A primer to single-particle cryo-electron microscopy.

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.

Structural snapshots of actively translating human ribosomes.

Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although 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 3D 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.

Structure of the rigor actin-tropomyosin-myosin complex.

Regulation of myosin and filamentous actin interaction by tropomyosin is a central feature of contractile events in muscle and nonmuscle cells. However, little is known about molecular interactions within the complex and the trajectory of tropomyosin movement between its "open" and "closed" positions on the actin filament. Here, we report the 8 Å resolution structure of the rigor (nucleotide-free) actin-tropomyosin-myosin complex determined by cryo-electron microscopy. The pseudoatomic model of the complex, obtained from fitting crystal structures into the map, defines the large interface involving two adjacent actin monomers and one tropomyosin pseudorepeat per myosin contact. Severe forms of hereditary myopathies are linked to mutations that critically perturb this interface. Myosin binding results in a 23 Å shift of tropomyosin along actin. Complex domain motions occur in myosin, but not in actin. Based on our results, we propose a structural model for the tropomyosin-dependent modulation of myosin binding to actin.

Cryo-EM Structure of Caspase-8 Tandem DED Filament Reveals Assembly and Regulation Mechanisms of the Death-Inducing Signaling Complex.

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.

Molecular imprinting as a signal-activation mechanism of the viral RNA sensor RIG-I.

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.

Structure of the F-actin-tropomyosin complex.

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 loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. 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 troponin. Although crystal structures for monomeric actin (G-actin) are available, 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 Å in complex with tropomyosin at a resolution of 6.5 Å, 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 Mg(2+) 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 structure 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.

Visualization of arrestin recruitment by a G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) are critically regulated by ß-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the ß2 adrenergic receptor (ß2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of ß-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-ß-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human ß2AR-ß-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between ß2AR and ß-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 ß-arrestin 1 to the ß2AR, 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 ß-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 ß-arrestin 1 when coupled to the ß2AR. A molecular model of the ß2AR-ß-arrestin signalling complex was made by docking activated ß-arrestin 1 and ß2AR 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.

Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy.

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.

Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites.

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 site. The translocation reaction is catalysed by elongation factor G (EF-G) in a GTP-dependent manner. 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.

Inward-facing conformation of the zinc transporter YiiP revealed by cryoelectron microscopy.

YiiP is a dimeric Zn(2+)/H(+) antiporter from Escherichia coli belonging to the cation diffusion facilitator family. We used cryoelectron microscopy to determine a 13-Å resolution structure of a YiiP homolog from Shewanella oneidensis within a lipid bilayer in the absence of Zn(2+). Starting from the X-ray structure in the presence of Zn(2+), we used molecular dynamics flexible fitting to build a model consistent with our map. Comparison of the structures suggests a conformational change that involves pivoting of a transmembrane, four-helix bundle (M1, M2, M4, and M5) relative to the M3-M6 helix pair. Although accessibility of transport sites in the X-ray model indicates that it represents an outward-facing state, our model is consistent with an inward-facing state, suggesting that the conformational change is relevant to the alternating access mechanism for transport. Molecular dynamics simulation of YiiP in a lipid environment was used to address the feasibility of this conformational change. Association of the C-terminal domains is the same in both states, and we speculate that this association is responsible for stabilizing the dimer that, in turn, may coordinate the rearrangement of the transmembrane helices.

Arrangement of the respiratory chain complexes in Saccharomyces cerevisiae supercomplex III2IV2 revealed by single particle cryo-electron microscopy.

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.

GTPase activation of elongation factor EF-Tu by the ribosome during decoding.

We have used single-particle reconstruction in cryo-electron microscopy to determine a structure of the Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF-Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin. This represents the state in the decoding process just after codon recognition by tRNA and the resulting GTP hydrolysis by EF-Tu, but before the release of EF-Tu from the ribosome. Progress in sample purification and image processing made it possible to reach a resolution of 6.4 A. Secondary structure elements in tRNA, EF-Tu and the ribosome, and even GDP and kirromycin, could all be visualized directly. The structure reveals a complex conformational rearrangement of the tRNA in the A/T state and the interactions with the functionally important switch regions of EF-Tu crucial to GTP hydrolysis. Thus, the structure provides insights into the molecular mechanism of signalling codon recognition from the decoding centre of the 30S subunit to the GTPase centre of EF-Tu.

Real-space processing of helical filaments in SPARX.

We present a major revision of the iterative helical real-space refinement (IHRSR) procedure and its implementation in the SPARX single particle image processing environment. We built on over a decade of experience with IHRSR helical structure determination and we took advantage of the flexible SPARX infrastructure to arrive at an implementation that offers ease of use, flexibility in designing helical structure determination strategy, and high computational efficiency. We introduced the 3D projection matching code which now is able to work with non-cubic volumes, the geometry better suited for long helical filaments, we enhanced procedures for establishing helical symmetry parameters, and we parallelized the code using distributed memory paradigm. Additional features include a graphical user interface that facilitates entering and editing of parameters controlling the structure determination strategy of the program. In addition, we present a novel approach to detect and evaluate structural heterogeneity due to conformer mixtures that takes advantage of helical structure redundancy.

Rubisco in complex with Rubisco large subunit methyltransferase.

SET domain protein lysine methyltransferases (PKMT) are a structurally unique class of enzymes that catalyze the specific methylation of lysine residues in a number of different substrates. Especially histone-specific SET domain PKMTs have received widespread attention because of their roles in the regulation of epigenetic gene expression and the development of some cancers. Rubisco large subunit methyltransferase (RLSMT) is a chloroplast-localized SET domain PKMT responsible for the formation of trimethyl-lysine-14 in the large subunit of Rubisco, an essential photosynthetic enzyme. Here, we have used cryoelectron microscopy to produce an 11-A density map of the Rubisco-RLSMT complex. The atomic model of the complex, obtained by fitting crystal structures of Rubisco and RLSMT into the density map, shows that the extensive contact regions between the 2 proteins are mainly mediated by hydrophobic residues and leucine-rich repeats. It further provides insights into potential conformational changes that may occur during substrate binding and catalysis. This study presents the first structural analysis of a SET domain PKMT in complex with its intact polypeptide substrate.

Structure of the ribosome-bound cricket paralysis virus IRES RNA.

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.

ERj1p uses a universal ribosomal adaptor site to coordinate the 80S ribosome at the membrane.

Ribosomes translating secretory and membrane proteins are targeted to the endoplasmic reticulum membrane and attach to the protein-conducting channel and ribosome-associated membrane proteins (RAMPs). Recently, a new RAMP, ERj1p, has been identified that recruits BiP to ribosomes and regulates translational activity. Here we present the cryo-EM structure of a ribosome-ERj1p complex, revealing how ERj1p coordinates the ribosome at the membrane and how allosteric effects may mediate ERj1p's regulatory activity.

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.

Reliable cryo-EM resolution estimation with modified Fourier shell correlation.

A modified Fourier shell correlation (mFSC) methodology is introduced that is aimed at addressing two fundamental problems that mar the use of the FSC: the strong influence of mask-induced artifacts on resolution estimation and the lack of assessment of FSC uncertainties stemming from the inability to determine the associated number of degrees of freedom. It is shown that by simply changing the order of the steps in which the FSC is computed, the correlations induced by masking of the input data can be eliminated. In addition, to further reduce artifacts, a smooth Gaussian window function is used to outline the regions of reciprocal space within which the mFSC is computed. Next, it is shown that the number of degrees of freedom (ndf) of the system is approximated well by combining the ndf associated with the Gaussian window in reciprocal space with further reduction of the ndf owing to the use of the mask in real space. It is demonstrated through the application of the mFSC to both single-particle and helical structures that the mFSC yields reliable, mask-induced artifact-free results as a result of the introduced modifications. Since the adverse effect of the mask is eliminated, it also becomes possible to compute robust local resolutions both per voxel of a 3D map as well as, in a newly developed approach, per functional subunit, segment or even larger secondary element of the studied complex.

MyD88 Death-Domain Oligomerization Determines Myddosome Architecture: Implications for Toll-like Receptor Signaling.

Toll-like receptors (TLRs) are pivotal in triggering the innate immune response to pathogen infection. Ligand binding induces receptor dimerization which facilitates the recruitment of other post-receptor signal transducers into a complex signalosome, the Myddosome. Central to this process is Myeloid differentiation primary response 88 (MyD88), which is required by almost all TLRs, and signaling is thought to proceed via the stepwise, sequential assembly of individual components. Here, we show that the death domains of human MyD88 spontaneously and reversibly associate to form helical filaments in vitro. A 3.1-Å cryoelectron microscopy structure reveals that the architecture of the filament is identical to that of the 6:4 MyD88-IRAK4-IRAK2 hetero-oligomeric Myddosome. Additionally, the death domain of IRAK4 interacts with the filaments to reconstitute the non-stoichiometric 6:4 MyD88-IRAK4 complex. Together, these data suggest that intracellularly, the MyD88 scaffold may be pre-formed and poised for recruitment of IRAKs on receptor activation and TIR engagement.

MicroED with the Falcon III direct electron detector.

Microcrystal electron diffraction (MicroED) combines crystallography and electron cryo-microscopy (cryo-EM) into a method that is applicable to high-resolution structure determination. In MicroED, nanosized crystals, which are often intractable using other techniques, are probed by high-energy electrons in a transmission electron microscope. Diffraction data are recorded by a camera in movie mode: the nanocrystal is continuously rotated in the beam, thus creating a sequence of frames that constitute a movie with respect to the rotation angle. Until now, diffraction-optimized cameras have mostly been used for MicroED. Here, the use of a direct electron detector that was designed for imaging is reported. It is demonstrated that data can be collected more rapidly using the Falcon III for MicroED and with markedly lower exposure than has previously been reported. The Falcon III was operated at 40 frames per second and complete data sets reaching atomic resolution were recorded in minutes. The resulting density maps to 2.1 Šresolution of the serine protease proteinase K showed no visible signs of radiation damage. It is thus demonstrated that dedicated diffraction-optimized detectors are not required for MicroED, as shown by the fact that the very same cameras that are used for imaging applications in electron microscopy, such as single-particle cryo-EM, can also be used effectively for diffraction measurements.