Jonathan M. Diprose

The atomic structure of the bluetongue virus core.

The structure of the core particle of bluetongue virus has been determined by X-ray crystallography at a resolution approaching 3.5 Å. This transcriptionally active compartment, 700 Å in diameter, represents the largest molecular structure determined in such detail. The atomic structure indicates how approximately 1,000 protein components self-assemble, using both the classical mechanism of quasi-equivalent contacts, which are achieved through triangulation, and a different method, which we term geometrical quasi-equivalence.

Insights into assembly from structural analysis of bacteriophage PRD1

The structure of the membrane-containing bacteriophage PRD1 has been determined by X-ray crystallography at about 4 Å resolution. Here we describe the structure and location of proteins P3, P16, P30 and P31. Different structural proteins seem to have specialist roles in controlling virus assembly. The linearly extended P30 appears to nucleate the formation of the icosahedral facets (composed of trimers of the major capsid protein, P3) and acts as a molecular tape-measure, defining the size of the virus and cementing the facets together. Pentamers of P31 form the vertex base, interlocking with subunits of P3 and interacting with the membrane protein P16. The architectural similarities with adenovirus and one of the largest known virus particles PBCV-1 support the notion that the mechanism of assembly of PRD1 is scaleable and applies across the major viral lineage formed by these viruses.

A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization

Crystallization trials at the Division of Structural Biology in Oxford are now almost exclusively carried out using a high‐throughput workflow implemented in the Oxford Protein Production Facility. Initial crystallization screening is based on nanolitre‐scale sitting‐drop vapour‐diffusion experiments (typically 100 nl of protein plus 100 nl of reservoir solution per droplet) which use standard crystallization screening kits and 96‐well crystallization plates. For 294 K crystallization trials the barcoded crystallization plates are entered into an automated storage system with a fully integrated imaging system. These plates are imaged in accordance with a pre‐programmed schedule and the resulting digital data for each droplet are harvested into a laboratory information‐management system (LIMS), scored by crystal recognition software and displayed for user analysis via a web‐based interface. Currently, storage for trials at 277 K is not automated and for imaging the crystallization plates are fed by hand into an imaging system from which the data enter the LIMS. The workflow includes two procedures for nanolitre‐scale optimization of crystallization conditions: (i) a protocol for variation of pH, reservoir dilution and protein:reservoir ratio and (ii) an additive screen. Experience based on 592 crystallization projects is reported.

The Highly Ordered Double-Stranded RNA Genome of Bluetongue Virus Revealed by Crystallography

The concentration of double-stranded RNA within the bluetongue virus core renders the genome segments liquid crystalline. Powder diffraction rings confirm this local ordering with a 30 A separation between strands. Determination of the structure of the bluetongue virus core serotype 10 and comparison with that of serotype 1 reveals most of the genomic double-stranded RNA, packaged as well-ordered layers surrounding putative transcription complexes at the apices of the particle. The outer layer of RNA is sufficiently well ordered by interaction with the capsid that a model can be built and extended to the less-ordered inner layers, providing a structural framework for understanding the mechanism of this complex transcriptional machine. We show that the genome segments maintain local order during transcription.

Membrane structure and interactions with protein and DNA in bacteriophage PRD1

Membranes are essential for selectively controlling the passage of molecules in and out of cells and mediating the response of cells to their environment. Biological membranes and their associated proteins present considerable difficulties for structural analysis. Although enveloped viruses have been imaged at about 9 Å resolution by cryo-electron microscopy and image reconstruction, no detailed crystallographic structure of a membrane system has been described. The structure of the bacteriophage PRD1 particle, determined by X-ray crystallography at about 4 Å resolution, allows the first detailed analysis of a membrane-containing virus. The architecture of the viral capsid and its implications for virus assembly are presented in the accompanying paper. Here we show that the electron density also reveals the icosahedral lipid bilayer, beneath the protein capsid, enveloping the viral DNA. The viral membrane contains about 26,000 lipid molecules asymmetrically distributed between the membrane leaflets. The inner leaflet is composed predominantly of zwitterionic phosphatidylethanolamine molecules, facilitating a very close interaction with the viral DNA, which we estimate to be packaged to a pressure of about 45 atm, factors that are likely to be important during membrane-mediated DNA translocation into the host cell. In contrast, the outer leaflet is enriched in phosphatidylglycerol and cardiolipin, which show a marked lateral segregation within the icosahedral asymmetric unit. In addition, the lipid headgroups show a surprising degree of order.

The nsp9 replicase protein of SARS-coronavirus, structure and functional insights.

As part of a high-throughput structural analysis of SARS-coronavirus (SARS-CoV) proteins, we have solved the structure of the non-structural protein 9 (nsp9). This protein, encoded by ORF1a, has no designated function but is most likely involved with viral RNA synthesis. The protein comprises a single β-barrel with a fold previously unseen in single domain proteins. The fold superficially resembles an OB-fold with a C-terminal extension and is related to both of the two subdomains of the SARS-CoV 3C-like protease (which belongs to the serine protease superfamily). nsp9 has, presumably, evolved from a protease. The crystal structure suggests that the protein is dimeric. This is confirmed by analytical ultracentrifugation and dynamic light scattering. We show that nsp9 binds RNA and interacts with nsp8, activities that may be essential for its function(s).

A procedure for setting up high-throughput nanolitre crystallization experiments. I. Protocol design and validation

A protocol for setting up nanolitre sitting-drop vapour-diffusion experiments is reported. The procedure uses standard crystallization screening kits and 96-well crystallization plates. Reservoir solutions are transferred from 96-deep-well blocks to crystallization plates in a single step with a Robbins-Hydra pipettor. Nanolitre droplets of protein as well as reservoir solution are dispensed by a Cartesian pipetting instrument. Experiments have been carried out to characterize the performance of this instrument. Adaptations to the Cartesian, which include an anti-evaporation cover plate, are described and tested. The protocol was designed for a high-throughput facility, but can be used in any standard crystallography laboratory.

Translocation portals for the substrates and products of a viral transcription complex: the bluetongue virus core

The bluetongue virus core is a molecular machine that simultaneously and repeatedly transcribes mRNA from 10 segments of viral double‐stranded RNA, packaged in a liquid crystalline array. To determine how the logistical problems of transcription within a sealed shell are solved, core crystals were soaked with various ligands and analysed by X‐ray crystallography. Mg2+ ions produce a slight expansion of the capsid around the 5‐fold axes. Oligonucleotide soaks demonstrate that the 5‐fold pore, opened up by this expansion, is the exit site for mRNA, whilst nucleotide soaks pinpoint a separate binding site that appears to be a selective channel for the entry and exit of substrates and by‐products. Finally, nucleotides also bind to the outer core layer, providing a substrate sink.

Recent advances in the production of proteins in insect and mammalian cells for structural biology

The production of proteins in sufficient quantity and of appropriate quality is an essential pre-requisite for structural studies. Escherichia coli remains the dominant expression system in structural biology with nearly 90% of the structures in the Protein Data Bank (PDB) derived from proteins produced in this bacterial host. However, many mammalian and eukaryotic viral proteins require post-translation modification for proper folding and/or are part of large multimeric complexes. Therefore expression in higher eukaryotic cell lines from both invertebrate and vertebrate is required to produce these proteins. Although these systems are generally more time-consuming and expensive to use than bacteria, there have been improvements in technology that have streamlined the processes involved. For example, the use of multi-host vectors, i.e., containing promoters for not only E. coli but also mammalian and baculovirus expression in insect cells, enables target genes to be evaluated in both bacterial and higher eukaryotic hosts from a single vector. Culturing cells in micro-plate format allows screening of large numbers of vectors in parallel and is amenable to automation. The development of large-scale transient expression in mammalian cells offers a way of rapidly producing proteins with relatively high throughput. Strategies for selenomethionine-labelling (important for obtaining phase information in crystallography) and controlling glycosylation (important for reducing the chemical heterogeneity of glycoproteins) have also been reported for higher eukaryotic cell expression systems.

Diffraction quality crystals of PRD1, a 66-MDa dsDNA virus with an internal membrane

It has proved difficult to obtain well diffracting single crystals of macromolecular complexes rich in lipid. We report here the path that has led to crystals of the bacteriophage PRD1, a particle containing approximately 2,000 protein subunits from 18 different protein species, around 10 of which are integral membrane proteins associated with a host-derived lipid bilayer of some 12,500 lipid molecules. These crystals are capable of diffracting X-rays to Bragg spacings below 4A. It is hoped that some lessons learned from PRD1 will be applicable to other lipidic systems and that these crystals will allow, as a proof of principle, the determination of the structure of the virus in terms of a detailed atomic model.