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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.

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.

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.

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.

Cryo-electron microscopy has been used to visualize elongation factor G (EF-G) on the 70S ribosome in GDP and GTP states. GTP hydrolysis is required for binding of all the domains of EF-G to the pretranslocational complex and for the completion of translocation. In addition, large conformational changes have been identified in the ribosome. The head of the 30S subunit shifts toward the L1 protein side, and the L7/L12 stalk becomes bifurcated upon EF-G binding. Upon GTP hydrolysis, the bifurcation is reversed and an arc-like connection is formed between the base of the stalk and EF-G.

The ribosome is formed by assembly of proteins and nucleic acids, and synthesizes proteins according to genetic instructions in all organisms. Many of the biochemical steps of this fundamental process are known, but a detailed understanding requires a well-defined structural model of the ribosome. Electron microscopy combined with image reconstruction of two-dimensional crystals or single ribosomes has been the most promising technique, but the resolution of the resulting models has been insufficient. Here we report a 25-A reconstruction of the ribosome from Escherichia coli, obtained by combining 4,300 projections of ice-embedded single particles. Our new reconstruction reveals a channel in the small ribosomal subunit and a bifurcating tunnel in the large subunit which may constitute pathways for the incoming message and the nascent polypeptide chain, respectively. Based on these new findings, a three-dimensional model of the basic framework of protein synthesis is presented.

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.

Fragmentation of molecules and their representation based on structural pattern is suggested in order to predict the retention characteristics in HPLC. The logarithm of the capacity factor is derived as a sum of logarithms of the structural fragments. The retention behaviour of a polycyclic aromatic hydrocarbon is related to the numerical representation of the individual compounds. In all cases a high correlation coefficient, ranging from 0.97 to 0.99 was obtained. The contribution of the molecule fragments to the capacity factor value is discussed.