Cryo-electron Microscopy Maps Research Articles

Regulation of the Mammalian Elongation Cycle by Subunit Rolling: A Eukaryotic-Specific Ribosome Rearrangement

The extent to which bacterial ribosomes and the significantly larger eukaryotic ribosomes share the same mechanisms of ribosomal elongation is unknown. Here, we present subnanometer resolution cryoelectron microscopy maps of the mammalian 80S ribosome in the posttranslocational state and in complex with the eukaryotic eEF1A⋅Val-tRNA⋅GMPPNP ternary complex, revealing significant differences in the elongation mechanism between bacteria and mammals. Surprisingly, and in contrast to bacterial ribosomes, a rotation of the small subunit around its long axis and orthogonal to the well-known intersubunit rotation distinguishes the posttranslocational state from the classical pretranslocational state ribosome. We term this motion "subunit rolling." Correspondingly, a mammalian decoding complex visualized in substates before and after codon recognition reveals structural distinctions from the bacterial system. These findings suggest how codon recognition leads to GTPase activation in the mammalian system and demonstrate that in mammalia subunit rolling occurs during tRNA selection.

Bacteriophage P22 tailspike: structure of the complete protein and function of the interdomain linker.

Attachment of phages to host cells, followed by phage DNA ejection, represents the first stage of viral infection of bacteria. Salmonella phage P22 has been extensively studied, serving as an experimental model for bacterial infection by phages. P22 engages bacteria by binding to the sugar moiety of lipopolysaccharides using the viral tailspike protein for attachment. While the structures of the N-terminal particle-binding domain and the major receptor-binding domain of the tailspike have been analyzed individually, the three-dimensional organization of the intact protein, including the highly conserved linker region between the two domains, remained unknown. A single amino-acid exchange in the linker sequence made it possible to crystallize the full-length protein. Two crystal structures of the linker region are presented: one attached to the N-terminal domain and the other present within the complete tailspike protein. Both retain their biological function, but the mutated full-length tailspike displays a retarded folding pathway. Fitting of the full-length tailspike into a published cryo-electron microscopy map of the P22 virion requires an elastic distortion of the crystal structure. The conservation of the linker suggests a role in signal transmission from the distal tip of the molecule to the phage head, eventually leading to DNA ejection.

Translation Initiation Factor eIF3b Contains a Nine-Bladed β-Propeller and Interacts with the 40S Ribosomal Subunit

The multisubunit eukaryotic translation initiation factor 3, among which the subunit b (eIF3b) is a major scaffold protein, plays essential roles in protein synthesis. Here, we report the crystal structure of the WD40 domain of Chaetomium thermophilum eIF3b, revealing a nine-bladed β-propeller fold. Sequence analysis indicates that this propeller architecture is common to all eIF3b orthologs. Revisiting the cryoelectron microscopy (cryo-EM) map of the 43S preinitiation complex suggests an interaction of the eIF3b with the 40S ribosomal subunit involving the ribosomal protein S9e and the 18S rRNA. This model is strongly supported by the direct binding of eIF3b to 40S ribosomes and to the isolated ribosomal protein rpS9e in vitro.

Finding the right fit: chiseling structures out of cryo-electron microscopy maps

Cryo-electron microscopy is a central tool for studying the architecture of macromolecular complexes at subnanometer resolution. Interpretation of an electron microscopy map requires its computational integration with data about the structure's components from all available sources, notably atomic models. Selecting a protocol for EM density-guided integrative structural modeling depends on the resolution and quality of the EM map as well as the available complimentary datasets. Here, we review rigid, flexible, and de novo integrative fitting into EM maps and provide guidelines and considerations for the design of modeling experiments. Finally, we discuss efforts towards establishing unified criteria for map and model assessment and validation.

Type 2 Ryanodine Receptor Domain A Contains a Unique and Dynamic α-Helix That Transitions to a β-Strand in a Mutant Linked with a Heritable Cardiomyopathy

Ryanodine receptors (RyRs) are large tetrameric calcium (Ca2+) release channels found on the sarcoplasmic reticulum that respond to dihydropyridine receptor activity through a direct conformational interaction and/or indirect Ca2+ sensitivity, propagating sarcoplasmic reticulum luminal Ca2+ release in the process of excitation–contraction coupling. There are three human RyR subtypes, and several debilitating diseases are linked to heritable mutations in RyR1 and RyR2 including malignant hypothermia, central core disease, catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Despite the recent appreciation that many disease-associated mutations within the N-terminal RyRABC domains (i.e., residues 1–559) are located in the putative interfaces mediating tetrameric channel assembly, the precise structural and dynamical consequences of the mutations are not well understood. We used solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography to examine the effect of ARVD2-associated (i.e., R176Q) and CPVT-associated [i.e., P164S, R169Q and delta exon 3 (Δ3)] mutations on the structure and dynamics of RyR2A. Our solution NMR data exposed a mobile α-helix, unique to type 2; further, this α2 helix rescues the β-strand lost in RyR2A Δ3 but remains dynamic in the hot-spot loop (HS-loop) P164S, R169Q and R176Q mutant proteins. Docking of our X-ray crystal/NMR hybrid structure into the RyR1 cryo-electron microscopy map revealed that this RyR2A α2 helix is in close proximity to dense “columns” projecting toward the channel pore. This is in contrast to the HS-loop mutations that cause structural changes largely localized to the intersubunit interface between adjacent ABC domains. Taken together, our data suggest that ARVD2 and CPVT mutations have at least two distinct structural consequences linked to channel dysfunction: perturbation of the HS-loop (i.e., domain A):domain B intersubunit interface and disruption of the communication between the N-terminal region and the channel domain.

A dodecameric ring-like structure of the N0 domain of the type II secretin from enterotoxigenic Escherichia coli

In many bacteria, secretins from the type II secretion system (T2SS) function as outer membrane gated channels that enable passage of folded proteins from the periplasm into the extracellular milieu. Cryo-electron microscopy of the T2SS secretin GspD revealed previously the dodecameric cylindrical architecture of secretins, and crystal structures of periplasmic secretin domains showed a modular domain organization. However, no high-resolution experimental data has as yet been provided about how the entire T2SS secretin or its domains are organized in a cylindrical fashion. Here we present a crystal structure of the N0 domain of the T2SS secretin GspD from enterotoxigenic Escherichia coli containing a helix with 12 subunits per turn. The helix has an outer diameter of ∼125Å and a pitch of only 24Å which suggests a model of a cylindrical dodecameric N0 ring whose dimensions correspond with the cryo-electron microscopy map of Vibrio cholerae GspD. The N0 domain is known to interact with the HR domain of the inner membrane T2SS protein GspC. When the new N0 ring model is combined with the known N0·HR crystal structure, a dodecameric double-ring of twelve N0-HR heterodimers is obtained. In contrast, the previously observed compact N0-N1 GspD module is not compatible with the N0 ring. Interestingly, a N0-N1 T3SS homolog is compatible with forming a N0-N1 dodecameric ring, due to a different N0-vs-N1 orientation. This suggests that the dodecameric N0 ring is an important feature of T2SS secretins with periplasmic domains undergoing considerable motions during exoprotein translocation.

Structural analyses at pseudo atomic resolution of Chikungunya virus and antibodies show mechanisms of neutralization.

A 5.3 Å resolution, cryo-electron microscopy (cryoEM) map of Chikungunya virus-like particles (VLPs) has been interpreted using the previously published crystal structure of the Chikungunya E1-E2 glycoprotein heterodimer. The heterodimer structure was divided into domains to obtain a good fit to the cryoEM density. Differences in the T = 4 quasi-equivalent heterodimer components show their adaptation to different environments. The spikes on the icosahedral 3-fold axes and those in general positions are significantly different, possibly representing different phases during initial generation of fusogenic E1 trimers. CryoEM maps of neutralizing Fab fragments complexed with VLPs have been interpreted using the crystal structures of the Fab fragments and the VLP structure. Based on these analyses the CHK-152 antibody was shown to stabilize the viral surface, hindering the exposure of the fusion-loop, likely neutralizing infection by blocking fusion. The CHK-9, m10 and m242 antibodies surround the receptor-attachment site, probably inhibiting infection by blocking cell attachment. DOI:http://dx.doi.org/10.7554/eLife.00435.001.

Macromolecular Structure Modeling and Electron Microscopy Fitting using 3D Zernike Descriptors

A novel computational method for fitting high-resolution structures of multiple proteins into a cryoelectron microscopy (EM) map is presented. The method named EMLZerD generates a pool of candidate complex conformations of component proteins using a novel multiple protein docking method, Multi-LZerD, which are later compared with a provided EM map to select the ones that fit well into the EM map (Esquivel-Rodriguez & Kihara, J. Phys Chem B, 2012). Multi-LZerD (Esquivel-Rodriguez & Kihara, Proteins, 2012) builds models of multimeric protein complexes by effectively reusing pairwise docking predictions of component proteins. The comparison of docking conformations and the EM map is performed using the 3D Zernike descriptor (3DZD), a mathematical series expansion of three-dimensional functions. The 3DZD provides a unified representation of the surface shape of multimeric protein complex models and EM maps, which allows a convenient, fast quantitative comparison of the three-dimensional structural data. Out of 19 multimeric complexes tested, near native complex structures with a RMSD of less than 2.5 A were obtained for 14 cases while medium range resolution structures with correct topology were computed for the additional 5 cases.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Modeling a Ryanodine Receptor Amino-Terminal Domain Connecting the Central Vestibule and the Corner Clamp Region

Ryanodine receptors (RyRs) form a class of intracellular calcium release channels in various excitable tissues and cells such as muscles and neurons. They are the major cellular mediators of the release of calcium ions from the sarcoplasmic reticulum, an essential step in muscle excitation-contraction coupling. Several crystal structures of skeletal muscle RyR1 peptide fragments have been solved, but these cover less than 15% of the full-length RyR1 sequence. In the present study, we obtain pseudo-atomic models for RyR fragments, consisting of residues 850-1,056 in rabbit RyR1 or residues 861-1,067 in mouse RyR2, by combining modeling techniques with sub-nanometer resolution cryo-electron microscopy (cryo-EM) maps. These fragments dock into a domain that connects the central vestibule and corner clamp region of RyR, with a good match between the secondary structure elements in the cryo-EM map and the pseudo-atomic models, and also consistent with our previous results of 3D cryo-EM RyR-GFP mapping and FRET measurement between RyR and FKBP. A combined model of the RyR fragment and FKBP docked into the cryo-EM map suggests that the fragment is positioned adjacent to the FKBP binding site. Its predicted binding interface with FKBP consists of primarily electrostatic contacts and contains several disease-associated mutations. A dynamic interaction between the fragment and a RyR phosphorylation domain, characterized using FRET data, also support the structural predictions of the pseudo-atomic models.

Modeling a Ryanodine Receptor N-terminal Domain Connecting the Central Vestibule and the Corner Clamp Region

Ryanodine receptors (RyRs) form a class of intracellular calcium release channels in various excitable tissues and cells such as muscles and neurons. They are the major cellular mediators of the release of calcium ions from the sarcoplasmic reticulum, an essential step in muscle excitation-contraction coupling. Several crystal structures of skeletal muscle RyR1 peptide fragments have been solved, but these cover less than 15% of the full-length RyR1 sequence. In this study, by combining modeling techniques with sub-nanometer resolution cryo-electron microscopy (cryo-EM) maps, we obtained pseudo-atomic models for RyR fragments consisting of residues 850-1,056 in rabbit RyR1 or residues 861-1,067 in mouse RyR2. These fragments are docked into a domain that connects the central vestibule and corner clamp region of RyR, resulting in a good match of the secondary structure elements in the cryo-EM map and the pseudo-atomic models, which is also consistent with our previous mappings of GFP insertions by cryo-EM and with FRET measurements involving RyR and FK506-binding protein (FKBP). A combined model of the RyR fragment and FKBP docked into the cryo-EM map suggests that the fragment is positioned adjacent to the FKBP-binding site. Its predicted binding interface with FKBP consists primarily of electrostatic contacts and contains several disease-associated mutations. A dynamic interaction between the fragment and an RyR phosphorylation domain, characterized by FRET experiments, also supports the structural predictions of the pseudo-atomic models.

Promiscuous behaviour of archaeal ribosomal proteins: Implications for eukaryotic ribosome evolution

In all living cells, protein synthesis occurs on ribonucleoprotein particles called ribosomes. Molecular models have been reported for complete bacterial 70S and eukaryotic 80S ribosomes; however, only molecular models of large 50S subunits have been reported for archaea. Here, we present a complete molecular model for the Pyrococcus furiosus 70S ribosome based on a 6.6 Å cryo-electron microscopy map. Moreover, we have determined cryo-electron microscopy reconstructions of the Euryarchaeota Methanococcus igneus and Thermococcus kodakaraensis 70S ribosomes and Crenarchaeota Staphylothermus marinus 50S subunit. Examination of these structures reveals a surprising promiscuous behavior of archaeal ribosomal proteins: We observe intersubunit promiscuity of S24e and L8e (L7ae), the latter binding to the head of the small subunit, analogous to S12e in eukaryotes. Moreover, L8e and L14e exhibit intrasubunit promiscuity, being present in two copies per archaeal 50S subunit, with the additional binding site of L14e analogous to the related eukaryotic r-protein L27e. Collectively, these findings suggest insights into the evolution of eukaryotic ribosomal proteins through increased copy number and binding site promiscuity.

RIBFIND: a web server for identifying rigid bodies in protein structures and to aid flexible fitting into cryo EM maps

To better analyze low-resolution cryo electron microscopy maps of macromolecular assemblies, component atomic structures frequently have to be flexibly fitted into them. Reaching an optimal fit and preventing the fitting process from getting trapped in local minima can be significantly improved by identifying appropriate rigid bodies (RBs) in the fitted component. Here we present the RIBFIND server, a tool for identifying RBs in protein structures. The server identifies RBs in proteins by calculating spatial proximity between their secondary structural elements. The RIBFIND web server and its standalone program are available at http://ribfind.ismb.lon.ac.uk. a.pandurangan@mail.cryst.bbk.ac.uk Supplementary data are available at Bioinformatics online.

Latest insights on adenovirus structure and assembly.

Adenovirus (AdV) capsid organization is considerably complex, not only because of its large size (~950 Å) and triangulation number (pseudo T = 25), but also because it contains four types of minor proteins in specialized locations modulating the quasi-equivalent icosahedral interactions. Up until 2009, only its major components (hexon, penton, and fiber) had separately been described in atomic detail. Their relationships within the virion, and the location of minor coat proteins, were inferred from combining the known crystal structures with increasingly more detailed cryo-electron microscopy (cryoEM) maps. There was no structural information on assembly intermediates. Later on that year, two reports described the structural differences between the mature and immature adenoviral particle, starting to shed light on the different stages of viral assembly, and giving further insights into the roles of core and minor coat proteins during morphogenesis [1,2]. Finally, in 2010, two papers describing the atomic resolution structure of the complete virion appeared [3,4]. These reports represent a veritable tour de force for two structural biology techniques: X-ray crystallography and cryoEM, as this is the largest macromolecular complex solved at high resolution by either of them. In particular, the cryoEM analysis provided an unprecedented clear picture of the complex protein networks shaping the icosahedral shell. Here I review these latest developments in the field of AdV structural studies.

Fitting multimeric protein complexes into electron microscopy maps using 3D Zernike descriptors.

A novel computational method for fitting high-resolution structures of multiple proteins into a cryoelectron microscopy map is presented. The method named EMLZerD generates a pool of candidate multiple protein docking conformations of component proteins, which are later compared with a provided electron microscopy (EM) density map to select the ones that fit well into the EM map. The comparison of docking conformations and the EM map is performed using the 3D Zernike descriptor (3DZD), a mathematical series expansion of three-dimensional functions. The 3DZD provides a unified representation of the surface shape of multimeric protein complex models and EM maps, which allows a convenient, fast quantitative comparison of the three-dimensional structural data. Out of 19 multimeric complexes tested, near native complex structures with a root-mean-square deviation of less than 2.5 Å were obtained for 14 cases while medium range resolution structures with correct topology were computed for the additional 5 cases.

Accurate Flexible Fitting of High-Resolution Protein Structures into cryo-Electron Microscopy Maps Using Coarse-Grained Pseudo-Energy Minimization

Accurate Flexible Fitting of High-Resolution Protein Structures into cryo-Electron Microscopy Maps Using Coarse-Grained Pseudo-Energy Minimization

The N-Terminal Disease Hot Spot of Ryanodine Receptors Forms a Cytoplasmic Vestibule

Ryanodine receptors (RyR) are ion channels that govern the release of Ca2+ from the endoplasmic reticulum. They thus regulate the contraction of skeletal and cardiac muscle. Mutations in RyR can lead to severe genetic conditions, including (but not limited to) malignant hyperthermia (MH) and catecholaminergic polymorphic ventricular tachycardia (CPVT). Despite the detailed investigation of the functional effects of the mutations, locating their position in the full-length channel structure has traditionally proven to be difficult. Here we present the 2.5 Ångström resolution crystal structure of a region spanning most of the N-terminal disease hot spot (residues 1-559), containing over 50 disease mutations in RyR1 and RyR2. The hot spot consists of three domains that interact through a predominantly hydrophilic interface. We have been able to dock the position of this hot spot into various RyR1 cryo electron microscopy maps, allowing its unambiguous positioning in the cytoplasmic portion of the channel, forming a 240-kDa ring around the fourfold symmetry axis. The disease mutations can be grouped into three different categories, either destabilizing the interfaces between the three N-terminal domains, affecting the folding of individual domains, or affecting one of six interfaces with other RyR parts. We propose a model whereby the opening of RyR coincides with allosterically coupled motions within the N-terminal domains. This can be affected by mutations that target various interfaces within and across subunits. We also propose a mechanism whereby RyRs are activated by redox modification through the destabilization of observed domain-domain interfaces. The structure provides a framework to understand the many disease mutations that have been studied using functional methods.

Accurate Flexible Fitting of High-Resolution Protein Structures into Cryo-Electron Microscopy Maps Using Coarse-Grained Pseudo-Energy Minimization

Cryo-electron microscopy (cryo-EM) has been widely used to explore conformational states of large biomolecular assemblies. The detailed interpretation of cryo-EM data requires the flexible fitting of a known high-resolution protein structure into a low-resolution cryo-EM map. To this end, we have developed what we believe is a new method based on a two-bead-per-residue protein representation, and a modified form of the elastic network model that allows large-scale conformational changes while maintaining pseudobonds and secondary structures. Our method minimizes a pseudo-energy which linearly combines various terms of the modified elastic network model energy with a cryo-EM-fitting score and a collision energy that penalizes steric collisions. Unlike previous flexible fitting efforts using the lowest few normal modes, our method effectively utilizes all normal modes so that both global and local structural changes can be fully modeled. We have validated our method for a diverse set of 10 pairs of protein structures using simulated cryo-EM maps with a range of resolutions and in the absence/presence of random noise. We have shown that our method is both accurate and efficient compared with alternative techniques, and its performance is robust to the addition of random noise. Our method is also shown to be useful for the flexible fitting of three experimental cryo-EM maps.

Structures of APC/CCdh1 with substrates identify Cdh1 and Apc10 as the D-box co-receptor

The ubiquitylation of cell cycle regulatory proteins by the large multimeric anaphase promoting complex (APC/C) controls sister chromatid segregation and the exit from mitosis 1,2. Selection of APC/C targets is achieved through recognition of destruction motifs, predominantly the D-box 3 and KEN-box 4. Although this process is known to involve a co-activator protein (either Cdc20 or Cdh1) together with core APC/C subunits 1,2, the structural basis for substrate recognition and ubiquitylation is not understood. Here, we investigated the APC/C using single particle electron microscopy (EM) and determined a cryo-EM map of APC/CCdh1 bound to a D-box peptide at ~10 Å resolution. We find that a combined catalytic and substrate recognition module is located within the central cavity of the APC/C assembled from Cdh1, Apc10 - a core APC/C subunit previously implicated in substrate recognition 5,6,7, and the cullin domain of Apc2. Cdh1 and Apc10, identified from difference maps, create a co-receptor for D-box following repositioning of Cdh1 towards Apc10. Using NMR spectroscopy we demonstrate specific D-box – Apc10 interactions, consistent with a role for Apc10 in directly contributing towards D-box recognition by the APC/CCdh1 complex. Our results rationalise the contribution of both co-activator and core APC/C subunits to D-box recognition 8,9 and provide a structural framework for understanding mechanisms of substrate recognition and catalysis by the APC/C.

RNA channelling by the eukaryotic exosome

The eukaryotic exosome is a key nuclease for the degradation, processing and quality control of a wide variety of RNAs. Here, we report electron microscopic reconstructions and pseudo-atomic models of the ten-subunit Saccharomyces cerevisiae exosome in the unbound and RNA-bound states. In the RNA-bound structures, extra density that is visible at the entry and exit sites of the exosome channel indicates that a substrate-threading mechanism is used by the eukaryotic exosome. This channelling mechanism seems to be conserved in exosome-like complexes from all domains of life, and might have been present in the most recent common ancestor.

The First Human Epitope Map of the Alphaviral E1 and E2 Proteins Reveals a New E2 Epitope with Significant Virus Neutralizing Activity

BackgroundVenezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 (binds receptor and elicits virus neutralizing antibodies). Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes (E2c,d,e,f,g,h) bound VEEV-neutralizing antibody and mapped to amino acids (aa) 182–207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.MethodsWe used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.FindingsUsing 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115–119. Using a 9 Å resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.ConclusionsThe VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.