Visualization of alpha-helices in a 6-angstrom resolution cryoelectron microscopy structure of adenovirus allows refinement of capsid protein assignments

The structure of adenovirus was determined to a resolution of 6 A by cryoelectron microscopy (cryoEM) single-particle image reconstruction. Docking of the hexon and penton base crystal structures into the cryoEM density established that alpha-helices of 10 or more residues are resolved as rods. A difference map was calculated by subtracting a pseudoatomic capsid from the cryoEM reconstruction. The resulting density was analyzed in terms of observed alpha-helices and secondary structure predictions for the additional capsid proteins that currently lack atomic resolution structures (proteins IIIa, VI, VIII, and IX). Protein IIIa, which is predicted to be highly alpha-helical, is assigned to a cluster of helices observed below the penton base on the inner capsid surface. Protein VI is present in approximately 1.5 copies per hexon trimer and is predicted to have two long alpha-helices, one of which appears to lie inside the hexon cavity. Protein VIII is cleaved by the adenovirus protease into two fragments of 7.6 and 12.1 kDa, and the larger fragment is predicted to have one long alpha-helix, in agreement with the observed density for protein VIII on the inner capsid surface. Protein IX is predicted to have one long alpha-helix, which also has a strongly indicated propensity for coiled-coil formation. A region of density near the facet edge is now resolved as a four-helix bundle and is assigned to four copies of the C-terminal alpha-helix from protein IX.     

The cryo-electron microscopy structure of feline calicivirus bound to junctional adhesion molecule A at 9-angstrom resolution reveals receptor-induced flexibility and two distinct conformational changes in the capsid protein VP1

Caliciviridae are small icosahedral positive-sense RNA-containing viruses and include the human noroviruses, a leading cause of infectious acute gastroenteritis and feline calicivirus (FCV), which causes respiratory illness and stomatitis in cats. FCV attachment and entry is mediated by feline junctional adhesion molecule A (fJAM-A), which binds to the outer face of the capsomere, inducing a conformational change in the capsid that may be important for viral uncoating. Here we present the results of our structural investigation of the virus-receptor interaction and ensuing conformational changes. Cryo-electron microscopy and three-dimensional image reconstruction were used to solve the structure of the virus decorated with a soluble fragment of the receptor at subnanometer resolution. In initial reconstructions, the P domains of the capsid protein VP1 and fJAM-A were poorly resolved. Sorting experiments led to improved reconstructions of the FCV-fJAM-A complex both before and after the induced conformational change, as well as in three transition states. These data showed that the P domain becomes flexible following fJAM-A binding, leading to a loss of icosahedral symmetry. Furthermore, two distinct conformational changes were seen; an anticlockwise rotation of up to 15° of the P domain was observed in the AB dimers, while tilting of the P domain away from the icosahedral 2-fold axis was seen in the CC dimers. A list of putative contact residues was calculated by fitting high-resolution coordinates for fJAM-A and VP1 to the reconstructed density maps, highlighting regions in both virus and receptor important for virus attachment and entry.     

Structure of a viral DNA gatekeeper at 10 A resolution by cryo-electron microscopy

In tailed bacteriophages and herpes viruses, the viral DNA is packaged through the portal protein channel. Channel closure is essential to prevent DNA release after packaging. Here we present the connector structure from bacteriophage SPP1 using cryo-electron microscopy and single particle analysis. The multiprotein complex comprises the portal protein gp6 and the head completion proteins gp15 and gp16. Although we show that gp6 in the connector has a fold similar to that of the isolated portal protein, we observe conformational changes in the region of gp6 exposed to the DNA-packaging ATPase and to gp15. This reorganization does not cause closure of the channel. The connector channel traverses the full height of gp6 and gp15, but it is closed by gp16 at the bottom of the complex. Gp16 acts as a valve whose closure prevents DNA leakage, while its opening is required for DNA release upon interaction of the virus with its host.     

The three-dimensional structure of Infectious flacherie virus capsid determined by cryo-electron microscopy

Cryo-electron microscopy and image reconstruction were used to determine the three-dimensional structure of Infectious flacherie virus (IFV). 5047 particles were selected for the final reconstruction. The FSC curve showed that the resolution of this capsid structure was 18 Å. The structure is a psuedo T=3 (P=3) icosahedral capsid with a diameter of 302.4 Å and a single shell thickness of 15 Å. The density map showed that IFV has a smooth surface without any prominent protrude or depression. Comparison of the IFV structure with those of the insect picorna-like virus-Cricket paralysis virus (CrPV)and human picornavirus-Human rhinovirus 14 (HRV 14) revealed that the IFV structure resembles the CrPV structure. The “Rossmann canyon” is absent in both IFV and CrPV particles. The polypeptide topology of IFV VP2, IFV VP3 was predicted and the subunit location at the capsid surface was further analyzed.     

The crystal structure of muscle phosphoglucomutase refined at 2.7-angstrom resolution.

A model of rabbit muscle phosphoglucomutase was refined at 2.7-A resolution by using two heavy atom derivatives for initial phasing and standard refinement procedures, including molecular replacement averaging about a 2-fold axis and dynamic simulation: final R-factor, 0.223 (no solvent modeling); RMS deviation from standard bond lengths and angles, 0.020 A and 3.6 degrees, respectively (all 8658 nonhydrogen atoms plus 36,953 reflections (F/sigma greater than or equal to 3) between 8- and 2.7-A resolutions); average of individually refined atomic B-factors, 40 A2 (all atoms) and 30 A2 (all atoms in domains I-III). An H-bonding scheme with 538 main chain H-bonds for the two monomers in the asymmetric unit and probable ligands for six uranyl ions in one heavy atom derivative is given. The monomer contains 42 strands/helices arranged into four alpha/beta-domains. Each of the first three domains contains an alpha 3 beta 4 alpha 1 motif, where the topology of beta 4 is 2,1,3,4:[arrows: see text] which is a topology not encountered in an extensive search among known protein structures. A spatial similarity is observed between corresponding residues in the three repetitions of this motif per monomer, but the minimal mutational distance between spatially corresponding residues is not statistically significant. The loop between the antiparallel strands in each of these domains is an important feature of the active site. In domain IV, beta-sheet topology is 2,1,3,4,5,6:[arrows:see text]. Noncovalent domain/domain interactions within the monomer are greatest between adjacent domains along the polypeptide chain, which are not substantially interdigitated and can be cleanly disengaged by altering the phi/psi torsional angles of three uniquely positioned residues in the model. The observed hierarchy of noncovalent interactions between structural units within the crystal, based on a semi-empirical paradigm, suggests that monomer-monomer contacts within the asymmetric unit are formed during growth of the lattice and provides a rationale for some of the diffraction characteristics of phosphoglucomutase crystals. An unusually deep crevice involving 58 residues is formed by the head-to-tail, twisted semicircular arrangement of the four domains of the monomer that places no atom more than 12 A from the water-accessible surface. The active site of the enzyme is extensively buried at the bottom of this crevice, at the approximate confluence of the four domains. Other features of the active site, including the surrounding helical dipoles, and the metal-ion binding pocket are described, together with structure/function comparisons with a number of other enzymes.     

Images of purple membrane at 2.8 A resolution obtained by cryo-electron microscopy

Improvements in technique have produced electron micrographs of purple membrane that provide, after computer analysis, reproducibly measurable diffraction peaks extending to 2.8 A (1 A = 0.1 nm). The improvements include better specimen preparation, a more stable cryo-electron microscope with better alignment and the addition of an image-processing step, which gives weights to local areas of the image according to the local strength of the periodic component of the image. These improvements have enabled the calculation of a directly phased projection map at 2.8 A resolution.     

Rapid increase of near atomic resolution virus capsid structures determined by cryo-electron microscopy

The recent technological advances in electron microscopes, detectors, as well as image processing and reconstruction software have brought single particle cryo-electron microscopy (cryo-EM) into prominence for determining structures of bio-molecules at near atomic resolution. This has been particularly true for virus capsids, ribosomes, and other large assemblies, which have been the ideal specimens for structural studies by cryo-EM approaches. An analysis of time series metadata of virus structures on the methods of structure determination, resolution of the structures, and size of the virus particles revealed a rapid increase in the virus structures determined by cryo-EM at near atomic resolution since 2010. In addition, the data highlight the median resolution (～3.0?Å) and size (～310.0?Å in diameter) of the virus particles determined by X-ray crystallography while no such limits exist for cryo-EM structures, which have a median diameter of 508?Å. Notably, cryo-EM virus structures in the last four years have a median resolution of 3.9?Å. Taken together with minimal sample requirements, not needing diffraction quality crystals, and being able to achieve similar resolutions of the crystal structures makes cryo-EM the method of choice for current and future virus capsid structure determinations.     

Visualization of membrane protein domains by cryo-electron microscopy of dengue virus

Improved technology for reconstructing cryo-electron microscopy (cryo-EM) images has now made it possible to determine secondary structural features of membrane proteins in enveloped viruses. The structure of mature dengue virus particles was determined to a resolution of 9.5 A by cryo-EM and image reconstruction techniques, establishing the secondary structural disposition of the 180 envelope (E) and 180 membrane (M) proteins in the lipid envelope. The alpha-helical 'stem' regions of the E molecules, as well as part of the N-terminal section of the M proteins, are buried in the outer leaflet of the viral membrane. The 'anchor' regions of E and the M proteins each form antiparallel E-E and M-M transmembrane alpha-helices, leaving their C termini on the exterior of the viral membrane, consistent with the predicted topology of the unprocessed polyprotein. This is one of only a few determinations of the disposition of transmembrane proteins in situ and shows that the nucleocapsid core and envelope proteins do not have a direct interaction in the mature virus.     

Crystal structure of the dengue virus RNA-dependent RNA polymerase catalytic domain at 1.85-angstrom resolution

Dengue fever, a neglected emerging disease for which no vaccine or antiviral agents exist at present, is caused by dengue virus, a member of the Flavivirus genus, which includes several important human pathogens, such as yellow fever and West Nile viruses. The NS5 protein from dengue virus is bifunctional and contains 900 amino acids. The S-adenosyl methionine transferase activity resides within its N-terminal domain, and residues 270 to 900 form the RNA-dependent RNA polymerase (RdRp) catalytic domain. Viral replication begins with the synthesis of minus-strand RNA from the dengue virus positive-strand RNA genome, which is subsequently used as a template for synthesizing additional plus-strand RNA genomes. This essential function for the production of new viral particles is catalyzed by the NS5 RdRp. Here we present a high-throughput in vitro assay partly recapitulating this activity and the crystallographic structure of an enzymatically active fragment of the dengue virus RdRp refined at 1.85-A resolution. The NS5 nuclear localization sequences, previously thought to fold into a separate domain, form an integral part of the polymerase subdomains. The structure also reveals the presence of two zinc ion binding motifs. In the absence of a template strand, a chain-terminating nucleoside analogue binds to the priming loop site. These results should inform and accelerate the structure-based design of antiviral compounds against dengue virus.     

Fibrinogen structure in projection at 18 A resolution. Electron density by co-ordinated cryo-electron microscopy and X-ray crystallography

Electron microscope images of frozen-hydrated crystals of a proteolytically modified fibrinogen show excellent preservation of the structure. An electron density map of the key centric projection of the crystal at 18 A resolution has been obtained by combining the phases derived from cryo-electron microscopy with X-ray amplitudes. Simulation methods developed in earlier studies have been used to interpret the map. In contrast to the earlier images, the map allows us to visualize the coiled-coil region of the molecule and possible substructure in the beta domains. The map also shows that there is a marked difference in density in the two regions corresponding to the molecular ends where the gamma domains interact. A possible interpretation of this finding is provided by assuming substructure in the gamma domains and the breaking of molecular symmetry where these domains interact. Some additional constraints useful for the determination of the three-dimensional structure were obtained from cryo-electron micrographs of a perpendicular view at 25 A resolution. Implications of this working model for the molecular length and contacts in the filaments in both the crystal and fibrin are described. The data used here will be valuable as a starting point for obtaining the three-dimensional structure.     

Unique structures in a tumor herpesvirus revealed by cryo-electron tomography and microscopy

Gammaherpesviruses, including the human pathogens Epstein–Barr virus and Kaposi’s sarcoma-associated herpesvirus, are causative agents of lymphomas and other malignancies. The structural characterization of these viruses has been limited due to difficulties in obtaining adequate amount of virion particles. Here we report the first three-dimensional structural characterization of a whole gammaherpesvirus virion by an emerging integrated approach of cryo-electron tomography combined with single-particle cryo-electron microscopy, using murine gammaherpesvirus-68 (MHV-68) as a model system. We found that the MHV-68 virion consists of distinctive envelope and tegument compartments, and a highly conserved nucleocapsid. Two layers of tegument are identified: an inner tegument layer tethered to the underlying capsid and an outer, flexible tegument layer conforming to the overlying, pleomorphic envelope, consistent with the sequential viral tegumentation process inside host cells. Surprisingly, comparison of the MHV-68 virion and capsid reconstructions shows that the interactions between the capsid and inner tegument proteins are completely different from those observed in alpha and betaherpesviruses. These observations support the notion that the inner layer tegument across different subfamilies of herpesviruses has evolved significantly to confer specific characteristics related to viral–host interactions, in contrast to a highly conserved capsid for genome encapsidation and protection.     

The three-dimensional structure of reovirus obtained by cryo-electron microscopy.

The structures of reovirus serotypes T2J (Jones), T3D (Dearing) and the T3D core particle have been determined by cryo-electron microscopy and image processing. At a resolution of 30 A the two serotypes have similar features. The core is visible within the virus structure. The outer surface of the virus particles contains 120 holes at T = 13.1 local 6-fold axes. The holes penetrate into the virus as far as the surface of the internal core shell. Protrusions extending 4 nm from the virus surface surround each hole on the outside of the virus. At the 5-fold axes on the surface of the virus flat 'penton craters' form covers over the underlying core spikes. The detailed structure of the reovirus shell is very different to that of rotavirus although both have holes at T = 13.1 axes. Little evidence was seen of reovirus fibres extending from the virus surface.     

The fine structure of nuclei as revealed by electron microscopy

Summary The process of nucleolus formation has been studied by electron microscopy in spermatogonia of new-born, 15-day-old mice. One of two heteropycnotic sex chromosomes is concerned with nucleolus formation in the type A spermatogonia. The evidence for such formation has been presented with regard to behaviour and fine structure of both sex chromosome and nucleolus, Nucleolar material appears at one of two heteropycnotic sex chromosomes which are closely attached to the nuclear envelope. The two sex chromosomes approach each other, and subsequently one of them migrates into the central part of the nucleoplasm, being related to the nucleolar material which develops to show a nucleolar configuration. The sex chromosomes are homogeneously electron dense during the nucleolus formation, but assume a vesicular form at the middle stage of its development. The nucleolus is mostly of fibrillar and amorphous components at early stages of its development, but the granular components increases in amount as development proceeds. The final, mature nucleolus is composed of irregularly twisted nucleolonemata consisting of granular components, separated from fibrillar and amorphous areas. The compactly dense sex chromosome remains closely connected with the mature nucleolus.     

The 13 angstroms structure of a chaperonin GroEL-protein substrate complex by cryo-electron microscopy

The 13 angstroms resolution structures of GroEL bound to a single monomer of the protein substrate glutamine synthetase (GS(m)), as well as that of unliganded GroEL have been determined from a heterogeneous image population using cryo-electron microscopy (cryo-EM) coupled with single-particle image classification and reconstruction techniques. We combined structural data from cryo-EM maps and dynamic modeling, taking advantage of the known X-ray crystallographic structure and normal mode flexible fitting (NMFF) analysis, to describe the changes that occur in GroEL structure induced by GS(m) binding. The NMFF analysis reveals that the molecular movements induced by GS(m) binding propagate throughout the GroEL structure. The modeled molecular motions show that some domains undergo en bloc movements, while others show more complex independent internal movements. Interestingly, the substrate-bound apical domains of both the cis (GS(m)-bound ring) and trans (the opposite substrate-free ring) show counterclockwise rotations, in the same direction (though not as dramatic) as those documented for the ATP-GroEL-induced structure changes. The structural changes from the allosteric substrate protein-induced negative cooperativity between the GroEL rings involves upward concerted movements of both cis and trans equatorial domains toward the GS(m)-bound ring, while the inter-ring distances between the heptamer contact residues are maintained. Furthermore, the NMFF analysis identifies the secondary structural elements that are involved in the observed approximately 5 angstroms reduction in the diameter of the cavity opening in the unbound trans ring. Understanding the molecular basis of these substrate protein-induced structural changes across the heptamer rings provides insight into the origins of the allosteric negative cooperative effects that are transmitted over long distances (approximately 140 angstroms).     

The architecture of CopA from Archeaoglobus fulgidus studied by cryo-electron microscopy and computational docking

CopA uses ATP to pump Cu(+) across cell membranes. X-ray crystallography has defined atomic structures of several related P-type ATPases. We have determined a structure of CopA at 10?? resolution by cryo-electron microscopy of a new crystal form and used computational molecular docking to study the interactions between the N-terminal metal-binding domain (NMBD) and other elements of the molecule. We found that the shorter-chain lipids used to produce these crystals are associated with movements of the cytoplasmic domains, with a novel dimer interface and with disordering of the NMBD, thus offering evidence for the transience of its interaction with the other cytoplasmic domains. Docking identified a binding site that matched the location of the NMBD in our previous structure by cryo-electron microscopy, allowing a more detailed view of its binding configuration and further support for its role in autoinhibition.     

Densely packed beta-structure at the protein-lipid interface of porin is revealed by high-resolution cryo-electron microscopy

Porin is an integral membrane protein that forms channels across the outer membrane of Escherichia coli. Electron microscopic studies of negatively stained two-dimensional porin crystals have shown three stain accumulations per porin trimer, revealing the locations of pores spanning the membrane. In this study, reconstituted porin lattices embedded in glucose were investigated using the low-dose technique on a cryo-electron microscope equipped with a helium-cooled superconducting objective lens. The specimen temperature was maintained at 5 K to yield an improved microscopic and specimen stability. Under these conditions, we obtained for the first time electron diffraction patterns from porin lattices to a resolution of 3.2 A and images showing optical diffraction up to a resolution of 4.9 A. Applying correlation averaging techniques to the digitized micrographs, we were able to reconstruct projected images of the porin trimer to a resolution of up to 3.5 A. In the final projection maps, amplitudes from electron diffraction and phases from these images were combined. The predominant feature is a high-density narrow band (about 6 A in thickness) that delineates the outer perimeter of the trimer. Since the molecule consists of almost exclusively beta-sheet structure, as revealed by spectroscopic data, we conclude that this band is a cylindrical beta-pleated sheet crossing the membrane nearly perpendicularly to its plane. Another intriguing finding is a low-density area (about 70 A2) situated in the centre of the trimer.