J. Bernard Heymann

The three-dimensional structure of aquaporin-1

The entry and exit of water from cells is a fundamental process of life. Recognition of the high water permeability of red blood cells led to the proposal that specialized water pores exist in the plasma membrane. Expression in Xenopus oocytes and functional studies of an erythrocyte integral membrane protein of relative molecular mass 28,000, identified it as the mercury-sensitive water channel, aquaporin-1 (AQP1). Many related proteins, all belonging to the major intrinsic protein (MIP) family, are found throughout nature. AQP1 is a homotetramer containing four independent aqueous channels. When reconstituted into lipid bilayers, the protein forms two-dimensional lattices with a unit cell containing two tetramers in opposite orientation. Here we present the three-dimensional structure of AQP1 determined at 6Å resolution by cryo-electron microscopy. Each AQP1 monomer has six tilted, bilayer-spanning α-helices which form a right-handed bundle surrounding a central density. These results, together with functional studies, provide a model that identifies the aqueous pore in the AQP1 molecule and indicates the organization of the tetrameric complex in the membrane.

Influenza virus pleiomorphy characterized by cryoelectron tomography.

Influenza virus remains a global health threat, with millions of infections annually and the impending threat that a strain of avian influenza may develop into a human pandemic. Despite its importance as a pathogen, little is known about the virus structure, in part because of its intrinsic structural variability (pleiomorphy): the primary distinction is between spherical and elongated particles, but both vary in size. Pleiomorphy has thwarted structural analysis by image reconstruction of electron micrographs based on averaging many identical particles. In this study, we used cryoelectron tomography to visualize the 3D structures of 110 individual virions of the X-31 (H3N2) strain of influenza A. The tomograms distinguish two kinds of glycoprotein spikes [hemagglutinin (HA) and neuraminidase (NA)] in the viral envelope, resolve the matrix protein layer lining the envelope, and depict internal configurations of ribonucleoprotein (RNP) complexes. They also reveal the stems that link the glycoprotein ectodomains to the membrane and interactions among the glycoproteins, the matrix, and the RNPs that presumably control the budding of nascent virions from host cells. Five classes of virions, four spherical and one elongated, are distinguished by features of their matrix layer and RNP organization. Some virions have substantial gaps in their matrix layer (“molecular fontanels”), and others appear to lack a matrix layer entirely, suggesting the existence of an alternative budding pathway in which matrix protein is minimally involved.

Three-dimensional cellular ultrastructure resolved by X-ray microscopy

We developed an X-ray microscope using partially coherent object illumination instead of previously used quasi-incoherent illumination. The design permitted the incorporation of a cryogenic tilt stage, enabling tomography of frozen-hydrated, intact adherent cells. We obtained three-dimensional reconstructions of mouse adenocarcinoma cells at ∼36-nm (Rayleigh) and ∼70-nm (Fourier ring correlation) resolution, which allowed us to visualize the double nuclear membrane, nuclear pores, nuclear membrane channels, mitochondrial cristae and lysosomal inclusions.

Atomic force microscopy of native purple membrane

Atomic force microscopy (AFM) allows the observation of surface structures of purple membrane (PM) in buffer solution with subnanometer resolution. This offers the possibility to classify the major conformations of the native bacteriorhodopsin (BR) surfaces and to map the variability of individual polypeptide loops connecting transmembrane alpha-helices of BR. The position, the variability and the flexibility of these loops depend on the packing arrangement of BR molecules in the lipid bilayer with significant differences observed between the trigonal and orthorhombic crystal forms. Cleavage of the Schiff base bond leads to a disassembly of the trigonal PM crystal, which is restored by regenerating the bleached PM. The combination of single molecule AFM imaging and single molecule force-spectroscopy provides an unique insight into the interactions between individual BR molecules and the PM, and between secondary structure elements within BR.

Capsid Structure of Kaposi's Sarcoma-Associated Herpesvirus, a Gammaherpesvirus, Compared to Those of an Alphaherpesvirus, Herpes Simplex Virus Type 1, and a Betaherpesvirus, Cytomegalovirus

ABSTRACT The capsid of Kaposis sarcoma-associated herpesvirus (KSHV) was visualized at 24-Å resolution by cryoelectron microscopy. Despite limited sequence similarity between corresponding capsid proteins, KSHV has the same T=16 triangulation number and much the same capsid architecture as herpes simplex virus (HSV) and cytomegalovirus (CMV). Its capsomers are hexamers and pentamers of the major capsid protein, forming a shell with a flat, close-packed, inner surface (the “floor”) and chimney-like external protrusions. Overlying the floor at trigonal positions are (αβ2) heterotrimers called triplexes. The floor structure is well conserved over all three viruses, and the most variable capsid features reside on the outer surface, i.e., in the shapes of the protrusions and triplexes, in which KSHV resembles CMV and differs from HSV. Major capsid protein sequences from the three subfamilies have some similarity, which is closer between KSHV and CMV than between either virus and HSV. The triplex proteins are less highly conserved, but sequence analysis identifies relatively conserved tracts. In alphaherpesviruses, the α-subunit (VP19c in HSV) has a 100-residue N-terminal extension and an insertion near the C terminus. The small basic capsid protein sequences are highly divergent: whereas the HSV and CMV proteins bind only to hexons, difference mapping suggests that the KSHV protein, ORF65, binds around the tips of both hexons and pentons.

One number does not fit all: Mapping local variations in resolution in cryo-EM reconstructions

The resolution of density maps from single particle analysis is usually measured in terms of the highest spatial frequency to which consistent information has been obtained. This calculation represents an average over the entire reconstructed volume. In practice, however, substantial local variations in resolution may occur, either from intrinsic properties of the specimen or for technical reasons such as a non-isotropic distribution of viewing orientations. To address this issue, we propose the use of a space-frequency representation, the short-space Fourier transform, to assess the quality of a density map, voxel-by-voxel, i.e. by local resolution mapping. In this approach, the experimental volume is divided into small subvolumes and the resolution determined for each of them. It is illustrated in applications both to model data and to experimental density maps. Regions with lower-than-average resolution may be mobile components or ones with incomplete occupancy or result from multiple conformational states. To improve the interpretability of reconstructions, we propose an adaptive filtering approach that reconciles the resolution to which individual features are calculated with the results of the local resolution map.

HIV-1 Maturation Inhibitor Bevirimat Stabilizes the Immature Gag Lattice

ABSTRACT Maturation of nascent virions, a key step in retroviral replication, involves cleavage of the Gag polyprotein by the viral protease into its matrix (MA), capsid (CA), and nucleocapsid (NC) components and their subsequent reorganization. Bevirimat (BVM) defines a new class of antiviral drugs termed maturation inhibitors. BVM acts by blocking the final cleavage event in Gag processing, the separation of CA from its C-terminal spacer peptide 1 (SP1). Prior evidence suggests that BVM binds to Gag assembled in immature virions, preventing the protease from accessing the CA-SP1 cleavage site. To investigate this hypothesis, we used cryo-electron tomography to examine the structures of (noninfectious) HIV-1 viral particles isolated from BVM-treated cells. We find that these particles contain an incomplete shell of density underlying the viral envelope, with a hexagonal honeycomb structure similar to the Gag lattice of immature HIV but lacking the innermost, NC-related, layer. We conclude that the shell represents a remnant of the immature Gag lattice that has been processed, except at the CA-SP1 sites, but has remained largely intact. We also compared BVM-treated particles with virions formed by the mutant CA5, in which cleavage between CA and SP1 is also blocked. Here, we find a thinner CA-related shell with no visible evidence of honeycomb organization, indicative of an altered conformation and further suggesting that binding of BVM stabilizes the immature lattice. In both cases, the observed failure to assemble mature capsids correlates with the loss of infectivity.

Procapsid Assembly, Maturation, Nuclear Exit: Dynamic Steps in the Production of Infectious Herpesvirions

Herpesviruses, a family of animal viruses with large (125-250 kbp) linear DNA genomes, are highly diversified in terms of host range; nevertheless, their virions conform to a common architecture. The genome is confined at high density within a thick-walled icosahedral capsid with the uncommon (among viruses, generally) but unvarying triangulation number T = 16. The envelope is a membrane in which some 11 different viral glycoproteins are implanted. Between the capsid and the envelope is a capacious compartment called the tegument that accommodates ∼20-40 different viral proteins (depending on which virus) destined for delivery into a host cell. A strong body of evidence supports the hypothesis that herpesvirus capsids and those of tailed bacteriophages stem from a distant common ancestor, whereas their radically different infection apparatuses - envelope on one hand and tail on the other - reflect subsequent coevolution with divergent hosts. Here we review the molecular components of herpesvirus capsids and the mechanisms that regulate their assembly, with particular reference to the archetypal alphaherpesvirus, herpes simplex virus type 1; assess their duality with the capsids of tailed bacteriophages; and discuss the mechanism whereby, once DNA packaging has been completed, herpesvirus nucleocapsids exit from the nucleus to embark on later stages of the replication cycle.

Three-dimensional structure of the toxin-delivery particle antifeeding prophage of Serratia entomophila.

Background: Antifeeding prophage (Afp) is a toxin-delivery bacteriophage tail-like particle. Results: The syringe-like three-dimensional structure, composed of a helical sheath formed by 10 disks, a baseplate, and a central tube displays 6-fold symmetry. Conclusion: Although similar to other type VI secretion systems, Afp possesses unique features. Significance: This is the first insight into the three-dimensional structure of a tailocin. The Serratia entomophila antifeeding prophage (Afp) is a bullet-shaped toxin-delivery apparatus similar to the R-pyocins of Pseudomonas aeruginosa. Morphologically it resembles the sheathed tail of bacteriophages such as T4, including a baseplate at one end. It also shares features with the type VI secretion systems. Cryo-electron micrographs of tilted Afp specimens (up to 60 degrees) were analyzed to determine the correct cyclic symmetry to overcome the limitation imposed by exclusively side views in nominally untilted specimens. An asymmetric reconstruction shows clear 6-fold cyclic symmetry contrary to a previous conclusion of 4-fold symmetry based on analysis of only the preferred side views (Sen, A., Rybakova, D., Hurst, M. R., and Mitra, A. K. (2010) J. Bacteriol. 192, 4522–4525). Electron tomography of negatively stained Afp revealed right-handed helical striations in many of the particles, establishing the correct hand. Higher quality micrographs of untilted specimens were processed to produce a reconstruction at 2.0-nm resolution with imposed 6-fold symmetry. The helical parameters of the sheath were determined to be 8.14 nm for the subunit rise along and 40.5° for the rotation angle around the helix. The sheath is similar to that of the T4 phage tail but with a different arrangement of the subdomain of the polymerizing sheath protein(s). The central tube is similar to the diameter and axial width of the Hcp1 hexamer of P. aeruginosa type VI secretion system. The tube extends through the baseplate into a needle resembling the “puncture device” of the T4 tail. The tube contains density that may be the toxin and/or a length-determining protein.

Initial Stages of V(D)J Recombination: The Organization of RAG1/2 and RSS DNA in the Postcleavage Complex

To obtain structural information on the early stages of V(D)J recombination, we isolated a complex of the core RAG1 and RAG2 proteins with DNA containing a pair of cleaved recombination signal sequences (RSS). Stoichiometric and molecular mass analysis established that this signal-end complex (SEC) contains two protomers each of RAG1 and RAG2. Visualization of the SEC by negative-staining electron microscopy revealed an anchor-shaped particle with approximate two-fold symmetry. Consistent with a parallel arrangement of DNA and protein subunits, the N termini of RAG1 and RAG2 are positioned at opposing ends of the complex, and the DNA chains beyond the RSS nonamer emerge from the same face of the complex, near the RAG1 N termini. These first images of the V(D)J recombinase in its postcleavage state provide a framework for modeling RAG domains and their interactions with DNA.