Faculty Profiles - TAMA Florence

Papers - TAMA Florence

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Consensus among flexible fitting approaches improves the interpretation of cryo-EM data Reviewed Open Access

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

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

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

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

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

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

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

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

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

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

Excited states of ribosome translocation revealed through integrative molecular modeling Reviewed Open Access

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

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

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

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

Osamu Miyashita, Christian Gorba, Florence Tama

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

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

Normal Mode Flexible Fitting of High-Resolution Structures of Biological Molecules Toward SAXS Data. International journal

Christian Gorba, Florence Tama

Bioinformatics and biology insights Vol. 4 page： 43 - 54 2010.6

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.

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

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

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

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

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

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

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

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

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

Marek Orzechowski, Florence Tama

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

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

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

Christian Gorba, Osamu Miyashita, Florence Tama

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

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

Normal Mode Analysis Techniques in Structural Biology. Invited Reviewed

Miyashita, O, Tama, F

eLS 2007

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

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

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

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

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

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

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

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

Electrostatic properties of cowpea chlorotic mottle virus and cucumber mosaic virus capsids Reviewed Open Access

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

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

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

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

Florence Tama, Charles L. Brooks

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

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'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

Structure of the E-coli protein-conducting channel bound to a translating ribosome Reviewed Open Access

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

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

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

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

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

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

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

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

F Tama, CL Brooks

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

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

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

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

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

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

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

F Tama, O Miyashita, CL Brooks

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

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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