A 11.5 A single particle reconstruction of GroEL using EMAN.

Single-particle analysis has become an increasingly important method for structural determination of large macromolecular assemblies. GroEL is an 800 kDa molecular chaperone, which, along with its co-chaperonin GroES, promotes protein folding both in vitro and in the bacterial cell. EMAN is a single-particle analysis software package, which was first publicly distributed in 2000. We present a three-dimensional reconstruction of native naked GroEL to approximately 11.5 A performed entirely with EMAN. We demonstrate that the single-particle reconstruction, X-ray scattering data and X-ray crystal structure all agree well at this resolution. These results validate the specific methods of image restoration, reconstruction and evaluation techniques implemented in EMAN. It also demonstrates that the single-particle reconstruction technique and X-ray crystallography will yield consistent structure factors, even at low resolution, when image restoration is performed correctly. A detailed comparison of the single-particle and X-ray structures exhibits some small variations in the equatorial domain of the molecule, likely due to the absence of crystal packing forces in the single-particle reconstruction.

The pattern of tegument-capsid interaction in the herpes simplex virus type 1 virion is not influenced by the small hexon-associated protein VP26.

Examination of the three-dimensional structure of intact herpes simplex virus type 1 (HSV-1) virions had revealed that the icosahedrally symmetrical interaction between the tegument and capsid involves the pentons but not the hexons (Z. H. Zhou, D. H. Chen, J. Jakana, F. J. Rixon, and W. Chiu, J. Virol. 73:3210-3218, 1999). To account for this, we postulated that the presence of the small capsid protein, VP26, on top of the hexons was masking potential binding sites and preventing tegument attachment. We have now tested this hypothesis by determining the structure of virions lacking VP26. Apart from the obvious absence of VP26 from the capsids, the structures of the VP26 minus and wild-type virions were essentially identical. Notably, they showed the same tegument attachment patterns, thereby demonstrating that VP26 is not responsible for the divergent tegument binding properties of pentons and hexons.

Electron cryomicroscopy and bioinformatics suggest protein fold models for rice dwarf virus.

The three-dimensional structure of rice dwarf virus was determined to 6.8 A resolution by single particle electron cryomicroscopy. By integrating the structural analysis with bioinformatics, the folds of the proteins in the double-shelled capsid were derived. In the outer shell protein, the uniquely orientated upper and lower domains are composed of similar secondary structure elements but have different relative orientations from that of bluetongue virus in the same Reoviridae family. Differences in both sequence and structure between these proteins may be important in defining virus-host interactions. The inner shell protein adopts a conformation similar to other members of Reoviridae, suggesting a common ancestor that has evolved to infect hosts ranging from plants to animals. Symmetry mismatch between the two shells results in nonequivalent, yet specific, interactions that contribute to the stability of this large macromolecular machine.

Fourier amplitude decay of electron cryomicroscopic images of single particles and effects on structure determination.

Several factors, including spatial and temporal coherence of the electron microscope, specimen movement, recording medium, and scanner optics, contribute to the decay of the measured Fourier amplitude in electron image intensities. We approximate the combination of these factors as a single Gaussian envelope function, the width of which is described by a single experimental B-factor. We present an improved method for estimating this B-factor from individual micrographs by combining the use of X-ray solution scattering and numerical fitting to the average power spectrum of particle images. A statistical estimation from over 200 micrographs of herpes simplex virus type-1 capsids was used to estimate the spread in the experimental B-factor of the data set. The B-factor is experimentally shown to be dependent on the objective lens defocus setting of the microscope. The average B-factor, the X-ray scattering intensity of the specimen, and the number of particles required to determine the structure at a lower resolution can be used to estimate the minimum fold increase in the number of particles that would be required to extend a single particle reconstruction to a specified higher resolution. We conclude that microscope and imaging improvements to reduce the experimental B-factor will be critical for obtaining an atomic resolution structure.

Seeing the herpesvirus capsid at 8.5 A.

Human herpesviruses are large and structurally complex viruses that cause a variety of diseases. The three-dimensional structure of the herpesvirus capsid has been determined at 8.5 angstrom resolution by electron cryomicroscopy. More than 30 putative alpha helices were identified in the four proteins that make up the 0.2 billion-dalton shell. Some of these helices are located at domains that undergo conformational changes during capsid assembly and DNA packaging. The unique spatial arrangement of the heterotrimer at the local threefold positions accounts for the asymmetric interactions with adjacent capsid components and the unusual co-dependent folding of its subunits.

Identification of additional coat-scaffolding interactions in a bacteriophage P22 mutant defective in maturation.

Scaffolding proteins play a critical role in the assembly of certain viruses by directing the formation and maturation of a precursor capsid. Using electron cryomicroscopy difference mapping, we have identified an altered arrangement of a mutant scaffolding within the bacteriophage P22 procapsid. This mutant scaffolding allows us to directly visualize scaffolding density within the P22 procapsid. Based on these observations we propose a model for why the mutant prevents scaffolding release and capsid maturation.

Visualization of the maturation transition in bacteriophage P22 by electron cryomicroscopy.

Large-scale conformational transitions are involved in the life-cycle of many types of virus. The dsDNA phages, herpesviruses, and adenoviruses must undergo a maturation transition in the course of DNA packaging to convert a scaffolding-containing precursor capsid to the DNA-containing mature virion. This conformational transition converts the procapsid, which is smaller, rounder, and displays a distinctive skewing of the hexameric capsomeres, to the mature virion, which is larger and more angular, with regular hexons. We have used electron cryomicroscopy and image reconstruction to obtain 15 A structures of both bacteriophage P22 procapsids and mature phage. The maturation transition from the procapsid to the phage results in several changes in both the conformations of the individual coat protein subunits and the interactions between neighboring subunits. The most extensive conformational transformation among these is the outward movement of the trimer clusters present at all strict and local 3-fold axes on the procapsid inner surface. As the trimer tips are the sites of scaffolding binding, this helps to explain the role of scaffolding protein in regulating assembly and maturation. We also observe DNA within the capsid packed in a manner consistent with the spool model. These structures allow us to suggest how the binding interactions of scaffolding and DNA with the coat shell may act to control the packaging of the DNA into the expanding procapsids.

The three-dimensional structure of the Limulus acrosomal process: a dynamic actin bundle.

Limulus sperm contains a dynamic macromolecular structure that rapidly extends a 50 microm process called the true discharge. The core of this structure is a bundle of ordered filaments composed of a complex of actin, scruin and calmodulin. We determined its structure by electron crystallographic reconstruction. The three-dimensional map reveals an actin-scruin helix that is azimuthally modulated by the influence of the interactions of a filament with its neighbors. There are a variety of density connections with neighboring filaments involving scruin. Scruin commonly contacts one neighbor, but we observe up to three interfilament connections involving both domains of the 28 scruin molecules in the unit cell. Our structure indicates that promiscuous scruin-scruin contacts are the major determinants of bundle stability in the true discharge. It also suggests that rearrangements would be permitted, which can facilitate the transition from the coiled to the true discharge form.

Roles of triplex and scaffolding proteins in herpes simplex virus type 1 capsid formation suggested by structures of recombinant particles.

Typical herpes simplex virus (HSV) capsids contain seven proteins that form a T=16 icosahedron of 1,250-A diameter. Infection of cells with recombinant baculoviruses expressing two of these proteins, VP5 (which forms the pentons and hexons in typical HSV capsids) and VP19C (a component of the triplexes that connect adjacent capsomeres), results in the formation of spherical particles of 880-A diameter. Electron cryomicroscopy and computer reconstruction revealed that these particles possess a T=7 icosahedral symmetry, having 12 pentons and 60 hexons. Among the characteristic structural features of the particle are the skewed appearance of the hexons and the presence of intercapsomeric mass densities connecting the middle domain of one hexon subunit to the lower domain of a subunit in the adjacent hexon. We interpret these connecting masses as being formed by VP19C. Comparison of the connecting masses with the triplexes, which occupy equivalent positions in the T=16 capsid, reveals the probable locations of the single VP19C and two VP23 molecules that make up the triplex. Their arrangement suggests that the two triplex proteins have different roles in controlling intercapsomeric interactions and capsid stability. The nature of these particles and of other aberrant forms made in the absence of scaffold demonstrates the conformational adaptability of the capsid proteins and illustrates how VP23 and the scaffolding protein modulate the nature of the VP5-VP19C network to ensure assembly of the functional T=16 capsid.

Visualization of tegument-capsid interactions and DNA in intact herpes simplex virus type 1 virions.

Herpes simplex virus type 1 virions were examined by electron cryomicroscopy, allowing the three-dimensional structure of the infectious particle to be visualized for the first time. The capsid shell is identical to that of B-capsids purified from the host cell nucleus, with the exception of the penton channel, which is closed. The double-stranded DNA genome is organized as regularly spaced ( approximately 26 A) concentric layers inside the capsid. This pattern suggests a spool model for DNA packaging, similar to that for some bacteriophages. The bulk of the tegument is not icosahedrally ordered. However, a small portion appears as filamentous structures around the pentons, interacting extensively with the capsid. Their locations and interactions suggest possible roles for the tegument proteins in regulating DNA transport through the penton channel and binding to cellular transport proteins during viral infection.

Visualization of protein-RNA interactions in cytoplasmic polyhedrosis virus.

Unlike the multiple-shelled organization of other Reoviridae members, cytoplasmic polyhedrosis virus (CPV) has a single-shelled capsid. The three-dimensional structures of full and empty CPV by electron cryomicroscopy show identical outer shells but differ inside. The outer surface reveals a T=1 icosahedral shell decorated with spikes at its icosahedral vertices. The internal space of the empty CPV is unoccupied except for 12 mushroom-shaped densities attributed to the transcriptional enzyme complexes. The ordered double-stranded RNA inside the full capsid forms spherical shells spaced 25 A apart. The RNA-protein interactions suggest a mechanism for RNA transcription and release.

Structure of double-shelled rice dwarf virus.

Rice dwarf virus (RDV), a member of the Reoviridae family, is a double-stranded RNA virus. Infection of rice plants with RDV reduces crop production significantly and can pose a major economic threat to Southeast Asia. A 25-A three-dimensional structure of the 700-A-diameter RDV capsid has been determined by 400-kV electron cryomicroscopy and computer reconstruction. The structure revealed two distinctive icosahedral shells: a T=13l outer icosahedral shell composed of 260 trimeric clusters of P8 (46 kDa) and an inner T=1 icosahedral shell of 60 dimers of P3 (114 kDa). Sequence and structural comparisons were made between the RDV outer shell trimer and the two crystal conformations (REF and HEX) of the VP7 trimer of bluetongue virus, an animal analog of RDV. The low-resolution structural match of the RDV outer shell trimer to the HEX conformation of VP7 trimer has led to the proposal that P8 consists of an upper domain of beta-sandwich motif and a lower domain of alpha helices. The less well fit REF conformation of VP7 to the RDV trimer may be due to the differences between VP7 and P8 in the sequence of the hinge region that connects the two domains. The additional mass density and the absence of a known signaling peptide on the surface of the RDV outer shell trimer may be responsible for the different interactions between plants and animal reoviruses.

Identification of the sites of interaction between the scaffold and outer shell in herpes simplex virus-1 capsids by difference electron imaging.

Formation of herpes simplex virus-1 capsids requires the presence of intact scaffolding proteins. The C terminus of the abundant scaffolding protein associates with the major capsid shell protein VP5 through hydrophobic interactions. After cleavage by the viral encoded protease, which removes their C-terminal 25 aa, the scaffolding proteins are released from the capsid. We have used electron cryomicroscopy and computer image processing to determine, to 13 A, the three-dimensional structures of capsids containing either cleaved or uncleaved scaffolding proteins. Detailed comparisons show that the structures of the outer icosahedral shells are almost identical in the two capsid types. Differences are apparent in the radial distribution of the density inside the capsid shell (within a radius of 460 ) which represents the scaffolding core. However, in both capsid types, the bulk of this internal density exhibits no icosahedral symmetry. Close examination revealed localized regions of icosahedrally arranged extra density at the interface between the outer shell and the scaffold of protease-minus capsids. Rod-like densities extending inwards for approximately 40 from the capsid shell are present under four of the six quasi-equivalent triplex positions. Under triplexes Tb, Tc, and Te, the major additional densities appear as pairs with the rods in each pair situated 37 apart. We propose that these rods are formed by the C-termini of the scaffolding proteins and represent the sites of interaction between the capsid shell and scaffold.

Refinement of herpesvirus B-capsid structure on parallel supercomputers.

Electron cryomicroscopy and icosahedral reconstruction are used to obtain the three-dimensional structure of the 1250-A-diameter herpesvirus B-capsid. The centers and orientations of particles in focal pairs of 400-kV, spot-scan micrographs are determined and iteratively refined by common-lines-based local and global refinement procedures. We describe the rationale behind choosing shared-memory multiprocessor computers for executing the global refinement, which is the most computationally intensive step in the reconstruction procedure. This refinement has been implemented on three different shared-memory supercomputers. The speedup and efficiency are evaluated by using test data sets with different numbers of particles and processors. Using this parallel refinement program, we refine the herpesvirus B-capsid from 355-particle images to 13-A resolution. The map shows new structural features and interactions of the protein subunits in the three distinct morphological units: penton, hexon, and triplex of this T = 16 icosahedral particle.

An atomic model of the outer layer of the bluetongue virus core derived from X-ray crystallography and electron cryomicroscopy.

BACKGROUND: Bluetongue virus (BTV), which belongs to the Reoviridae family and orbivirus genus, is a non-enveloped, icosahedral, double-stranded RNA virus. Several protein layers enclose its genome; upon cell entry the outer layer is stripped away leaving a core, the surface of which is composed of VP7. The structure of the trimeric VP7 molecule has previously been determined using X-ray crystallography. The articulated VP7 subunit consists of two domains, one which is largely alpha-helical and the other, smaller domain, is a beta barrel with jelly-roll topology. The relative orientations of these two domains vary in different crystal forms. The structure of VP7 and the organizations of 780 subunits of this molecule in the core of virus is central to the assembly and function of BTV. RESULTS: A 23 A resolution map of the core, determined using electron cryomicroscopy (cryoEM) data, reveals that the 260 trimers of VP7 are organized on a rather precise T = 13 laevo icosahedral lattice, in accordance with the theory of quasi-equivalence. The VP7 layer occupies a shell that is between 260 A and 345 A from the centre of the core. Below this radius (230-260 A) lies the T = 1 layer of 120 molecules of VP3. By fitting the X-ray structure of an individual VP7 trimer onto the cryoEM BTV core structure, we have generated an atomic model of the VP7 layer of BTV. This demonstrates that one of the molecular structures seen in crystals of the isolated VP7 corresponds to the in vivo conformation of the molecule in the core. CONCLUSIONS: The beta-barrel domains of VP7 are external to the core and interact with protein in the outer layer of the mature virion. The lower, alpha-helical domains of VP7 interact with VP3 molecules which form the inner layer of the BTV core. Adjacent VP7 trimer-trimer interactions in the T = 13 layer are mediated principally through well-defined regions in the broader lower domains, to form a structure that conforms well with that expected from the theory of quasi-equivalence with no significant conformational changes within the individual trimers. The VP3 layer determines the particle size and forms a rather smooth surface upon which the two-dimensional lattice of VP7 trimers is laid down.

A strategy for electron tomographic data collection and crystallographic reconstruction of biological bundles.

Structures of highly ordered biological bundles have unique features which call for special experimental and computational methods in electron cryomicroscopy. They can be considered as three-dimensional quasi-crystals and reconstructed using a crystallographic approach. However, they are neither "infinitely" large with respect to the borders of the bundle, nor are they a single unit cell in thickness along the viewing direction. Also, because of their shape, bundles do not generally have a preferred azimuthal orientation, which poses challenges for orientation estimation and refinement. We developed a strategy for recording and processing electron cryomicroscopic images that differs from classical two-dimensional crystalline reconstruction techniques. These developments allowed us to merge data from tomographic tilt series of ice-embedded acrosomal bundles. The goal is to determine accurately amplitudes and phases at the diffraction maxima in terms of hkl indices, and compute a three-dimensional map from the diffraction data.

Visualization of ordered genomic RNA and localization of transcriptional complexes in rotavirus.

In double-stranded-RNA (dsRNA) viruses found in animals, bacteria and yeast, the genome is transcribed within the structurally intact core of the virion with extraordinary efficiency. The structural organization of the genome and the enzymes involved in the transcription inside any of these viruses, critical for understanding this process, is not known. Here we report what we believe is the first three-dimensional characterization of the viral genome and the transcription complex in a prototypical dsRNA virus. Rotavirus is a large (diameter 1,000 A) icosahedral virus composed of three capsid protein layers and 11 dsRNA segments. It is the most important cause of gastroenteritis in children, accounting for over a million deaths annually. We show that viral dsRNA forms a dodecahedral structure in which the RNA double helices, interacting closely with the inner capsid layer, are packed around the enzyme complex located at the icosahedral 5-fold axes. The ordered RNA accounts for about 4,500 out of a total 18,525 base pairs in the genome, the largest amount of icosahedrally ordered RNA observed in any virus structure to date. We propose that the observed organization of the dsRNA is conducive for an orchestrated movement of the RNA relative to the enzyme complex during transcription.

Three-dimensional structure of scaffolding-containing phage p22 procapsids by electron cryo-microscopy.

The procapsids of bacterial viruses are the products of the polymerization of coat and scaffolding subunits, as well as the precursors in DNA packaging. Electron cryo-microscopy has been used to study the three-dimensional structures of bacteriophage P22 procapsids containing wild-type and mutant scaffolding proteins. The scaffolding mutant structure has been resolved to 19 A resolution and agrees with the 22 A resolution wild-type procapsid reconstruction. Both procapsid reconstructions contain an outer icosahedral coat protein shell and an inner scaffolding protein core. The outer core protein forms a T = 7 icosahedral lattice with distinctive channels present at the centers of the pentons and hexons. In addition, the hexons display a prominent skew. Computational isolation of the skewed hexon shows the presence of a local 2-fold axis that reduces the number of unique conformations in the asymmetric unit to four at this resolution. We have classified the four unique subunits into three distinct classes, based upon the shape of the upper domain and the presence of a channel leading to the inner coat protein surface. In addition, at the inner surface of the coat protein, finger-like regions that extend towards the scaffolding protein core are present in two of the subunits. The finger-like regions suggest the presence of an ordered interaction between the inner coat protein and the scaffolding protein. However, an icosahedral scaffolding protein shell is not formed, and the innermost scaffolding protein core does not pack with icosahedral symmetry.

Mycobacterium tuberculosis 16-kDa antigen (Hsp16.3) functions as an oligomeric structure in vitro to suppress thermal aggregation.

Tuberculosis continues to be a major disease threatening millions of lives worldwide. Several antigens of Mycobacterium tuberculosis, identified by monoclonal antibodies, have been cloned and are being exploited in the development of improved vaccines and diagnostic reagents. We have expressed and purified the 16-kDa antigen, an immunodominant antigen with serodiagnostic value, which has been previously cloned and shown to share low sequence homology with the alpha-crystallin-related small heat shock protein family. Sedimentation equilibrium analytical ultracentrifugation and dynamic light scattering demonstrate the formation of a specific oligomer, 149 +/- 8 kDa, consisting of approximately nine monomers. In 4 M urea, a smaller oligomer of 47 +/- 6 kDa (or trimer) is produced. Analysis by electron cryomicroscopy reveals a triangular shaped oligomeric structure arising from the presence of three subparticles or globules. Taken together, the data suggest an antigen complex structure of a trimer of trimers. This antigen, independent of ATP addition, effectively suppresses the thermal aggregation of citrate synthase at 40 degrees C, indicating that it can function as a molecular chaperone in vitro. A complex between the antigen and heat-denatured citrate synthase can be detected and isolated using high performance liquid chromatography. We propose to rename the 16-kDa antigen Hsp16.3 to be consistent with other members of the small heat shock protein family.

CTF determination of images of ice-embedded single particles using a graphics interface.

We implement a graphical user interface in an X-window/UNIX environment to compute and display the incoherently averaged Fourier transforms of electron images of single particles embedded in ice and the simulated contrast transfer function with or without envelope functions. This interface provides an easy and efficient operation for the determination of defocus value and the evaluation of the extent of Fourier amplitude falloff. This computational procedure is crucial for prescreening image data and performing image correction of contrast transfer function in high-resolution three-dimensional reconstruction of single particles.