Florence Tama

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.

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 SecYEβ 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.

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.

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.

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.

Removal of Divalent Cations Induces Structural Transitions in Red Clover Necrotic Mosaic Virus, Revealing a Potential Mechanism for RNA Release

ABSTRACT The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 Å 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-Å-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.

Excited states of ribosome translocation revealed through integrative molecular modeling

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.

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

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 overfitting can be avoided by assessing the quality of the fitted model in terms of correlation coefficient and secondary structure preservation.

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

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 Å × 90 Å × 75 Å. 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 β1 H-NOX and α1 PAS domains are in contact and form the core signaling complex, while the α1 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.

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

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-resolution 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 20A. 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.