Search

eLife 7, e38017 (2018). doi:10.7554/eLife.38017, α-Xenorhabdolysins (Xax) are α-pore-forming toxins (α-PFT) that form 1–1.3 MDa large pore complexes to perforate the host cell membrane. PFTs are used by a variety of bacterial pathogens to attack host cells. Due to the lack of structural information, the molecular mechanism of action of Xax toxins is poorly understood. Here, we report the cryo-EM structure of the XaxAB pore complex from Xenorhabdus nematophila and the crystal structures of the soluble monomers of XaxA and XaxB. The structures reveal that XaxA and XaxB are built similarly and appear as heterodimers in the 12–15 subunits containing pore, classifying XaxAB as bi-component α-PFT. Major conformational changes in XaxB, including the swinging out of an amphipathic helix are responsible for membrane insertion. XaxA acts as an activator and stabilizer for XaxB that forms the actual transmembrane pore. Based on our results, we propose a novel structural model for the mechanism of Xax intoxication., Published by eLife Sciences Publications, Cambridge

Electron cryomicroscopy reveals the three-dimensional structure of F-actin at a resolution of 3.7 A in complex with tropomyosin at a resolution of 6.5 A; the stabilizing interactions and the effects of disease-causing mutants are also investigated. Filamentous actin (F-actin) — a main component of the cytoskeleton — is the major protein of thin filaments in the muscle. The binding of the motor protein myosin to F-actin is mediated by another protein called tropomyosin, which also binds to F-actin in smooth muscle and in non-muscle cells, stabilizing and regulating these filaments. Using electron cryomicroscopy, Stefan Raunser and colleagues have obtained the first high-resolution, three-dimensional structure of F-actin in complex with tropomyosin. The structure reveals the interactions that stabilize the F-actin and sheds light on the possible effect of prominent disease-causing mutations. Comparison of the F-actin structure with the crystal structure of monomeric (G)-actin reveals conformational changes associated with filament formation. Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss1, familial thoracic aortic aneurysms and dissections2, and multiple variations of myopathies3. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin4,5. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin6. Although crystal structures for monomeric actin (G-actin) are available7, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 A in complex with tropomyosin at a resolution of 6.5 A, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg2+ and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin–tropomyosin with its position in our previously determined F-actin–tropomyosin–myosin structure8 reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted development of drugs.

Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, a reaction essential for DNA replication and repair. Human RNR requires two subunits for activity, the α subunit contains the active site, and the β subunit houses the radical cofactor. Here, we present a 3.3-Å resolution structure by cryo-electron microscopy (EM) of a dATP-inhibited state of human RNR. This structure, which was determined in the presence of substrate CDP and allosteric regulators ATP and dATP, has three α2 units arranged in an α6 ring. At near-atomic resolution, these data provide insight into the molecular basis for CDP recognition by allosteric specificity effectors dATP/ATP. Additionally, we present lower-resolution EM structures of human α6 in the presence of both the anticancer drug clofarabine triphosphate and β2. Together, these structures support a model for RNR inhibition in which β2 is excluded from binding in a radical transfer competent position when α exists as a stable hexamer., eLife digest Cells often need to make more DNA, for example when they are about to divide or need to repair their genetic information. The building blocks of DNA – also called deoxyribonucleotides – are created through a series of biochemical reactions. Among the enzymes that accomplish these reactions, ribonucleotide reductases (or RNRs, for short) perform a key irreversible step. One prominent class of RNR contains two basic units, named alpha and beta. The active form of these RNRs is made up of a pair of alpha units (α2), which associates with a pair of beta units (β2) to create an α2β2 structure. α2 captures molecules called ribonucleotides and, with the help of β2, converts them to deoxyribonucleotides that after futher processing will be used to create DNA. As RNR produces deoxyribonucleotides, levels of DNA building blocks in the cell rise. To avoid overstocking the cell, RNR contains an ‘off switch’ that is triggered when levels of one of the DNA building blocks, dATP, is high enough to occupy a particular site on the alpha unit. Binding of dATP to this site results in three pairs of alpha units getting together to form a stable ring of six units (called α6). How the formation of this stable α6 ring actually turns off RNR was an open question. Here, Brignole, Tsai et al. use a microscopy method called cryo-EM to reveal the three-dimensional structure of the inactive human RNR almost down to the level of individual atoms. When the alpha pairs form an α6 ring, the hole in the center of this circle is smaller than β2, keeping β2 away from α2. This inaccessibility leads to RNR being switched off. If RNR is inactive, DNA synthesis is impaired and cells cannot divide. In turn, controlling whether or not cells proliferate is key to fighting diseases like cancer (where ‘rogue’ cells keep replicating) or bacterial infections. Certain cancer treatments already target RNR, and create the inactive α6 ring structure. In addition, in bacteria, the inactive form of RNR is different from the human one and forms an α4β4 ring,rather than an α6 ring. Understanding the structure of the human inactive RNR could help scientists to find both new anticancer and antibacterial drugs.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 A structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

RIG-I activates interferon signaling pathways by promoting filament formation of the adaptor molecule, MAVS. Assembly of the MAVS filament is mediated by its CARD domain (CARD(MAVS)), and requires its interaction with the tandem CARDs of RIG-I (2CARD(RIG-I)). However, the precise nature of the interaction between 2CARD(RIG-I) and CARD(MAVS), and how this interaction leads to CARD(MAVS) filament assembly, has been unclear. Here we report a 3.6 Å electron microscopy structure of the CARD(MAVS) filament and a 3.4 Å crystal structure of the 2CARD(RIG-I):CARD(MAVS) complex, representing 2CARD(RIG-I) "caught in the act" of nucleating the CARD(MAVS) filament. These structures, together with functional analyses, show that 2CARD(RIG-I) acts as a template for the CARD(MAVS) filament assembly, by forming a helical tetrameric structure and recruiting CARD(MAVS) along its helical trajectory. Our work thus reveals that signal activation by RIG-I occurs by imprinting its helical assembly architecture on MAVS, a previously uncharacterized mechanism of signal transmission.

Single-particle electron microscopy and hydrogen–deuterium exchange mass spectrometry are used to characterize the structure and dynamics of a G-protein-coupled receptor–arrestin complex. Much has been learned about the structure of G-protein-coupled receptors (GCPRs) over the past seven years, but we still don't know what an activated GPCR looks like when it is bound to a β-arrestin. (Arrestins are cellular mediators with a broad range of functions, many of them involving GPCRs.) In this study the authors use single-particle electron microscopy and hydrogen–deuterium exchange mass spectrometry to characterize the structure and dynamics of a GPCR–arrestin complex. Their data support a 'biphasic' mechanism, in which the arrestin initially interacts with the phosphorylated carboxy terminus of the GPCR before re-arranging to more fully engage the membrane protein in a signalling-competent conformation. G-protein-coupled receptors (GPCRs) are critically regulated by β-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling1,2,3,4,5. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)–G-protein complex, has provided novel insights into the structural basis of receptor activation6,7,8,9,10,11. However, complementary information has been lacking on the recruitment of β-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor–β-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human β2AR–β-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between β2AR and β-arrestin 1 using hydrogen–deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of β-arrestin 1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of β-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of β-arrestin 1 when coupled to the β2AR. A molecular model of the β2AR–β-arrestin signalling complex was made by docking activated β-arrestin 1 and β2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor–arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.

Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.

Here we present for the first time a three-dimensional cryo-EM map of the Saccharomyces cerevisiae respiratory supercomplex composed of dimeric complex III flanked on each side by one monomeric complex IV. A precise fit of the existing atomic x-ray structures of complex III from yeast and complex IV from bovine heart into the cryo-EM map resulted in a pseudo-atomic model of the three-dimensional structure for the supercomplex. The distance between cytochrome c binding sites of complexes III and IV is about 6 nm, which supports proposed channeling of cytochrome c between the individual complexes. The opposing surfaces of complexes III and IV differ considerably from those reported for the bovine heart supercomplex as determined by cryo-EM. A closer association between the individual complex domains at the aqueous membrane interface and larger spaces between the membrane-embedded domains where lipid molecules may reside are also demonstrated. The supercomplex contains about 50 molecules of cardiolipin (CL) with a fatty acid composition identical to that of the inner membrane CL pool, consistent with CL-dependent stabilization of the supercomplex.

SummaryWe present the codimensional principal component analysis (PCA), a novel and straightforward method for resolving sample heterogeneity within a set of cryo-EM 2D projection images of macromolecular assemblies. The method employs PCA of resampled 3D structures computed using subsets of 2D data obtained with a novel hypergeometric sampling scheme. PCA provides us with a small subset of dominating “eigenvolumes” of the system, whose reprojections are compared with experimental projection data to yield their factorial coordinates constructed in a common framework of the 3D space of the macromolecule. Codimensional PCA is unique in the dramatic reduction of dimensionality of the problem, which facilitates rapid determination of both the plausible number of conformers in the sample and their 3D structures. We applied the codimensional PCA to a complex data set of Thermus thermophilus 70S ribosome, and we identified four major conformational states and visualized high mobility of the stalk base region.

During translation, transfer RNAs enter the ribosome and then move sequentially through three sites, known as A, P and E, as they transfer their attached amino acids onto the growing peptide chain. How the ribosome facilitates tRNA translocation between the sites remains largely unknown. Christian Spahn and colleagues have used multiparticle cryoelectron microscopy of a ribosome bound to the translation elongation factor, EF-G, to get information about tRNA movement. They identify two new sub-states and conclude that, following spontaneous inter-subunit ratcheting, translocation is the direct result of head swivelling and unratcheting of the 30S ribosomal subunit. During translation, tRNAs enter the ribosome and then move sequentially through three sites, known as A, P and E, as they transfer their attached amino acids onto the growing peptide chain. How the ribosome facilitates tRNA translocation between the sites remains largely unknown. Now a study uses multiparticle cryoelectron microscopy of a ribosome bound to the translation elongation factor, EF-G, to get information about tRNA movement. It identifies two new substates and sees that translocation is linked to unratcheting of the 30S ribosomal subunit. The elongation cycle of protein synthesis involves the delivery of aminoacyl-transfer RNAs to the aminoacyl-tRNA-binding site (A site) of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A site1,2. The translocation reaction is catalysed by elongation factor G (EF-G) in a GTP-dependent manner3. Despite the availability of structures of various EF-G–ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryoelectron microscopy analysis to resolve two previously unseen subpopulations within Thermus thermophilus EF-G–ribosome complexes at subnanometre resolution, one of them with a partly translocated tRNA. Comparison of these substates reveals that translocation of tRNA on the 30S subunit parallels the swivelling of the 30S head and is coupled to unratcheting of the 30S body. Because the tRNA maintains contact with the peptidyl-tRNA-binding site (P site) on the 30S head and simultaneously establishes interaction with the exit site (E site) on the 30S platform, a novel intra-subunit ‘pe/E’ hybrid state is formed. This state is stabilized by domain IV of EF-G, which interacts with the swivelled 30S-head conformation. These findings provide direct structural and mechanistic insight into the ‘missing link’ in terms of tRNA intermediates involved in the universally conserved translocation process.

Single particle cryo-electron microscopy (cryo-EM) is a technique aimed at structure determination of large macromolecular complexes in their unconstrained, physiological conditions. The power of the method has been demonstrated in selected studies where for highly symmetric molecules the resolution attained permitted backbone tracing. However, most molecular complexes appear to exhibit intrinsic conformational variability necessary to perform their functions. Therefore, it is now increasingly recognized that sample heterogeneity constitutes a major methodological challenge for cryo-EM. To overcome it dedicated experimental and particularly computational multi-particle approaches have been developed. Their applications point to the future of cryo-EM as an experimental method uniquely suited to visualize the conformational modes of large macromolecular complexes and machines.

We describe the Transform Class in SPARX/EMAN2 that is designed to handle rigid body motions of two- and three-dimensional data. We describe relationships between Eulerian angles conventions used in different electron microscopy software packages, as well as give examples of the simple scripts that execute conversions of Eulerian angles between these packages and handle other related tasks. The Transform Class is also responsible for generating point-group symmetry operations as well as generating quasi-evenly distributed points on the sphere (which is used in creating reference projections for structure refinement procedures in single particle reconstruction). We discuss how this is carried out internally in the code, and how symmetry operations are accessed through the SPARX interactive interface. We present a comprehensive description of symmetry operations for all point-group symmetries, as implemented in the class. Finally, we provide solutions to a number of typical problems associated with rotation operations-alignment of markers for dual-axis tomography, delineations of asymmetric subunits, quasi-uniform distribution of projection directions and such, and provide examples how these problems are solved using operations in the Transform Class.

We describe the strategies and implementation details we employed to parallelize the SPIDER software package on distributed-memory parallel computers using the message passing interface (MPI). The MPI-enabled SPIDER preserves the interactive command line and batch interface used in the sequential version of SPIDER, thus does not require users to modify their existing batch programs. We show the excellent performance of the MPI-enabled SPIDER when it is used to perform multi-reference alignment and 3-D reconstruction operations on a number of different computing platforms. We point out some performance issues when the MPI-enabled SPIDER is used for a complete 3-D projection matching refinement run, and propose several ways to further improve the parallel performance of SPIDER on distributed-memory machines.

Members of the AAA family of ATPases assemble into hexameric double rings and perform vital functions, yet their molecular mechanisms remain poorly understood. Here, we report structures of the Pex1/Pex6 complex; mutations in these proteins frequently cause peroxisomal diseases. The structures were determined in the presence of different nucleotides by cryo-electron microscopy. Models were generated using a computational approach that combines Monte Carlo placement of structurally homologous domains into density maps with energy minimization and refinement protocols. Pex1 and Pex6 alternate in an unprecedented hexameric double ring. Each protein has two N-terminal domains, N1 and N2, structurally related to the single N domains in p97 and N-ethylmaleimide sensitive factor (NSF); N1 of Pex1 is mobile, but the others are packed against the double ring. The N-terminal ATPase domains are inactive, forming a symmetric D1 ring, whereas the C-terminal domains are active, likely in different nucleotide states, and form an asymmetric D2 ring. These results suggest how subunit activity is coordinated and indicate striking similarities between Pex1/Pex6 and p97, supporting the hypothesis that the Pex1/Pex6 complex has a role in peroxisomal protein import analogous to p97 in ER-associated protein degradation.

Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. While their mode of action is often compared to that of mechanical machines, a crucial difference is that at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and three-dimensional variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements.

Cryo-electron microscopy (cryo-EM) of single-particle specimens is used to determine the structure of proteins and macromolecular complexes without the need for crystals. Recent advances in detector technology and software algorithms now allow images of unprecedented quality to be recorded and structures to be determined at near-atomic resolution. However, compared with X-ray crystallography, cryo-EM is a young technique with distinct challenges. This primer explains the different steps and considerations involved in structure determination by single-particle cryo-EM to provide an overview for scientists wishing to understand more about this technique and the interpretation of data obtained with it, as well as a starting guide for new practitioners.

Internal ribosome entry sites (IRESs) facilitate an alternative, end-independent pathway of translation initiation. A particular family of dicistroviral IRESs can assemble elongation-competent 80S ribosomal complexes in the absence of canonical initiation factors and initiator transfer RNA. We present here a cryo-EM reconstruction of a dicistroviral IRES bound to the 80S ribosome. The resolution of the cryo-EM reconstruction, in the subnanometer range, allowed the molecular structure of the complete IRES in its active, ribosome-bound state to be solved. The structure, harboring three pseudoknot-containing domains, each with a specific functional role, shows how defined elements of the IRES emerge from a compactly folded core and interact with the key ribosomal components that form the A, P and E sites, where tRNAs normally bind. Our results exemplify the molecular strategy for recruitment of an IRES and reveal the dynamic features necessary for internal initiation.

Summary The structure of the endosomal-associated protein, Hrs, has been determined with cryo-electron microscopy. Hrs interacts with a number of proteins, including SNAP-25 and STAM1, forming a complex that binds ubiquitin moieties. Analytical ultracentrifugation studies revealed that Hrs exists as a hexamer. The symmetry and the structure of the hexameric form of Hrs were determined with the single-particle reconstruction method. Hrs comprises three antiparallel dimers with a central core and distinct caps on either end. Crystal structures of VHS and FYVE domains fit into the Hrs end caps in the EM density map. Thus, the location of domains that interact with the endosomal membrane, the VHS, FYVE, and C-terminal domains, facilitates the anchorage of Hrs to the membrane, initiating the functional processes of Hrs on the endosome. Based on our model, the Hrs hexamer interacts with the membrane and acts as a "master molecule" that presents multiple sites for protein binding.

We describe an algorithm for simultaneous refinement of a three-dimensional (3-D) density map and of the orientation parameters of two-dimensional (2-D) projections that are used to reconstruct this map. The application is in electron microscopy, where the 3-D structure of a protein has to be determined from a set of 2-D projections collected at random but initially unknown angles. The design of the algorithm is based on the assumption that initial low resolution approximation of the density map and reasonable guesses for orientation parameters are available. Thus, the algorithm is applicable in final stages of the structure refinement, when the quality of the results is of main concern. We define the objective function to be minimized in real space and solve the resulting nonlinear optimization problem using a Quasi-Newton algorithm. We calculate analytical derivatives with respect to density distribution and the finite difference approximations of derivatives with respect to orientation parameters. We demonstrate that calculation of derivatives is robust with respect to noise in the data. This is due to the fact that noise is annihilated by the back-projection operations. Our algorithm is distinguished from other orientation refinement methods (i) by the simultaneous update of the density map and orientation parameters resulting in a highly efficient computational scheme and (ii) by the high quality of the results produced by a direct minimization of the discrepancy between the 2-D data and the projected views of the reconstructed 3-D structure. We demonstrate the speed and accuracy of our method by using simulated data.

A framework for constructing circulant and block circulant preconditioners ($C$) for a symmetric linear system $Ax=b$ arising from certain signal and image processing applications is presented in this paper. The proposed scheme does not make explicit use of matrix elements of $A$. It is ideal for applications in which $A$ only exists in the form of a matrix vector multiplication routine, and in which the process of extracting matrix elements of $A$ is costly. The proposed algorithm takes advantage of the fact that for many linear systems arising from signal or image processing applications, eigenvectors of $A$ can be well represented by a small number of Fourier modes. Therefore, the construction of $C$ can be carried out in the frequency domain by carefully choosing its eigenvalues so that the condition number of $C^TAC$ can be reduced significantly. We illustrate how to construct the spectrum of $C$ in a way such that the smallest eigenvalues of $C^TAC$ overlaps with those of $A$ extremely well while the largest eigenvalues of $C^TAC$ are smaller than those of $A$ by several orders of magnitude. Numerical examples are provided to demonstrate the effectiveness of the preconditioner on accelerating the solution of linear systems arising from image reconstruction application.

An 11.7-Å-resolution cryo-EM map of the yeast 80S·eEF2 complex in the presence of the antibiotic sordarin was interpreted in molecular terms, revealing large conformational changes within eEF2 and the 80S ribosome, including a rearrangement of the functionally important ribosomal intersubunit bridges. Sordarin positions domain III of eEF2 so that it can interact with the sarcin–ricin loop of 25S rRNA and protein rpS23 (S12p). This particular conformation explains the inhibitory action of sordarin and suggests that eEF2 is stalled on the 80S ribosome in a conformation that has similarities with the GTPase activation state. A ratchet-like subunit rearrangement (RSR) occurs in the 80S·eEF2·sordarin complex that, in contrast to Escherichia coli 70S ribosomes, is also present in vacant 80S ribosomes. A model is suggested, according to which the RSR is part of a mechanism for moving the tRNAs during the translocation reaction.

Manual selection of single particles in images acquired using cryo-electron microscopy (cryoEM) will become a significant bottleneck when datasets of a hundred thousand or even a million particles are required for structure determination at near atomic resolution. Algorithm development of fully automated particle selection is thus an important research objective in the cryoEM field. A number of research groups are making promising new advances in this area. Evaluation of algorithms using a standard set of cryoEM images is an essential aspect of this algorithm development. With this goal in mind, a particle selection "bakeoff" was included in the program of the Multidisciplinary Workshop on Automatic Particle Selection for cryoEM. Twelve groups participated by submitting the results of testing their own algorithms on a common dataset. The dataset consisted of 82 defocus pairs of high-magnification micrographs, containing keyhole limpet hemocyanin particles, acquired using cryoEM. The results of the bakeoff are presented in this paper along with a summary of the discussion from the workshop. It was agreed that establishing benchmark particles and using bakeoffs to evaluate algorithms are useful in promoting algorithm development for fully automated particle selection, and that the infrastructure set up to support the bakeoff should be maintained and extended to include larger and more varied datasets, and more criteria for future evaluations.

A three-dimensional (3D) version of the spectral signal-to-noise ratio (SSNR)-based resolution measure is introduced. The measure is defined for a class of 3D reconstruction algorithms that use interpolation in Fourier space. The statistical properties of the SSNR are discussed and related to the properties of another resolution measure, the Fourier shell correlation (FSC). The new measure was tested on 3D structures calculated from a simulated set of quasi-evenly spaced 2D projections using a nearest-neighbor interpolation and a gridding algorithm. In the latter case, the results agree very well with the FSC-based estimate, with the exception of very high SSNR values. The main applicability of the 3D SSNR is tomography, where due to the small number of projections collected, FSC cannot be used. The new measure was applied to three sets of tomographic data. It was demonstrated that the measure is sufficiently sensitive to yield theoretically expected results. Therefore, the 3D SSNR opens up the possibility of evaluating the quality of tomographic reconstructions in an objective manner. The 3D distribution of SSNR is of major interest in single-particle analysis. It is shown that the new measure can be used to evaluate the anisotropy of 3D reconstructions. The distribution of SSNR is characterized by three anisotropy indices derived from principal axes of the 3D inertia covariance matrix of the SSNR. These indices are used to construct a 3D Fourier filter which, when applied to a 3D reconstruction of a macromolecule, maximizes the SNR in real space and minimizes real-space artifacts caused by uneven distribution of 2D projections.

Robust ab initio structure determination and outcome validation are significant challenges that limit the potential of single particle structural EM. Stochastic Hill Climbing (SHC) can improve the convergence properties of widely-used projection matching (PM) algorithms and facilitate ab initio structure determination, but outcome correctness remains in question as SHC is an local optimizer. We propose a protocol, VIPER, for determination and validation of ab initio structures based on an evolutionary approach. The outcomes of parallel independent runs are brought into register, monitored for consistency, and crossovers are conducted to improve overall convergence behavior. After each generation, the best candidates are selected according to their L2 norm values used as a goal function. Screening based on reproducibility of individual image parameters allows for outlier elimination. This new approach introduces rigorous outcome validation into EM ab initio structure determination.

As a process that brings together, in close proximity, two linear structures of considerable length (mRNA and the nascent polypeptide chain) and large protein factors (EF-G and aminoacyl-tRNA·EF-Tu·GTP ternary complex), protein synthesis poses a logistic problem of traffic control: how to guarantee uninterrupted, high-precision performance without steric interference and entanglement of the various ligands. Since cryo-electron microscopy (cryo-EM) visualization provided the first detailed three-dimensional (3-D) images of the ribosome, much work has gone into the mapping of tRNA and elongation factors bound to the ribosome at various stages of the elongation cycle. A recent study of a 70S ribosome carrying a genetically inserted tRNA-like RNA fragment furnished a higher-resolution (17-A) map of the vacant ribosome, and the use of this new map in the subtraction produced a linear mass distribution covering the platform side segment of the mRNA path, as well as a mass hovering just at the entrance of the 30S subunit channel. tRNA bound to the ribosome has been directly visualized by 3-D cryo-EM in various tRNA-ribosome complexes. Visualization of the ribosome-bound tRNA is still a challenging task because the smallest dimension of the molecule is on the order of the resolution of cryo-EM and the occupancy of some tRNA binding states is intrinsically low.

Recently, the everimproving cryo-electron microscopy maps of the 70S ribosome have increased the overall accuracy of the modeling studies. This chapter talks about three important structure-function relationships. First, it briefly reviews the recently published model of interactions between the 16S rRNA decoding site and the mRNAtRNA complex. Second, it discusses the functional implications of the placement of that model in the electron density map. Finally, it identifies regions that the authors believe compose the ‘‘gate’’ that closes to form the channel in which the mRNA is located. The second-generation models incorporate atomic-resolution data for those components whose structures have been determined by crystallography or nuclear magnetic resonance (NMR), and for selected components the authors have modeled at atomic detail because of their functional significance. The authors therefore predict that the density in the channel will rise when tRNA is bound at the A site. Another interesting aspect of this model is that the axis of the decoding-site RNA lies perpendicular to the long axis of the 30S ribosomal subunit. Cross-linking studies between mRNA and the 16S rRNA also suggest that these two regions of the 16S rRNA are close together in the ribosomal subunit. Multiscale modeling permits investigation of structure-function relationships at atomic detail within the low-resolution framework of those regions that are less well defined and less functionally significant.

In structural electron microscopy, the accurate estimation of the Contrast Transfer Function (CTF) parameters, particularly defocus and astigmatism, is of utmost importance for both initial evaluation of micrograph quality and for subsequent structure determination. Due to increases in the rate of data collection on modern microscopes equipped with new generation cameras, it is also important that the CTF estimation can be done rapidly and with minimal user intervention. Finally, in order to minimize the necessity for manual screening of the micrographs by a user it is necessary to provide an assessment of the errors of fitted parameters values. In this work we introduce CTER, a CTF parameters estimation method distinguished by its computational efficiency. The efficiency of the method makes it suitable for high-throughput EM data collection, and enables the use of a statistical resampling technique, bootstrap, that yields standard deviations of estimated defocus and astigmatism amplitude and angle, thus facilitating the automation of the process of screening out inferior micrograph data. Furthermore, CTER also outputs the spatial frequency limit imposed by reciprocal space aliasing of the discrete form of the CTF and the finite window size. We demonstrate the efficiency and accuracy of CTER using a data set collected on a 300 kV Tecnai Polara (FEI) using the K2 Summit DED camera in super-resolution counting mode. Using CTER we obtained a structure of the 80S ribosome whose large subunit had a resolution of 4.03 A without, and 3.85 A with, inclusion of astigmatism parameters.

In vitro assembled yeast ribosome-nascent chain complexes (RNCs) containing a signal sequence in the nascent chain were immunopurified and reconstituted with the purified protein-conducting channel (PCC) of yeast endoplasmic reticulum, the Sec61 complex. A cryo-EM reconstruction of the RNC-Sec61 complex at 15.4 Å resolution shows a tRNA in the P site. Distinct rRNA elements and proteins of the large ribosomal subunit form four connections with the PCC across a gap of about 10–20 Å. Binding of the PCC influences the position of the highly dynamic rRNA expansion segment 27. The RNC-bound Sec61 complex has a compact appearance and was estimated to be a trimer. We propose a binary model of cotranslational translocation entailing only two basic functional states of the translating ribosome-channel complex.

A cryo-EM reconstruction of the translating yeast 80S ribosome was analyzed. Computationally separated rRNA and protein densities were used for docking of appropriately modified rRNA models and homology models of yeast ribosomal proteins. The core of the ribosome shows a remarkable degree of conservation. However, some significant differences in functionally important regions and dramatic changes in the periphery due to expansion segments and additional ribosomal proteins are evident. As in the bacterial ribosome, bridges between the subunits are mainly formed by RNA contacts. Four new bridges are present at the periphery. The position of the P site tRNA coincides precisely with its prokaryotic counterpart, with mainly rRNA contributing to its molecular environment. This analysis presents an exhaustive inventory of an eukaryotic ribosome at the molecular level.

Tet(O) belongs to a class of ribosomal protection proteins that mediate tetracycline resistance. It is a G protein that shows significant sequence similarity to elongation factor EF-G. Here we present a cryo-electron microscopic reconstruction, at 16 A resolution, of its complex with the E. coli 70S ribosome. Tet(O) was bound in the presence of a noncleavable GTP analog to programmed ribosomal complexes carrying fMet-tRNA in the P site. Tet(O) is directly visible as a mass close to the A-site region, similar in shape and binding position to EF-G. However, there are important differences. One of them is the different location of the tip of domain IV, which in the Tet(O) case, does not overlap with the ribosomal A site but is directly adjacent to the primary tetracycline binding site. Our findings give insights into the mechanism of tetracycline resistance.

The extracellular giant hemoglobin from the earthworm Lumbricus terrestris was reconstructed at 14.9-A resolution from cryo-electron microscope images, using a new procedure for estimating parameters of the contrast transfer (CTF) function. In this approach, two important CTF parameters, defocus and amplitude contrast ratio, can be refined iteratively within the framework of 3D projection alignment procedure, using minimization of sign disagreement between theoretical CTF and cross-resolution curves. The 3D cryo-EM map is in overall good agreement with the recent X-ray crystallography map of Royer et al. (2000, Proc. Natl. Acad. Sci. USA 97, 7107-7111), and it reveals the local threefold arrangement of the three linker chains present within each 1/12 of the complex. The 144 globin chains and 36 linker chains within the complex are clearly visible, and the interdigitation of the 12 coiled-coil helical spokes forming the central toroidal piece is confirmed. Based on these findings, two mechanisms of the dodecameric unit assembly are proposed and termed "zigzag" and "pairwise" polymerizations. However, the detection by cryo-EM of 12 additional rod-like bodies within the toroid raises the possibility that the architecture of the toroid is more complex than previously thought or that yet unknown ligands or allosteric effectors for this oxygen carrier are present.

Background: This study addresses the general problem of dividing a density map of a nucleic-acid–protein complex obtained by cryo-electron microscopy (cryo-EM) or X-ray crystallography into its two components. When the resolution of the density map approaches ∼3 A it is generally possible to interpret its shape (i.e., the envelope obtained for a standard choice of threshold) in terms of molecular structure, and assign protein and nucleic acid elements on the basis of their known sequences. The interpretation of low-resolution maps in terms of proteins and nucleic acid elements of known structure is of increasing importance in the study of large macromolecular complexes, but such analyses are difficult. Results: Here we show that it is possible to separate proteins from nucleic acids in a cryo-EM density map, even at 11.5 A resolution. This is achieved by analysing the (continuous-valued) densities using the difference in scattering density between protein and nucleic acids, the contiguity constraints that the image of any nucleic acid molecule must obey, and the knowledge of the molecular volumes of all proteins. Conclusions: The new method, when applied to an 11.5 A cryo-EM map of the Escherichia coli 70S ribosome, reproduces boundary assignments between rRNA and proteins made from higher-resolution X-ray maps of the ribosomal subunits with a high degree of accuracy. Plausible predictions for the positions of as yet unassigned proteins and RNA components are also possible. One of the conclusions derived from this separation is that 23S rRNA is solely responsible for the catalysis of peptide bond formation. Application of the separation method to any nucleoprotein complex appears feasible.

Over 73,000 projections of the E. coli ribosome bound with formyl-methionyl initiator tRNA f 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 α-sarcin-ricin loop. Analysis of the bridging connections between the subunits provides insight into the dynamic signaling mechanism between the ribosomal subunits.

Three-dimensional cryomaps have been reconstructed for tRNA–ribosome complexes in pre- and posttranslocational states at 17-Å resolution. The positions of tRNAs in the A and P sites in the pretranslocational complexes and in the P and E sites in the posttranslocational complexes have been determined. Of these, the P-site tRNA position is the same as seen earlier in the initiation-like fMet-tRNAfMet-ribosome complex, where it was visualized with high accuracy. Now, the positions of the A- and E-site tRNAs are determined with similar accuracy. The positions of the CCA end of the tRNAs at the A site are different before and after peptide bond formation. The relative positions of anticodons of P- and E-site tRNAs in the posttranslocational state are such that a codon–anticodon interaction at the E site appears feasible.