The structure of an elongation factor G-ribosome complex captured in the absence of inhibitors.

During translation's elongation cycle, elongation factor G (EF-G) promotes messenger and transfer RNA translocation through the ribosome. Until now, the structures reported for EF-G-ribosome complexes have been obtained by trapping EF-G in the ribosome. These results were based on use of non-hydrolyzable guanosine 5'-triphosphate (GTP) analogs, specific inhibitors or a mutated EF-G form. Here, we present the first cryo-electron microscopy structure of EF-G bound to ribosome in the absence of an inhibitor. The structure reveals a natural conformation of EF-G·GDP in the ribosome, with a previously unseen conformation of its third domain. These data show how EF-G must affect translocation, and suggest the molecular mechanism by which fusidic acid antibiotic prevents the release of EF-G after GTP hydrolysis.

The complex of tmRNA-SmpB and EF-G on translocating ribosomes.

Bacterial ribosomes stalled at the 3' end of malfunctioning messenger RNAs can be rescued by transfer-messenger RNA (tmRNA)-mediated trans-translation. The SmpB protein forms a complex with the tmRNA, and the transfer-RNA-like domain (TLD) of the tmRNA then enters the A site of the ribosome. Subsequently, the TLD-SmpB module is translocated to the P site, a process that is facilitated by the elongation factor EF-G, and translation is switched to the mRNA-like domain (MLD) of the tmRNA. Accurate loading of the MLD into the mRNA path is an unusual initiation mechanism. Despite various snapshots of different ribosome-tmRNA complexes at low to intermediate resolution, it is unclear how the large, highly structured tmRNA is translocated and how the MLD is loaded. Here we present a cryo-electron microscopy reconstruction of a fusidic-acid-stalled ribosomal 70S-tmRNA-SmpB-EF-G complex (carrying both of the large ligands, that is, EF-G and tmRNA) at 8.3 Å resolution. This post-translocational intermediate (TI(POST)) presents the TLD-SmpB module in an intrasubunit ap/P hybrid site and a tRNA(fMet) in an intrasubunit pe/E hybrid site. Conformational changes in the ribosome and tmRNA occur in the intersubunit space and on the solvent side. The key underlying event is a unique extra-large swivel movement of the 30S head, which is crucial for both tmRNA-SmpB translocation and MLD loading, thereby coupling translocation to MLD loading. This mechanism exemplifies the versatile, dynamic nature of the ribosome, and it shows that the conformational modes of the ribosome that normally drive canonical translation can also be used in a modified form to facilitate more complex tasks in specialized non-canonical pathways.

Methods for three-dimensional reconstruction of heterogeneous assemblies.

Electron microscopy (EM) has developed into an important method for determining the three-dimensional (3D) structures of biological complexes, in particular of isolated macromolecular complexes in vitrified solution (cryo-EM of "single particles"). One of the consequences of studying complexes in solution rather than in a crystal lattice is that they are less constrained to adopt a single conformation. It is a common problem in single-particle analysis that samples of purified macromolecules can be structurally heterogeneous, with molecules adopting different conformations, corresponding to different functional states. In the case of multisubunit assemblies, there may also be heterogeneity of assembly or ligand binding. Heterogeneity limits the accuracy and resolution of 3D structures, since different conformations will contribute to a single 3D map and variable parts of the structure will be smeared out. Therefore, a new group of image processing methods has been developed to deal with the problems of detecting and sorting structural heterogeneity. The basic problem is to discriminate the source of image variations, and then to separate the images into homogeneous subsets for separate reconstruction. Variations in image features can arise from different particle orientations, variations in conformation and/or ligand binding, and noise fluctuations in the low signal-to-noise ratio images typical of cryo-EM. Here, we present a review of approaches developed to deal with these problems, along with examples of the application of a method based on multivariate statistical analysis to both model and real data. The methods have been used to discriminate small differences in size, conformation and ligand binding, and to obtain high quality, reliable reconstructions of multiple structures from mixed data sets.

Introducing robustness to maximum-likelihood refinement of electron-microscopy data.

An expectation-maximization algorithm for maximum-likelihood refinement of electron-microscopy images is presented that is based on fitting mixtures of multivariate t-distributions. The novel algorithm has intrinsic characteristics for providing robustness against atypical observations in the data, which is illustrated using an experimental test set with artificially generated outliers. Tests on experimental data revealed only minor differences in two-dimensional classifications, while three-dimensional classification with the new algorithm gave stronger elongation factor G density in the corresponding class of a structurally heterogeneous ribosome data set than the conventional algorithm for Gaussian mixtures.

Heterogeneity of large macromolecular complexes revealed by 3D cryo-EM variance analysis.

Macromolecular structure determination by cryo-electron microscopy (EM) and single-particle analysis are based on the assumption that imaged molecules have identical structure. With the increased size of processed data sets, it becomes apparent that many complexes coexist in a mixture of conformational states or contain flexible regions. We describe an implementation of the bootstrap resampling technique that yields estimates of voxel-by-voxel variance of a structure reconstructed from the set of its projections. We introduce a highly efficient reconstruction algorithm that is based on direct Fourier inversion and that incorporates correction for the transfer function of the microscope, thus extending the resolution limits of variance estimation. We also describe a validation method to determine the number of resampled volumes required to achieve stable estimate of the variance. The proposed bootstrap method was applied to a data set of 70S ribosome complexed with tRNA and the elongation factor G. The proposed method of variance estimation opens new possibilities for single-particle analysis, by extending applicability of the technique to heterogeneous data sets of macromolecules and to complexes with significant conformational variability.

Structural basis for interaction of the ribosome with the switch regions of GTP-bound elongation factors.

Elongation factor G (EF-G) catalyzes tRNA translocation on the ribosome. Here a cryo-EM reconstruction of the 70S*EF-G ribosomal complex at 7.3 A resolution and the crystal structure of EF-G-2*GTP, an EF-G homolog, at 2.2 A resolution are presented. EF-G-2*GTP is structurally distinct from previous EF-G structures, and in the context of the cryo-EM structure, the conformational changes are associated with ribosome binding and activation of the GTP binding pocket. The P loop and switch II approach A2660-A2662 in helix 95 of the 23S rRNA, indicating an important role for these conserved bases. Furthermore, the ordering of the functionally important switch I and II regions, which interact with the bound GTP, is dependent on interactions with the ribosome in the ratcheted conformation. Therefore, a network of interaction with the ribosome establishes the active GTP conformation of EF-G and thus facilitates GTP hydrolysis and tRNA translocation.

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.

Three-dimensional electron cryomicroscopy of ribosomes.

Single particle electron cryomicroscopy is nowadays routinely used to generate three-dimensional structural information of ribosomal complexes without the need of crystallization. A large number of structures of functional important ribosomal complexes have thus been determined using this technique. In E. coli 70S ribosomes all three tRNA binding sites could be localized. The ternary complex of EF-TutRNAGTP that delivers the tRNA to the ribosome was directly visualized in a ribosomal complex blocked by the antibiotic kirromycin. Three different functional states of translocation have been studied and the respective EF-G binding sites have been mapped. The level of resolution achievable with electron cryomicroscopy allows conformational changes in the domain structures of elongation factors to be modelled in terms of rigid body movements. Structural information on eukaryotic ribosomes is also available for yeast and mammalian 80S ribosomes. The structural differences between rabbit 80S and E. coli 70S ribosomes could be interpreted in terms of ribosomal RNA expansion segments in the 18S and 23S RNA. The EF-G homologue EF2 was mapped analysing the structure of an 80SEF2sodarin complex and most recently the binding of a hepatitis C virus IRES element to a yeast 40S subunit has been studied. The first electron cryomicroscopical 3D reconstructions have further been used to overcome the initial phasing problems in X-ray crystallographic studies of the ribosome facilitating structure determination of the recent atomic resolution structures of the 30S and 50S ribosomal subunits. In turn, the knowledge of the atomic structure of the ribosome makes detailed interpretations of cryo-EM maps possible at approximately 20 A resolution.

Domain motions of EF-G bound to the 70S ribosome: insights from a hand-shaking between multi-resolution structures.

Molecular modeling and information processing techniques were combined to refine the structure of translocase (EF-G) in the ribosome-bound form against data from cryoelectron microscopy (cryo-EM). We devised a novel multi-scale refinement method based on vector quantization and force-field methods that gives excellent agreement between the flexibly docked structure of GDP. EF-G and the cryo-EM density map at 17 A resolution. The refinement reveals a dramatic "induced fit" conformational change on the 70S ribosome, mainly involving EF-G's domains III, IV, and V. The rearrangement of EF-G's structurally preserved regions, mediated and guided by flexible linkers, defines the site of interaction with the GTPase-associated center of the ribosome.

Solution structure of the E. coli 70S ribosome at 11.5 A resolution.

Over 73,000 projections of the E. coli ribosome bound with formyl-methionyl initiator tRNAf(Met) were used to obtain an 11.5 A cryo-electron microscopy map of the complex. This map allows identification of RNA helices, peripheral proteins, and intersubunit bridges. Comparison of double-stranded RNA regions and positions of proteins identified in both cryo-EM and X-ray maps indicates good overall agreement but points to rearrangements of ribosomal components required for the subunit association. Fitting of known components of the 50S stalk base region into the map defines the architecture of the GTPase-associated center and reveals a major change in the orientation of the alpha-sarcin-ricin loop. Analysis of the bridging connections between the subunits provides insight into the dynamic signaling mechanism between the ribosomal subunits.

Large-scale movement of elongation factor G and extensive conformational change of the ribosome during translocation.

Elongation factor (EF) G promotes tRNA translocation on the ribosome. We present three-dimensional reconstructions, obtained by cryo-electron microscopy, of EF-G-ribosome complexes before and after translocation. In the pretranslocation state, domain 1 of EF-G interacts with the L7/12 stalk on the 50S subunit, while domain 4 contacts the shoulder of the 30S subunit in the region where protein S4 is located. During translocation, EF-G experiences an extensive reorientation, such that, after translocation, domain 4 reaches into the decoding center. The factor assumes different conformations before and after translocation. The structure of the ribosome is changed substantially in the pretranslocation state, in particular at the head-to-body junction in the 30S subunit, suggesting a possible mechanism of translocation.