray structures toward a conformation that agrees with the low-resolution data. This method uses a linear combination of low-frequency normal modes from elastic-network description of the molecule in an iterative manner to deform the structure optimally to conform to the target pair distribution function. A simple function, pair distance distribution function between atoms, is chosen as a test model to establish computational algorithms-optimization algorithm and scoring function-that can utilize low-resolution 1 D data. To select a correct structural model based on less information, we developed a scoring function that takes into account a characteristic of pair distribution functions. In addition, we employ a new optimization algorithm, the trusted region method, that relies on both first and second derivatives of the scoring function. Illustrative results of our studies on simulated 1D data from five different proteins, for which large conformational changes are known to occur, are presented.

    DOI: 10.1529/biophysj.107.122218

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  61. Normal Mode Analysis Techniques in Structural Biology. Invited Reviewed

    Miyashita, O, Tama, F

    eLS     2007

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    DOI: 10.1002/9780470015902.a0020204

  62. Removal of divalent cations induces structural transitions in Red clover necrotic mosaic virus, revealing a potential mechanism for RNA release Reviewed

    Michael B. Sherman, Richard H. Guenther, Florence Tama, Tim L. Sit, Charles L. Brooks, Albert M. Mikhailov, Elena V. Orlova, Timothy S. Baker, Steven A. Lommel

    JOURNAL OF VIROLOGY   Vol. 80 ( 21 ) page: 10395 - 10406   2006.11

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    The structure of Red clover necrotic mosaic virus (RCNW), an icosahedral plant virus, was resolved to 8.5 by cryoelectron microscopy. The virion capsid has prominent surface protrusions and subunits with a clearly defined shell and protruding domains. The structures of both the individual capsid protein (CP) subunits and the entire virion capsid are consistent with other species in the Tombusviridae family. Within the RCNNW capsid, there is a clearly defined inner cage formed by complexes of genomic RNA and the amino termini of CP subunits. An RCNNW virion has approximately 390 +/- 30 Ca2+ ions bound to the capsid and 420 +/- 25 Mg2+ ions thought to be in the interior of the capsid. Depletion of both Ca2+ and Mg2+ ions from RCNMV leads to significant structural changes, including (i) formation of 11- to 13-A-diameter channels that extend through the capsid and (ii) significant reorganization within the interior of the capsid. Genomic RNA within native capsids containing both Ca2+ and Mg2+ ions is extremely resistant to nucleases, but depletion of both of these cations results in nuclease sensitivity, as measured by a significant reduction in RCNMV infectivity. These results indicate that divalent cations play a central role in capsid dynamics and suggest a mechanism for the release of viral RNA in low-divalent-cation environments such as those found within the cytoplasm of a cell.

    DOI: 10.1128/JVI.01137-06

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  63. Structure of the E-coli protein-conducting channel bound to a translating ribosome (vol 438, pg 318, 2005) Reviewed

    Kakoli Mitra, Christiane Schaffitzel, Tanvir Shaikh, Florence Tama, Simon Jenni, Charles L. Brooks, Nenad Ban, Joachim Frank

    NATURE   Vol. 443 ( 7112 ) page: 718 - 718   2006.10

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    DOI: 10.1038/nature05086

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  64. Model of the toxic complex of anthrax: Responsive conformational changes in both the lethal factor and the protective antigen heptamer Reviewed

    Florence Tama, Gang Ren, Charles L. Brooks, Alok K. Mitra

    PROTEIN SCIENCE   Vol. 15 ( 9 ) page: 2190 - 2200   2006.9

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    The toxic complex of anthrax is formed when the monomeric protective antigen (PA) (83 kDa), while bound to its cell-surface receptor, is first converted to PA63 heptamers (PA63h) following N-terminal proteolytic cleavage, and then lethal (LF) (90 kDa) or edema factor (EF) binds to the heptamer. We report a "pseudoatomic'' model for the complex of PA63h and full-length LF determined by applying the normal-mode flexible fitting procedure to a similar to 18 angstrom cryo-electron microscopy (EM) density map of the complex. The model describes the interacting surface that buries a total area of similar to 10,140 angstrom(2) comprising; similar to 40% charged, and; 30% each of polar and hydrophobic residues. For the heptamer, the buried surface, composed of similar to 110 residues, involves primarily three monomers and includes for two, similar stretches of the polypeptide chain from domain 1. For LF, the interface again involves; 110 residues, mostly from the N-terminal domain I (LFN), and the structurally homologous C-terminal domain IV. Most interestingly, bound LF displays a marked conformational change resulting from a ``collapse'' of domains I, III, and IVon domain II, with the largest movement of similar to 9 angstrom noted for domain I. On the other hand, primarily, rigid-body movements, larger than; 10 A for three PA63 monomers, cause the hourglass-shaped heptamer lumen to enlarge by as much as similar to 50% near the middle of the molecule. Such concerted structural rearrangements in LF and the heptamer can facilitate ingress of the ligand into the heptamer lumen prior to unfolding and release through the PA63h channel formed in the acidic late endosomal membrane.

    DOI: 10.1110/ps.062293906

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  65. Electrostatic properties of cowpea chlorotic mottle virus and cucumber mosaic virus capsids Reviewed

    R Konecny, J Trylska, F Tama, DQ Zhang, NA Baker, CL Brooks, JA McCammon

    BIOPOLYMERS   Vol. 82 ( 2 ) page: 106 - 120   2006.6

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    Electrostatic properties of cowpea chlorotic mottle virus (CCMV) and cucumber mosaic virus (CMV) were investigated using numerical solutions to the Poisson-Boltzmann equation. Experimentally, it has been shown that CCMV particles swell in the absence of divalent cations when the pH is raised from 5 to 7. CMV, although structurally homologous, does not undergo this transition. An analysis of the calculated electrostatic potential confirms that a strong electrostatic repulsion at the calcium-binding sites in the CCMV capsid is most likely the driving force for the capsid swelling process during the release of calcium. The binding interaction between the encapsulated genome material (RNA) inside of the capsid and the inner capsid shell is weakened during the swelling transition. This probably aids in the RNA release process, but it is unlikely that the RNA is released through capsid openings due to unfavorable electrostatic interaction between the RNA and capsid inner shell residues at these openings. Calculations of the calcium binding energies show that Ca2+ can bind both to the native and swollen forms of the CCMV virion. Favorable binding to the swollen form suggests that Ca2+ ions can induce the capsid contraction and stabilize the native form. (c) 2005 Wiley Periodicals, Inc.

    DOI: 10.1002/bip.20409

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  66. Symmetry, form, and shape: Guiding principles for robustness in macromolecular machines Reviewed

    Florence Tama, Charles L. Brooks

    ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE   Vol. 35   page: 115 - 133   2006

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    Computational studies of large macromolecular assemblages have come a long way during the past 10 years. With the explosion of computer power and parallel computing, timescales of molecular dynamics simulations have been extended far beyond the hundreds of picoseconds timescale. However, limitations remain for studies of large-scale conformational changes occurring on timescales beyond nanoseconds, especially for large macromolecules. In this review, we describe recent methods based on normal mode analysis that have enabled us to study dynamics on the microsecond timescale for large macromolecules using different levels of coarse graining, from atomically detailed models to those employing only low-resolution structural information. Emerging from such studies is a control principle for robustness in Nature's machines. We discuss this idea in the context of large-scale functional reorganization of the ribosome, virus particles, and the muscle protein myosin.

    DOI: 10.1146/annurev.biophys.35.040405.102010

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  67. Structure of the E-coli protein-conducting channel bound to a translating ribosome Reviewed

    K Mitra, C Schaffitzel, T Shaikh, F Tama, S Jenni, CL Brooks, N Ban, J Frank

    NATURE   Vol. 438 ( 7066 ) page: 318 - 324   2005.11

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    Secreted and membrane proteins are translocated across or into cell membranes through a protein-conducting channel (PCC). Here we present a cryo-electron microscopy reconstruction of the Escherichia coli PCC, SecYEG, complexed with the ribosome and a nascent chain containing a signal anchor. This reconstruction shows a messenger RNA, three transfer RNAs, the nascent chain, and detailed features of both a translocating PCC and a second, non-translocating PCC bound to mRNA hairpins. The translocating PCC forms connections with ribosomal RNA hairpins on two sides and ribosomal proteins at the back, leaving a frontal opening. Normal mode-based flexible fitting of the archaeal SecYE beta structure into the PCC electron microscopy densities favours a front-to-front arrangement of two SecYEG complexes in the PCC, and supports channel formation by the opening of two linked SecY halves during polypeptide translocation. On the basis of our observation in the translocating PCC of two segregated pores with different degrees of access to bulk lipid, we propose a model for co-translational protein translocation.

    DOI: 10.1038/nature04133

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  68. The 13 angstrom structure of a chaperonin GroEL-protein substrate complex by cryo-electron microscopy Reviewed

    S Falke, F Tama, CL Brooks, EP Gogol, MT Fisher

    JOURNAL OF MOLECULAR BIOLOGY   Vol. 348 ( 1 ) page: 219 - 230   2005.4

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    The 13 angstrom resolution structures of GroEL bound to a single monomer of the protein substrate glutamine synthetase (GS(m)), as well as that of unliganded GroEL have been determined from a heterogeneous image population using cryo-electron microscopy (cryo-EM) coupled with single-particle image classification and reconstruction techniques. We combined structural data from cryo-EM maps and dynamic modeling, taking advantage of the known X-ray crystallographic structure and normal mode flexible fitting (NMFF) analysis, to describe the changes that occur in GroEL structure induced by GS(m) binding. The NMFF analysis reveals that the molecular movements induced by GS(m) binding propagate throughout the GroEL structure. The modeled molecular motions show that some domains undergo en bloc movements, while others show more complex independent internal movements. Interestingly, the substrate-bound apical domains of both the cis (GS(m)-bound ring) and trans (the opposite substrate-free ring) show counterclockwise rotations, in the same direction (though not as dramatic) as those documented for the ATP-GroEL-induced structure changes. The structural changes from the allosteric substrate protein-induced negative cooperativity between the GroEL rings involves upward concerted movements of both cis and trans equatorial domains toward the GS(m)-bound ring, while the inter-ring distances between the heptamer contact residues are maintained. Furthermore, the NMFF analysis identifies the secondary structural elements that are involved in the observed similar to 5 angstrom reduction in the diameter of the cavity opening in the unbound trans ring. Understanding the molecular basis of these substrate protein-induced structural changes across the heptamer rings provides insight into the origins of the allosteric negative cooperative effects that are transmitted over long distances (similar to 140 angstrom). (c) 2005 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.jmb.2005.02.027

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  69. Diversity and identity of mechanical properties of icosahedral viral capsids studied with elastic network normal mode analysis Reviewed

    F Tama, CL Brooks

    JOURNAL OF MOLECULAR BIOLOGY   Vol. 345 ( 2 ) page: 299 - 314   2005.1

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    We analyze the mechanical properties and putative dynamical fluctuations of a variety of viral capsids comprising different sizes and quasi-equivalent symmetries by performing normal mode analysis using the elastic network model. The expansion of the capsid to a swollen state is studied using normal modes and is compared with the experimentally observed conformational change for three of the viruses for which experimental data exist. We show that a combination of one or two normal modes captures remarkably well the overall translation that dominates the motion between the two conformational states, and reproduces the overall conformational change. We observe for all of the viral capsids that the nature of the modes is different. In particular for the T=7 virus, HK97, for which the shape of the capsid changes from spherical to faceted polyhedra, two modes are necessary to accomplish the conformational transition. In addition, we extend our study to viral capsids with other T numbers, and discuss the similarities and differences in the features of virus capsid conformational dynamics. We note that the pentamers generally have higher flexibility and propensity to move freely from the other capsomers, which facilitates the shape adaptation that may be important in the viral life cycle. (C) 2004 Elsevier ttd. All rights reserved.

    DOI: 10.1016/j.jmb.2004.10.054

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  70. The requirement for mechanical coupling between head and S2 domains in smooth muscle myosin ATPase regulation and its implications for dimeric motor function Reviewed

    F Tama, M Feig, J Liu, CL Brooks, KA Taylor

    JOURNAL OF MOLECULAR BIOLOGY   Vol. 345 ( 4 ) page: 837 - 854   2005.1

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    A combination of experimental structural data, homology modelling and elastic network normal mode analysis is used to explore how coupled motions between the two myosin heads and the dimerization domain (S2) in smooth muscle myosin 11 determine the domain movements required to achieve the inhibited state of this ATP-dependent molecular motor. These physical models rationalize the empirical requirement for at least two heptads of non-coiled a-helix at the junction between the myosin heads and S2, and the dependence of regulation on S2 length. The results correlate well with biochemical data regarding altered conformational-dependent solubility and stability. Structural models of the conformational transition between putative active states and the inhibited state show that torsional flexibility of the S2 a-helices is a key mechanical requirement for myosin 11 regulation. These torsional motions of the myosin heads about their coiled coil a-helices affect the S2 domain structure, which reciprocally affects the motions of the myosin heads. This inter-relationship may explain a large body of data on function of molecular motors that form dimers through a coiled-coil domain. (C) 2004 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.jmb.2004.10.084

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  71. Normal mode based flexible fitting of high-resolution structure into low-resolution experimental data from cryo-EM Reviewed

    F Tama, O Miyashita, CL Brooks

    JOURNAL OF STRUCTURAL BIOLOGY   Vol. 147 ( 3 ) page: 315 - 326   2004.9

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    A new method for the flexible fitting of high-resolution structures into low-resolution maps of macromolecular complexes from electron microscopy has been recently described in applications to simulated electron density maps. This method uses a linear combination of low-frequency normal modes in an iterative manner to deform the structure optimally to conform to the low-resolution electron density map. Gradient-following techniques in the coordinate space of collective normal modes are used to optimize the overall correlation coefficient between computed and measured electron densities. With this approach, multi-scale flexible fitting can be performed using all-atoms or Cot atoms. In this paper, illustrative studies of normal mode based flexible fitting to experimental cryo-EM maps are presented for three different systems. Large, functionally relevant conformational changes for elongation factor G bound to the ribosome, Escherichia coli RNA polymerase and cowpea chlorotic mottle virus are elucidated as the result of the application of NMFF from high-resolution structures to cryo-electron microscopy maps. (C) 2004 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.jsb.2004.03.002

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  72. Ribosome motions modulate electrostatic properties Reviewed

    J Trylska, R Konecny, F Tama, CL Brooks, JA McCammon

    BIOPOLYMERS   Vol. 74 ( 6 ) page: 423 - 431   2004.8

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    The electrostatic properties of the 70S ribosome of Thermus thermophilus were studied qualitatively by solving the Poisson-Boltzmann (PB) equation in aqueous solution and with physiological ionic strength. The electrostatic potential was calculated for conformations of the ribosome derived by recent normal mode analysis (Tama, F., et al. Proc Natl Acad Sci USA 2003 1001, 9319-9323) of the ratchet-like reorganization that occurs during translocation (Frank, J.; Agrawal, R. K. Nature 2000 406, 318-322). To solve the PB equation, effective parameters (charges and radii), applicable to a highly charged backbone model of the ribosome, were developed. Regions of positive potential were found at the binding site of the elongation factors G and Tu, as well as where the release factors bind. Large positive potential areas are especially pronounced around the L11 and L6 proteins. The region around the L1 protein is also positively charged, supporting the idea that L1 may interact with the E-site tRNA during its release from the ribosome after translocation. Functional rearrangement of the ribosome leads to electrostatic changes which may help the translocation of the tRNAs during the elongation stage. (C) 2004 Wiley Periodicals, Inc.

    DOI: 10.1002/bip.20093

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  73. Flexible multi-scale fitting of atomic structures into low-resolution electron density maps with elastic network normal mode analysis Reviewed

    F Tama, O Miyashita, CL Brooks

    JOURNAL OF MOLECULAR BIOLOGY   Vol. 337 ( 4 ) page: 985 - 999   2004.4

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    A novel method is presented for the quantitative flexible docking of high-resolution structure into low-resolution maps of macromolecular complexes from electron microscopy. This method uses a linear combination of low-frequency normal modes from elastic network description of the molecular framework in an iterative manner to deform the structure optimally to conform to the low-resolution electron density map. The methodology utilizes gradient following techniques in collective normal modes to locally optimize the overall correlation coefficient between computed and measured electron density. To evaluate the performance of our approach, several proteins, which undergo large conformational changes, have been studied. We demonstrate that refinement based on normal mode analysis provides an accurate and fast alternative for the flexible fitting of high-resolution structure into a low-resolution density map. Additionally, we show that lower resolution (multi-scale) structural models can be used for the normal mode searching in lieu of fully atomic models with little loss of overall accuracy. (C) 2004 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.jmb.2004.01.048

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  74. Topology representing neural networks reconcile biomolecular shape, structure, and dynamics Reviewed

    W Wriggers, P Chacon, JA Kovacs, F Tama, S Birmanns

    NEUROCOMPUTING   Vol. 56   page: 365 - 379   2004.1

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    Topology-representing networks (TRNs) generate reduced models of biomolecules and thereby facilitate the fitting of molecular fragments into large macromolecular complexes. The components of such complexes undergo a wide range of motions, and shapes observed at low resolution often deviate from the known atomic structures. What is required for the modeling of such motions is a combination of global shape constraints based on the low-resolution data with a local modeling of atomic interactions. We present a novel Motion Capture Network that freezes inessential degrees of freedom to maintain the stereochemistry of an atomic model. TRN-based deformable models retain much of the mechanical properties of biological macromolecules. The elastic models yield a decomposition of the predicted motion into vibrational normal modes and are amenable to interactive manipulation with haptic rendering software. (C) 2003 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.neucom.2003.09.007

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  75. Dynamic reorganization of the functionally active ribosome explored by normal mode analysis and cryo-electron microscopy Reviewed

    F Tama, M Valle, J Frank, CL Brooks

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 100 ( 16 ) page: 9319 - 9323   2003.8

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    Combining structural data for the ribosome from x-ray crystallography and cryo-electron microscopy with dynamic models based on elastic network normal mode analysis, an atomically detailed picture of functionally important structural rearrangements that occur during translocation is elucidated. The dynamic model provides a near-atomic description of the ratchet-like rearrangement of the 70S ribosome seen in cryo-electron microscopy, and permits the identification of bridging interactions that either facilitate the conformational switching or maintain structural integrity of the 50S/30S interface. Motions of the tRNAs residing in the A and P sites also suggest the early stages of tRNA translocation as a result of this ratchet-like movement. Displacement of the L1 stalk, alternately closing and opening the intersubunit space near the E site, is observed in the dynamic model, in line with growing experimental evidence for the role of this structural component in facilitating the exiting of tRNA. Finally, a hinge-like transition in the 30S ribosomal subunit, similar to that observed in crystal structures of this complex, is also manifest as a dynamic mode of the ribosome. The coincidence of these dynamic transitions with the individual normal modes of the ribosome and the good correspondence between these motions and those observed in experiment suggest an underlying principle of nature to exploit the shape of molecular assemblies such as the ribosome to provide robustness to functionally important motions.

    DOI: 10.1073/pnas.1632476100

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  76. Normal mode analysis with simplified models to investigate the global dynamics of biological systems Reviewed

    F Tama

    PROTEIN AND PEPTIDE LETTERS   Vol. 10 ( 2 ) page: 119 - 132   2003.4

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    Dynamical properties of macromolecules are increasingly being recognized as significantly contributing to biological functions, including catalysis, regulation of activity, etc. In this review, theoretical approaches to the study of dynamics of biological systems and their application are discussed. In particular, simplified models for the normal mode analysis are described.

    DOI: 10.2174/0929866033479077

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  77. Mega-Dalton biomolecular motion captured from electron microscopy reconstructions Reviewed

    P Chacon, F Tama, W Wriggers

    JOURNAL OF MOLECULAR BIOLOGY   Vol. 326 ( 2 ) page: 485 - 492   2003.2

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    The vibrational analysis of elastic models suggests that the essential motions of large biomolecular assemblies can be captured efficiently at an intermediate scale without requiring knowledge of the atomic structure. While prior work has established a theoretical foundation for this analysis, we demonstrate here on experimental electron microscopy maps that vibrational modes indeed describe functionally relevant movements of macromolecular machines. The clamp closure in bacterial RNA polymerase, the ratcheting of 30 S and 50 S subunits of the ribosome, and the dynamic flexibility of chaperonin CCT are extracted directly from single electron microscopy structures at 15-27 Angstrom resolution. The striking agreement of the presented results with experimentally observed motions suggests that the motion of the large scale machinery in the cell is surprisingly independent of detailed atomic interactions and can be quite reasonably described as a motion of elastic bodies. (C) 2003 Elsevier Science Ltd. All rights reserved.

    DOI: 10.1016/S0022-2836(02)01426-2

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  78. Exploring global distortions of biological macromolecules and assemblies from low-resolution structural information and elastic network theory Reviewed

    F Tama, W Wriggers, CL Brooks

    JOURNAL OF MOLECULAR BIOLOGY   Vol. 321 ( 2 ) page: 297 - 305   2002.8

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    A theory of elastic normal modes is described for the exploration of global distortions of biological structures and their assemblies based upon low-resolution image data. Structural information at low resolution, e.g. from density maps measured by cryogenic electron microscopy (cryo-EM), is used to construct discrete multi-resolution models for the electron density using the techniques of vector quantization. The elastic normal modes computed based on these discretized low-resolution models are found to compare well with the normal modes obtained at atomic resolution. The quality of the normal modes describing global displacements of the molecular system is found to depend on the resolution of the synthetic EM data and the extent of reductionism in the discretized representation. However, models that reproduce the functional rearrangements of our test set of molecules are achieved for realistic values of experimental resolution. Thus large conformational changes as occur during the functioning of biological macromolecules and assemblies can be elucidated directly from low-resolution structural data through the application of elastic normal mode theory and vector quantization. (C) 2002 Elsevier Science Ltd. All rights reserved.

    DOI: 10.1016/S0022-2836(02)00627-7

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  79. The mechanism and pathway of pH induced swelling in cowpea chlorotic mottle virus Reviewed

    F Tama, CL Brooks

    JOURNAL OF MOLECULAR BIOLOGY   Vol. 318 ( 3 ) page: 733 - 747   2002.5

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    Normal mode analysis based on a simplified energy function was used to study the swelling process of the icosahedral virus, cowpea chlorotic mottle virus (CCMV). Native state virus particles (coat proteins) of this T = 3 icosahedral virus have been shown to undergo a large conformational change to a swollen state when metal ions are removed or the pH is raised. A normal mode analysis based on the native state capsid showed one preferential direction, a breathing mode, that explains the majority of the structural rearrangement necessary to bring the native structure close to the swollen state.
    From the native form of CCMV, the structure can be displaced along the direction of a single breathing mode by different amounts to create several candidate swollen structures and a putative pathway for virus expansion. The R-factor between these predicted swollen capsid structures and experimental electron density from cryoelectron microscopy (cryo-EM) measurements is then calculated to indicate how well each structure satisfies the experimental measurements on the swollen capsid state. A decrease of the crystallographic R-factor value from similar to72% to similar to49% was observed for these simple incremental displacements along the breathing mode. The simultaneous displacement of the native structure along other relevant (symmetric, non-degenerate) modes produce a structure with an R-factor of 45%, which is further reduced to 43.9% after minimization: a value in good accord with models based on the EM data at 28 Angstrom resolution.
    Based on the incrementally expanded structures, a pathway for the swelling process has been proposed. Analysis of the intermediate structures along this pathway indicates a significant loss of interactions at the quasi-3-fold interfaces occurs in the initial stages of the swelling process and this serves as a trigger for the compact to swollen transition. Furthermore, the pH dependent swelling appears to be triggered by the titration of a single residue with an anomalous pK(a) value in the unswollen particle. (C) 2002 Elsevier Science Ltd. All rights reserved.

    DOI: 10.1016/S0022-2836(02)00135-3

    Web of Science

  80. Conformational change of proteins arising from normal mode calculations Reviewed

    F Tama, YH Sanejouand

    PROTEIN ENGINEERING   Vol. 14 ( 1 ) page: 1 - 6   2001.1

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    A normal mode analysis of 20 proteins in 'open' or 'closed' forms was performed using simple potential and protein models. The quality of the results was found to depend upon the form of the protein studied, normal modes obtained with the open form of a given protein comparing better with the conformational change than those obtained with the closed form. Moreover, when the motion of the protein is a highly collective one, then, in all cases considered, there is a single low-frequency normal mode whose direction compares well with the conformational change. When it is not, in most cases there is still a single low-frequency normal mode giving a good description of the pattern of the atomic displacements, as they are observed experimentally during the conformational change. Hence a lot of information on the nature of the conformational change of a protein is often found in a single low-frequency normal mode of its open form. Since this information can be obtained through the normal mode analysis of a model as simple as that used in the present study, it is likely that the property captured by such an analysis is for the most part a property of the shape of the protein itself. One of the points that has to be clarified now is whether or not amino acid sequences have been selected in order to allow proteins to follow a single normal mode direction, as least at the very beginning of their conformational change.

    DOI: 10.1093/protein/14.1.1

    Web of Science

  81. Building-block approach for determining low-frequency normal modes of macromolecules Reviewed

    F Tama, FX Gadea, O Marques, YH Sanejouand

    PROTEINS-STRUCTURE FUNCTION AND GENETICS   Vol. 41 ( 1 ) page: 1 - 7   2000.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-LISS  

    Normal mode analysis of proteins of various sizes, ranging from 46 (crambin) up to 858 residues (dimeric citrate synthase) were performed, by using standard approaches, as well as a recently proposed method that rests on the hypothesis that low-frequency normal modes of proteins can be described as pure rigid-body motions of blocks of consecutive amino-acid residues. Such a hypothesis is strongly supported by our results, because we show that the latter method, named RTB, yields very accurate approximations for the low-frequency normal modes of all proteins considered. Moreover, the quality of the normal modes thus obtained depends very little on the way the polypeptidic chain is split into blocks, Noteworthy, with six aminoacids per block, the normal modes are almost as accurate as with a single amino-acid per block. In this case, for a protein of n residues and N atoms, the RTB method requires the diagonalization of an n x n matrix, whereas standard procedures require the diagonalization of a 3N x 3N matrix. Being a fast method, our approach can be useful for normal mode analyses of large systems, paving the way for further developments and applications in contexts for which the normal modes are needed frequently, as for example during molecular dynamics calculations. Proteins 2000;41:1-7. (C) 2000 Wiley-Liss, Inc.

    Web of Science

  82. Molecular dynamics simulation shows large volume fluctuations of proteins Reviewed

    F Tama, O Miyashita, A Kitao, N Go

    EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS   Vol. 29 ( 7 ) page: 472 - 480   2000

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:SPRINGER-VERLAG  

    In this paper we present a new approach to study the volume fluctuations of proteins. From a 1 ns molecular dynamics simulation, the volume fluctuation of human lysozyme has been calculated. We used two different ways for the calculation. In the first one, the volume fluctuation is extracted directly from the trajectory. For the second one, a newly developed formalism based on principal component analysis is used. The r.m.s. volume fluctuations obtained from the two analyses agree well with each other. The isothermal intrinsic compressibility was found to be larger than the one reported by experiment. The difference is discussed and suggested to exist in the assumed uncertainty of the compressibility of hydrated water to deduce the isothermal intrinsic compressibility from the experimental value. Spectral analysis shows that low-frequency dynamics dominate the total volume fluctuation. The same aspect is found in the study using principal component analysis. This low-frequency region is related to large and slow motions of proteins. Therefore a long time dynamics simulation is necessary to describe the volume fluctuations of proteins.

    DOI: 10.1007/s002490000103

    Web of Science

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KAKENHI (Grants-in-Aid for Scientific Research) 4

  1. コンピューティングを活用した寄生植物ストライガを抑制する生命機能分子の探索

    Grant number:17F17819  2017.11 - 2018.3

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    Authorship:Other 

    Based on X-ray crystal structure of Striga receptor ShHTL5, homology models of ShHTL7 were developed and validated. In addition, models of a collection of experimentally investigated HTL7 mutants (amino acid residue changes in the receptor’s active site) were created. These structures were submitted to MD simulations to explore their structural flexibility. The analyses of resulting MD trajectories showed significantly higher plasticity of HTL7 protein helices that surround the active site of HTLs compared to HTL5. The degree of flexibility decreases with increasing number of single residue changes from HTL7 towards HTL5. The increased flexibility in HTL7 is found to be in accordance with experimental assay results that indicate that HTL7 is more promiscuous with regard to ligand binding compared to other HTLs.
    The developed homology models of HTL7 and the published structure of HTL5 were further used as basis for induced fit docking studies with experimentally validated active molecules. The purpose of these docking studies was to (i) computationally assess the structure-activity relationship of tested small molecules and Striga receptors and to identify essential protein-ligand interactions that lead to receptor modulation, and (ii) to create an ensemble of structural complexes with highly potent molecules for subsequent protein-ligand MD simulations. These could be used for the development of 3D dynophore models that would allow virtual screening of chemical libraries.
    29年度が最終年度であるため、記入しない。
    29年度が最終年度であるため、記入しない。

  2. Database of Molecular Shapes and Diffraction Patterns for X-ray Free Electron Laser Data Analysis

    Grant number:17K07305  2017.4 - 2020.3

    Tama Florence

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    Authorship:Principal investigator 

    Grant amount:\4810000 ( Direct Cost: \3700000 、 Indirect Cost:\1110000 )

    In this project, we have been developing new efficient approaches to find 3D biological shapes from a few EM or XFEL images to serve as a starting point for further data analyses. In this approach, databases of known molecular shapes are assembled and numerical algorithms are used to identify the shapes that are consistent with a few query images.
    We had first developed the protocol for using EM real space images as inputs to test the feasibility of the approach. We showed that a small number of images can be sufficient as query images to identify similar 3D shapes. Then we have been developing algorithms to use XFEL diffraction patterns as inputs. Since simulations of XFEL diffraction patterns is time-consuming, we have developed a new approach to use Gaussian mixture model to model the structure. We have also the algorithms to automatically identify the region with strong information in the diffraction patterns to identify matches.

  3. Support activities aimed at international network formation in the research area of dynamic structural life sciences

    Grant number:15K21711  2015.11 - 2019.3

    Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

    Kohda Daisuke

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    Authorship:Coinvestigator(s) 

    We aimed to expand the scope of the academic research activities conducted in our Grant‐in‐Aid for Scientific Research on Innovative Area, entitled “Novel measurement techniques for visualizing ‘live’ protein molecules at work” internationally. During the four-year period, five invited speakers, 13 joint researchers including postdoctoral fellows and students, and one sabbatical professor were accepted from abroad. Our four members stayed in a laboratory in Germany for a joint experiment. Three Japan-residing foreign students were dispatched to an international conference held in Japan. We organized an international version of the annual technical AFM workshops during the last three years and accepted 31 researchers and students from abroad. Finally, overseas travel expenses were supported for 11 members of our research activity.

  4. Molecular dynamics simulations of protein complexes based on multi-resolution models

    Grant number:26119006  2014.7 - 2019.3

    Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

    Sugita Yuji

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    Authorship:Coinvestigator(s) 

    We developed new methods combining experimental information with simulation data to obtain reliable structure-dynamics relationship of proteins. Generally, experimental data tend to be low-resolution and low-dimension, whereas simulation can provide high-resolution structural information. In our methods, we combined cryo-EM with MD simulation in flexible fitting calculation and utilized machine learning for combining single-molecule analysis with MD simulation. The developed methods were installed into GENESIS software package and can be used together with enhanced sampling methods and multi-scale/multi-resolution molecular models. We applied these methods to understand structure-dynamics relationships of membrane proteins or protein/nucleic acid complexes.