Biomedical subjects

Florence Tama

Publications and source records attributed to Florence Tama.

16 recordsLinked to original sources

PubMed2006

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

The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 A 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 RCNMV capsid, there is a clearly defined inner cage formed by complexes of genomic RNA and the amino termini of CP subunits. An RCNMV 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.

Amino Acid Sequence↗

PubMed2006

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

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 Ca(2+) can bind both to the native and swollen forms of the CCMV virion. Favorable binding to the swollen form suggests that Ca(2+) ions can induce the capsid contraction and stabilize the native form.

Binding Sites↗

PubMed2006

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

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 approximately 18 A cryo-electron microscopy (EM) density map of the complex. The model describes the interacting surface that buries a total area of approximately 10,140 A2 comprising approximately 40% charged, and approximately 30% each of polar and hydrophobic residues. For the heptamer, the buried surface, composed of approximately 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 approximately 110 residues, mostly from the N-terminal domain I (LF(N)), 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 IV on domain II, with the largest movement of approximately 9 A noted for domain I. On the other hand, primarily, rigid-body movements, larger than approximately 10 A for three PA63 monomers, cause the hourglass-shaped heptamer lumen to enlarge by as much as approximately 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.

Antigens, Bacterial↗

PubMed2006

Symmetry, form, and shape: guiding principles for robustness in macromolecular machines.

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.

Algorithms↗

PubMed2005

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

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 SecYEbeta 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.

Cell Membrane↗

PubMed2005

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

The 13 angstroms 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 approximately 5 angstroms 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 (approximately 140 angstroms).

Chaperonin 60↗

PubMed2005

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

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 II 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 alpha-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 alpha-helices is a key mechanical requirement for myosin II regulation. These torsional motions of the myosin heads about their coiled coil alpha-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.

Animals↗

PubMed2005

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

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.

Bacteriophages↗

PubMed2004

Ribosome motions modulate electrostatic properties.

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 100, 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.

Binding Sites↗

PubMed2004

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

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.

Algorithms↗

PubMed2004

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

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 Calpha 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.

Cryoelectron Microscopy↗

PubMed2003

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

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.

Binding Sites↗

PubMed2003

Mega-Dalton biomolecular motion captured from electron microscopy reconstructions.

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 30S and 50S subunits of the ribosome, and the dynamic flexibility of chaperonin CCT are extracted directly from single electron microscopy structures at 15-27 A 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.

Chaperonin Containing TCP-1↗

PubMed2003

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

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.

Data Interpretation, Statistical↗

PubMed2002

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

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.

Adenylate Kinase↗

PubMed2002

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

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 approximately 72% to approximately 49% 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 A 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.

Binding Sites↗