Helical ordering of envelope-associated proteins and glycoproteins in respiratory syncytial virus.

Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three-dimensional reconstruction, we show that there is extensive helical ordering of the envelope-associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 Å resolution sub-tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M-dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M-layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly.

High-resolution structure of the presynaptic RAD51 filament on single-stranded DNA by electron cryo-microscopy.

Homologous DNA recombination (HR) by the RAD51 recombinase enables error-free DNA break repair. To execute HR, RAD51 first forms a presynaptic filament on single-stranded (ss) DNA, which catalyses pairing with homologous double-stranded (ds) DNA. Here, we report a structure for the presynaptic human RAD51 filament at 3.5-5.0Å resolution using electron cryo-microscopy. RAD51 encases ssDNA in a helical filament of 103Å pitch, comprising 6.4 protomers per turn, with a rise of 16.1Å and a twist of 56.2°. Inter-protomer distance correlates with rotation of an α-helical region in the core catalytic domain that is juxtaposed to ssDNA, suggesting how the RAD51-DNA interaction modulates protomer spacing and filament pitch. We map Fanconi anaemia-like disease-associated RAD51 mutations, clarifying potential phenotypes. We predict binding sites on the presynaptic filament for two modules present in each BRC repeat of the BRCA2 tumour suppressor, a critical HR mediator. Structural modelling suggests that changes in filament pitch mask or expose one binding site with filament-inhibitory potential, rationalizing the paradoxical ability of the BRC repeats to either stabilize or inhibit filament formation at different steps during HR. Collectively, our findings provide fresh insight into the structural mechanism of HR and its dysregulation in human disease.

Electron cryo-microscopy of TPPS4⋠2HCl tubes reveals a helical organisation explaining the origin of their chirality.

A widely studied achiral porphyrin, which is highly soluble in aqueous solutions (TPPS4), is shown to self-assemble into helical nanotubes. These were imaged by electron cryo-microscopy and a state-of-the-art image analysis allows building a map at ∼5 Šresolution, one of the highest obtained so far for molecular materials. The authors were able to trace the apparent symmetry breaking to existing nuclei in the "as received samples", while carefully purified samples show that both handnesses occur in equal amounts.

High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy.

Three-dimensional (3D) structure determination by single particle electron cryomicroscopy (cryoEM) involves the calculation of an initial 3D model, followed by extensive iterative improvement of the orientation determination of the individual particle images and the resulting 3D map. Because there is much more noise than signal at high resolution in the images, this creates the possibility of noise reinforcement in the 3D map, which can give a false impression of the resolution attained. The balance between signal and noise in the final map at its limiting resolution depends on the image processing procedure and is not easily predicted. There is a growing awareness in the cryoEM community of how to avoid such over-fitting and over-estimation of resolution. Equally, there has been a reluctance to use the two principal methods of avoidance because they give lower resolution estimates, which some people believe are too pessimistic. Here we describe a simple test that is compatible with any image processing protocol. The test allows measurement of the amount of signal and the amount of noise from overfitting that is present in the final 3D map. We have applied the method to two different sets of cryoEM images of the enzyme beta-galactosidase using several image processing packages. Our procedure involves substituting the Fourier components of the initial particle image stack beyond a chosen resolution by either the Fourier components from an adjacent area of background, or by simple randomisation of the phases of the particle structure factors. This substituted noise thus has the same spectral power distribution as the original data. Comparison of the Fourier Shell Correlation (FSC) plots from the 3D map obtained using the experimental data with that from the same data with high-resolution noise (HR-noise) substituted allows an unambiguous measurement of the amount of overfitting and an accompanying resolution assessment. A simple formula can be used to calculate an unbiased FSC from the two curves, even when a substantial amount of overfitting is present. The approach is software independent. The user is therefore completely free to use any established method or novel combination of methods, provided the HR-noise test is carried out in parallel. Applying this procedure to cryoEM images of beta-galactosidase shows how overfitting varies greatly depending on the procedure, but in the best case shows no overfitting and a resolution of ~6 Å. (382 words).

Three-dimensional structure of TspO by electron cryomicroscopy of helical crystals.

The 18 kDa TSPO protein is a polytopic mitochondrial outer membrane protein involved in a wide range of physiological functions and pathologies, including neurodegeneration and cancer. The pharmacology of TSPO has been extensively studied, but little is known about its biochemistry, oligomeric state, and structure. We have expressed, purified, and characterized a homologous protein, TspO from Rhodobacter sphaeroides, and reconstituted it as helical crystals. Using electron cryomicroscopy and single-particle helical reconstruction, we have determined a three-dimensional structure of TspO at 10 A resolution. The structure suggests that monomeric TspO comprises five transmembrane alpha helices that form a homodimer, which is consistent with the dimeric state observed in detergent solution. Furthermore, the arrangement of transmembrane domains of individual TspO subunits indicates a possibility of two substrate translocation pathways per dimer. The structure provides the first insight into the molecular architecture of TSPO/PBR protein family that will serve as a framework for future studies.

The BRC repeats of human BRCA2 differentially regulate RAD51 binding on single- versus double-stranded DNA to stimulate strand exchange.

The breast and ovarian cancer suppressor BRCA2 controls the enzyme RAD51 during homologous DNA recombination (HDR) to preserve genome stability. BRCA2 binds to RAD51 through 8 conserved BRC repeat motifs dispersed in an 1127-residue region (BRCA2([BRC1-8])). Here, we show that BRCA2([BRC1-8]) exerts opposing effects on the binding of RAD51 to single-stranded (ss) versus double-stranded (ds) DNA substrates, enhancing strand exchange. BRCA2([BRC1-8]) alters the electrophoretic mobility of RAD51 bound to an ssDNA substrate, accompanied by an increase in ssDNA-bound protein assemblies, revealed by electron microscopy. Single-molecule fluorescence spectroscopy shows that BRCA2([BRC1-8]) promotes RAD51 loading onto ssDNA. In contrast, BRCA2([BRC1-8]) has a different effect on RAD51 assembly on dsDNA; it suppresses and slows this process. When homologous ssDNA and dsDNA are both present, BRCA2([BRC1-8]) stimulates strand exchange, with delayed RAD51 loading onto dsDNA accompanying the appearance of joint molecules representing recombination products. Collectively, our findings suggest that BRCA2([BRC1-8]) targets RAD51 to ssDNA while inhibiting dsDNA binding and that these contrasting activities together bolster one another to stimulate HDR. Our work provides fresh insight into the mechanism of HDR in humans, and its regulation by the BRCA2 tumor suppressor.

Structure of hepatitis B surface antigen from subviral tubes determined by electron cryomicroscopy.

Hepatitis B virus consists of an icosahedral core containing the double-stranded DNA genome, enveloped by a membrane with embedded surface proteins. The crystal structure of the core protein has been solved but little information about the structure of the surface proteins has so far been available. There are three sizes of surface protein, small (S), medium (M) and large (L), which form disulfide-bonded homo- and heterodimers. The three proteins, expressed from different start sites in the coding sequence, share the common C-terminal S region; the M protein contains an additional preS2 sequence N-terminal to S, and the L protein a further preS1 sequence N-terminal to M. In infected individuals, the surface proteins are produced in huge excess over the amount needed for viral envelopment and are secreted as a heterogeneous mixture of isometric and tubular subviral particles. We have used electron cryomicroscopy to study tubular particles extracted from human serum. Helical Fourier-Bessel analysis was used to calculate a low-resolution map, although it showed that the tubes were quite disordered. From the symmetry derived from this analysis, we used single-particle methods to improve the resolution. We found that the tubes had a diameter of approximately 250 A, with spike-like features projecting from the membrane. In the plane of the membrane the proteins appear to be close packed. We propose a model for the packing arrangement of surface protein dimers in the tubes.

Structure and disassembly of filaments formed by the ESCRT-III subunit Vps24.

The ESCRT machinery mediates sorting of ubiquitinated transmembrane proteins to lysosomes via multivesicular bodies (MVBs) and also has roles in cytokinesis and viral budding. The ESCRT-III subunits are metastable monomers that transiently assemble on membranes. However, the nature of these assemblies is unknown. Among the core yeast ESCRT-III subunits, Snf7 and Vps24 spontaneously form ordered polymers in vitro. Single-particle EM reconstruction of helical Vps24 filaments shows both parallel and head-to-head subunit arrangements. Mutations of regions involved in intermolecular assembly in vitro result in cargo-sorting defects in vivo, suggesting that these homopolymers mimic interactions formed by ESCRT-III heteropolymers during MVB biogenesis. The C terminus of Vps24 is at the surface of the filaments and is not required for filament assembly. When this region is replaced by the MIT-interacting motif from the Vps2 subunit of ESCRT-III, the AAA-ATPase Vps4 can both bundle and disassemble the chimeric filaments in a nucleotide-dependent fashion.

SLEUTH--a fast computer program for automatically detecting particles in electron microscope images.

A method has been developed to locate biological complexes in a digitized electron micrograph by matching small windows to a set of reference images using a series of simple criteria. From the reference images, the program calculates parameters such as the radius of gyration, the density sum and variance. It compares them with corresponding values from a moving square window of densities extracted from the micrograph and records the coordinates of successfully matched candidate squares. Since the same particle is detected in a series of overlapping windows, candidates found to be within close proximity are grouped and the best-fitting one is selected from each cluster. The user is required only to select a small stack of boxed reference images and provide a few parameters, such as the particle radius and the minimum acceptable distance between particle centres. Micrograph labels and other areas that do not contain appropriate specimens are automatically ignored in order to minimize false positives. The program has been tested successfully on a variety of different biological structures, from both negatively stained and ice-embedded specimens.