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

DOI： 10.1016/j.jmb.2023.167951

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1063/4.0000175

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DOI： 10.1145/3605573.3605636

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DOI： 10.1073/pnas.2207558119

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

DOI： 10.1073/pnas.2203936119

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Optica Publishing Group  

DOI： 10.1364/OPTICA.457352

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

In bifidobacteria, phosphoketolase (PKT) plays a key role in the central hexose fermentation pathway called "bifid shunt." The three-dimensional structure of PKT from Bifidobacterium longum with co-enzyme thiamine diphosphate (ThDpp) was determined at 2.1 Å resolution by cryo-EM single-particle analysis using 196,147 particles to build up the structural model of a PKT octamer related by D4 symmetry. Although the cryo-EM structure of PKT was almost identical to the X-ray crystal structure previously determined at 2.2 Å resolution, several interesting structural features were observed in the cryo-EM structure. Because this structure was solved at relatively high resolution, it was observed that several amino acid residues adopt multiple conformations. Among them, Q546-D547-H548-N549 (the QN-loop) demonstrate the largest structural change, which seems to be related to the enzymatic function of PKT. The QN-loop is at the entrance to the substrate binding pocket. The minor conformer of the QN-loop is similar to the conformation of the QN-loop in the crystal structure. The major conformer is located further from ThDpp than the minor conformer. Interestingly, the major conformer in the cryo-EM structure of PKT resembles the corresponding loop structure of substrate-bound Escherichia coli transketolase. That is, the minor and major conformers may correspond to "closed" and "open" states for substrate access, respectively. Moreover, because of the high-resolution analysis, many water molecules were observed in the cryo-EM structure of PKT. Structural features of the water molecules in the cryo-EM structure are discussed and compared with water molecules observed in the crystal structure.

DOI： 10.1016/j.jsb.2022.107842

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

Protein kinases are responsible for healthy cellular processes and signalling pathways, and their dysfunction is the basis of many pathologies. There are numerous small molecule inhibitors of protein kinases that systemically regulate dysfunctional signalling processes. However, attaining selectivity in kinase inhibition within the complex human kinome is still a challenge that inspires unconventional approaches. One of those approaches is photopharmacology, which uses light-controlled bioactive molecules to selectively activate drugs only at the intended space and time, thereby avoiding side effects outside of the irradiated area. Still, in the context of kinase inhibition, photopharmacology has thus far been rather unsuccessful in providing light-controlled drugs. Here, we present the discovery and optimisation of a photoswitchable inhibitor of casein kinase 1δ (CK1δ), important for the control of cell differentiation, circadian rhythm, DNA repair, apoptosis, and numerous other signalling processes. Varying the position at which the light-responsive azobenzene moiety has been introduced into a known CK1δ inhibitor, LH846, revealed the preferred regioisomer for efficient photo-modulation of inhibitory activity, but the photoswitchable inhibitor suffered from sub-optimal (photo)chemical properties. Replacement of the bis-phenyl azobenzene group with the arylazopyrazole moiety yielded a superior photoswitch with very high photostationary state distributions, increased solubility and a 10-fold difference in activity between irradiated and thermally adapted samples. The reasons behind those findings are explored with molecular docking and molecular dynamics simulations. Results described here show how the evaluation of privileged molecular architecture, followed by the optimisation of the photoswitchable unit, is a valuable strategy for the challenging design of the photoswitchable kinase inhibitors.

DOI： 10.3390/ijms23105326

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

Atomic models of cryo electron microscopy (cryo-EM) maps of biomolecular conformations are often obtained by flexible fitting of the maps with available atomic structures of other conformations (e.g., obtained by X-ray crystallography). This article presents a new flexible fitting method, NMMD, which combines normal mode analysis (NMA) and molecular dynamics simulation (MD). Given an atomic structure and a cryo-EM map to fit, NMMD simultaneously estimates global atomic displacements based on NMA and local displacements based on MD. NMMD was implemented by modifying EMfit, a flexible fitting method using MD only, in GENESIS 1.4. As EMfit, NMMD can be run with replica exchange umbrella sampling procedure. The new method was tested using a variety of EM maps (synthetic and experimental, with different noise levels and resolutions). The results of the tests show that adding normal modes to MD-based fitting makes the fitting faster (40% in average) and, in the majority of cases, more accurate.

DOI： 10.1016/j.jmb.2022.167483

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

Pannexin (PANX) family proteins form large-pore channels that mediate purinergic signaling. We analyzed the cryo-EM structures of human PANX1 in lipid nanodiscs to elucidate the gating mechanism and its regulation by the amino terminus in phospholipids. The wild-type channel has an amino-terminal funnel in the pore, but in the presence of the inhibitor probenecid, a cytoplasmically oriented amino terminus and phospholipids obstruct the pore. Functional analysis using whole-cell patch-clamp and oocyte voltage clamp showed that PANX1 lacking the amino terminus did not open and had a dominant negative effect on channel activity, thus confirming that the amino-terminal domain played an essential role in channel opening. These observations suggest that dynamic conformational changes in the amino terminus of human PANX1 are associated with lipid movement in and out of the pore. Moreover, the data provide insight into the gating mechanism of PANX1 and, more broadly, other large-pore channels.

DOI： 10.1126/scisignal.abg6941

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DOI： 10.3389/fmolb.2022.913860

DOI： 10.3389/fmolb.2022.913860

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DOI： 10.1109/eScience55777.2022.00052

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DOI： 10.1109/eScience55777.2022.00017

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X-ray free-electron laser (XFEL) scattering promises to probe single biomolecular complexes without crystallization, enabling the study of biomolecular structures under near-physiological conditions at room temperature. However, such structural determination of biomolecules is extremely challenging thus far. In addition to the large numbers of diffraction patterns required, the orientation of each diffraction pattern needs to be accurately estimated and the missing phase information needs to be recovered for three-dimensional (3D) structure reconstruction. Given the current limitations to the amount and resolution of the data available from single-particle XFEL scattering experiments, we propose an alternative approach to find plausible 3D biological shapes from a limited number of diffraction patterns to serve as a starting point for further analyses. In our proposed strategy, small sets of input (e.g., five) XFEL diffraction patterns were matched against a library of diffraction patterns simulated from 1628 electron microscopy (EM) models to find potential matching 3D models that are consistent with the input diffraction patterns. This approach was tested for three example cases: EMD-3457 (Thermoplasma acidophilum 20S proteasome), EMD-5141 (Escherichia coli 70S ribosome complex), and EMD-5152 (budding yeast Nup84 complex). We observed that choosing the best strategy to define matching regions on the diffraction patterns is critical for identifying correctly matching diffraction patterns. While increasing the number of input diffraction patterns improved the matches in some cases, we found that the resulting matches are more dependent on the uniqueness or complexity of the shape as captured in the individual input diffraction patterns and the availability of a similar 3D biological shape in the search library. The protocol could be useful for finding candidate models for a limited amount of low-resolution data, even when insufficient for reconstruction, performing a quick exploration of new data upon collection, and the analysis of the conformational heterogeneity of the particle of interest as captured within the diffraction patterns.

DOI： 10.1021/acs.jcim.1c00602

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Language：English   Publishing type：Research paper (conference, symposium, etc.)  

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The circadian clock is a biological timekeeper that operates through transcription-translation feedback loops in mammals. Cryptochrome 1 (CRY1) and Cryptochrome 2 (CRY2) are highly conserved core clock components having redundant and distinct functions. We recently identified the CRY1- and CRY2-selective compounds KL101 and TH301, respectively, which provide useful tools for the exploration of isoform-selective CRY regulation. However, intrinsic differences in the compound-binding FAD (flavin adenine dinucleotide) pockets between CRY1 and CRY2 are not well understood, partly because of nonoptimal properties of previously reported apo form structures in this particular region constituted by almost identical sequences. Here, we show unliganded CRY1 and CRY2 crystal structures with well-defined electron densities that are largely free of crystal contacts at the FAD pocket and nearby lid loop. We revealed conformational isomerism in key residues. In particular, CRY1 W399 and corresponding CRY2 W417 in the FAD pocket had distinct conformations ("out" for CRY1 and "in" for CRY2) by interacting with the lid loop residues CRY1 Q407 and CRY2 F424, respectively, resulting in different overall lid loop structures. Molecular dynamics simulations supported that these conformations were energetically favorable to each isoform. Isoform-selective compounds KL101 and TH301 preferred intrinsic "out" and "in" conformations of the tryptophan residue in CRY1 and CRY2, respectively, while the nonselective compound KL001 fit to both conformations. Mutations of lid loop residues designed to perturb their isoform-specific interaction with the tryptophan resulted in reversed responses of CRY1 and CRY2 to KL101 and TH301. We propose that these intrinsic structural differences of CRY1 and CRY2 can be targeted for isoform-selective regulation.

DOI： 10.1073/pnas.2026191118

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The circadian clock controls daily rhythms of physiological processes. The presence of the clock mechanism throughout the body is hampering its local regulation by small molecules. A photoresponsive clock modulator would enable precise and reversible regulation of circadian rhythms using light as a bio-orthogonal external stimulus. Here we show, through judicious molecular design and state-of-the-art photopharmacological tools, the development of a visible light-responsive inhibitor of casein kinase I (CKI) that controls the period and phase of cellular and tissue circadian rhythms in a reversible manner. The dark isomer of photoswitchable inhibitor 9 exhibits almost identical affinity towards the CKIα and CKIδ isoforms, while upon irradiation it becomes more selective towards CKIδ, revealing the higher importance of CKIδ in the period regulation. Our studies enable long-term regulation of CKI activity in cells for multiple days and show the reversible modulation of circadian rhythms with a several hour period and phase change through chronophotopharmacology.

DOI： 10.1038/s41467-021-23301-x

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

ClpB belongs to the cellular disaggretase machinery involved in rescuing misfolded or aggregated proteins during heat or other cellular shocks. The function of this protein relies on the interconversion between different conformations in its native condition. A recent high-speed-atomic-force-microscopy (HS-AFM) experiment on ClpB from Thermus thermophilus shows four predominant conformational classes, namely, open, closed, spiral, and half-spiral. Analyses of AFM images provide only partial structural information regarding the molecular surface, and thus computational modeling of three-dimensional (3D) structures of these conformations should help interpret dynamical events related to ClpB functions. In this study, we reconstruct 3D models of ClpB from HS-AFM images in different conformational classes. We have applied our recently developed computational method based on a low-resolution representation of 3D structure using a Gaussian mixture model, combined with a Monte-Carlo sampling algorithm to optimize the agreement with target AFM images. After conformational sampling, we obtained models that reflect conformational variety embedded within the AFM images. From these reconstructed 3D models, we described, in terms of relative domain arrangement, the different types of ClpB oligomeric conformations observed by HS-AFM experiments. In particular, we highlighted the slippage of the monomeric components around the seam. This study demonstrates that such details of information, necessary for annotating the different conformational states involved in the ClpB function, can be obtained by combining HS-AFM images, even with limited resolution, and computational modeling.

DOI： 10.3389/fmolb.2021.704274

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Language：Japanese  

DOI： 10.1107/S0108767320098785

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Language：English   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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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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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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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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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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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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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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Single-particle imaging using X-ray free-electron lasers (XFELs) is a promising technique for observing nanoscale biological samples under near-physiological conditions. However, as the sample's orientation in each diffraction pattern is unknown, advanced algorithms are required to reconstruct the 3D diffraction intensity volume and subsequently the sample's density model. While most approaches perform 3D reconstruction via determining the orientation of each diffraction pattern, a correlation-based approach utilizes the averaged spatial correlations of diffraction intensities over all patterns, making it well suited for processing experimental data with a poor signal-to-noise ratio of individual patterns. Here, a method is proposed to determine the 3D structure of a sample by analyzing the double, triple and quadruple spatial correlations in diffraction patterns. This ab initio method can reconstruct the basic shape of an irregular unsymmetric 3D sample without requiring any prior knowledge of the sample. The impact of background and noise on correlations is investigated and corrected to ensure the success of reconstruction under simulated experimental conditions. Additionally, the feasibility of using the correlation-based approach to process incomplete partial diffraction patterns is demonstrated. The proposed method is a variable addition to existing algorithms for 3D reconstruction and will further promote the development and adoption of XFEL single-particle imaging techniques.

DOI： 10.1107/S2052252523009831

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

Circadian clocks, biological timekeepers that are present in almost every cell of our body, are complex systems whose disruption is connected to various diseases. Controlling cellular clock function with high temporal resolution in an inducible manner would yield an innovative approach for the circadian rhythm regulation. In the present study, we present structure-guided incorporation of photoremovable protecting groups into a circadian clock modifier, longdaysin, which inhibits casein kinase I (CKI). Using photodeprotection by UV or visible light (400 nm) as the external stimulus, we have achieved quantitative and light-inducible control over the CKI activity accompanied by an accurate regulation of circadian period in cultured human cells and mouse tissues, as well as in living zebrafish. This research paves the way for the application of photodosing in achieving precise temporal control over the biological timing and opens the door for chronophotopharmacology to deeper understand the circadian clock system.

DOI： 10.1021/jacs.9b05445

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DOI： 10.1038/s41477-019-0440-x

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DOI： 10.1016/j.bpj.2018.11.3139

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DOI： 10.1016/j.str.2018.09.004

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Compounds targeting the circadian clock have been identified as potential treatments for clock-related diseases, including cancer. Our cell-based phenotypic screen revealed uncharacterized clock-modulating compounds. Through affinity-based target deconvolution, we identified GO289, which strongly lengthened circadian period, as a potent and selective inhibitor of CK2. Phosphoproteomics identified multiple phosphorylation sites inhibited by GO289 on clock proteins, including PER2 S693. Furthermore, GO289 exhibited cell type-dependent inhibition of cancer cell growth that correlated with cellular clock function. The x-ray crystal structure of the CK2α-GO289 complex revealed critical interactions between GO289 and CK2-specific residues and no direct interaction of GO289 with the hinge region that is highly conserved among kinases. The discovery of GO289 provides a direct link between the circadian clock and cancer regulation and reveals unique design principles underlying kinase selectivity.

DOI： 10.1126/sciadv.aau9060

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DOI： 10.1002/prot.25625

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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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DOI： 10.1049/iet-ipr.2018.5145

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DOI： 10.3390/ijms19113401

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DOI： 10.1364/OE.26.026734

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

Background: Advancements in biophysical experimental techniques have pushed the limits in terms of the types of phenomena that can be characterized, the amount of data that can be produced and the resolution at which we can visualize them. Single particle techniques such as Electron Microscopy (EM) and X-ray free electron laser (XFEL) scattering require a large number of 2D images collected to resolve three-dimensional (3D) structures. In this study, we propose a quick strategy to retrieve potential 3D shapes, as low-resolution models, from a few 2D experimental images by searching a library of 2D projection images generated from existing 3D structures.Results: We developed the protocol to assemble a non-redundant set of 3D shapes for generating the 2D image library, and to retrieve potential match 3D shapes for query images, using EM data as a test. In our strategy, we disregard differences in volume size, giving previously unknown structures and conformations a greater number of 3D biological shapes as possible matches. We tested the strategy using images from three EM models as query images for searches against a library of 22750 2D projection images generated from 250 random EM models. We found that our ability to identify 3D shapes that match the query images depends on how complex the outline of the 2D shapes are and whether they are represented in the search image library.Conclusions: Through our computational method, we are able to quickly retrieve a 3D shape from a few 2D projection images. Our approach has the potential for exploring other types of 2D single particle structural data such as from XFEL scattering experiments, for providing a tool to interpret low-resolution data that may be insufficient for 3D reconstruction, and for estimating the mixing of states or conformations that could exist in such experimental data.

DOI： 10.1186/s12859-018-2358-0

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：INT UNION CRYSTALLOGRAPHY  

Three-dimensional (3D) structures of biomolecules provide insight into their functions. Using X-ray free-electron laser (XFEL) scattering experiments, it was possible to observe biomolecules that are difficult to crystallize, under conditions that are similar to their natural environment. However, resolving 3D structure from XFEL data is not without its challenges. For example, strong beam intensity is required to obtain sufficient diffraction signal and the beam incidence angles to the molecule need to be estimated for diffraction patterns with significant noise. Therefore, it is important to quantitatively assess how the experimental conditions such as the amount of data and their quality affect the expected resolution of the resulting 3D models. In this study, as an example, the restoration of 3D structure of ribosome from two-dimensional diffraction patterns created by simulation is shown. Tests are performed using the diffraction patterns simulated for different beam intensities and using different numbers of these patterns. Guidelines for selecting parameters for slice-matching 3D reconstruction procedures are established. Also, the minimum requirements for XFEL experimental conditions to obtain diffraction patterns for reconstructing molecular structures to a high-resolution of a few nanometers are discussed.

DOI： 10.1107/S1600577518005568

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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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DOI： 10.1007/978-981-13-2200-6_13

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PubMed

Language：Japanese   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

Web of Science

PubMed

Web of Science

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

Web of Science

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

DOI： 10.1016/j.sbi.2017.04.009

Web of Science

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

<p>Hybrid approaches combine computational modeling techniques with low-resolution structural data. Such approaches have proven to be powerful tools to obtain new 3D structural and dynamical information on biological systems. Currently, major applications focus on cryo-EM data. Methods and some applications to construct atomic structural models of new functional states will be reviewed. In addition, possible extension to data from X-ray free electron laser, which currently provides low-resolution data, will be discussed.</p>

DOI： 10.2142/biophys.57.090

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

Web of Science

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

Web of Science

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

DOI： 10.1093/nar/gkv1133

Web of Science

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

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

Web of Science

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

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

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

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

Web of Science

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

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

Web of Science

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

Web of Science

Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

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.

Web of Science

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

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

Web of Science

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

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

Web of Science

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

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

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

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

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

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

Web of Science

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

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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Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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

Web of Science

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

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

Web of Science

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

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

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

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

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

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

Web of Science

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

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

Web of Science

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

We present a method to reconstruct a three-dimensional protein structure from an atomic pair distribution function derived from the scattering intensity profile from SAXS data by flexibly fitting known x-ray structures. This method uses a linear combination of low-frequency normal modes from an elastic network description of the molecule in an iterative manner to deform the structure to conform optimally to the target pair distribution function derived from SAXS data. For computational efficiency, the protein and water molecules included in the protein first hydration shell are coarse-grained. In this paper, we demonstrate the validity of our coarse-graining approach to study SAXS data. Illustrative results of our flexible fitting studies on simulated SAXS data from five different proteins are presented.

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

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

Web of Science

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

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

Web of Science

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

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

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOPHYSICAL SOC  

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

DOI： 10.1002/9780470015902.a0020204

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

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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Language：English   Publishing type：Part of collection (book)   Publisher：ANNUAL REVIEWS  

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&apos;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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

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

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

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

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

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

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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Language：English   Publisher：BENTHAM SCIENCE PUBL LTD  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

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

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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

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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.

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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

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