Geoffrey C. Sutton

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.

Proteoglycan-specific molecular switch for RPTPσ clustering and neuronal extension.

One receptor binds two different types of proteoglycan at the same site but with divergent outcomes. Heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) regulate numerous cell surface signaling events, with typically opposite effects on cell function. CSPGs inhibit nerve regeneration through receptor protein tyrosine phosphatase sigma (RPTPσ). Here we report that RPTPσ acts bimodally in sensory neuron extension, mediating CSPG inhibition and HSPG growth promotion. Crystallographic analyses of a shared HSPG-CSPG binding site reveal a conformational plasticity that can accommodate diverse glycosaminoglycans with comparable affinities. Heparan sulfate and analogs induced RPTPσ ectodomain oligomerization in solution, which was inhibited by chondroitin sulfate. RPTPσ and HSPGs colocalize in puncta on sensory neurons in culture, whereas CSPGs occupy the extracellular matrix. These results lead to a model where proteoglycans can exert opposing effects on neuronal extension by competing to control the oligomerization of a common receptor.

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.

A procedure for setting up high-throughput nanolitre crystallization experiments. II. Crystallization results

An initial tranche of results from day-to-day use of a robotic system for setting up 100 nl-scale vapour-diffusion sitting-drop protein crystallizations has been surveyed. The database of over 50 unrelated samples represents a snapshot of projects currently at the stage of crystallization trials in Oxford research groups and as such encompasses a broad range of proteins. The results indicate that the nanolitre-scale methodology consistently identifies more crystallization conditions than traditional hand-pipetting-style methods; however, in a number of cases successful scale-up is then problematic. Crystals grown in the initial 100 nl-scale drops have in the majority of cases allowed useful characterization of x-ray diffraction, either in-house or at synchrotron beamlines. For a significant number of projects, full x-ray diffraction data sets have been collected to 3 A resolution or better (either in-house or at the synchrotron) from crystals grown at the 100 nl scale. To date, five structures have been determined by molecular replacement directly from such data and a further three from scale-up of conditions established at the nanolitre scale.

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.

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.

Eukaryotic expression: developments for structural proteomics

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high‐throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus‐infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein‐production pipelines are reported. Strategies for co‐expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.

Crystal structure of the GluR2 amino-terminal domain provides insights into the architecture and assembly of ionotropic glutamate receptors.

Ionotropic glutamate receptors are functionally diverse but have a common architecture, including the 400-residue amino-terminal domain (ATD). We report a 1.8-A resolution crystal structure of human GluR2-ATD. This dimeric structure provides a mechanism for how the ATDs can drive receptor assembly and subtype-restricted composition. Lattice contacts in a 4.1-A resolution crystal form reveal a tetrameric (dimer-dimer) arrangement consistent with previous cellular and cryo-electron microscopic data for full-length AMPA receptors.

A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering

At low emittance synchrotron sources it has become possible to perform structure determinations from the measurement of multiple microcrystals which were previously considered too small for diffraction experiments. Conventional mounting techniques do not fulfill the requirements of these new experiments. They significantly contribute to background scattering and it is difficult to locate the crystals, making them incompatible with automated serial crystallography. We have developed a micro-fabricated sample holder from single crystalline silicon with micropores, which carries up to thousands of crystals and significantly reduces the background scattering level. For loading, the suspended microcrystals are pipetted onto the chip and excess mother liquor is subsequently soaked off through the micropores. Crystals larger than the pore size are retained and arrange themselves according to the micropore pattern. Using our chip we were able to collect 1.5 Å high resolution diffraction data from protein microcrystals with sizes of 4 micrometers and smaller.

The Core of Bluetongue Virus Binds Double-Stranded RNA

ABSTRACT Double-stranded RNA (dsRNA) viruses conceal their genome from the host to avoid triggering unfavorable cellular responses. The crystal structure of the core of one such virus, bluetongue virus, reveals an outer surface festooned with dsRNA. This may represent a deliberate strategy to sequester dsRNA released from damaged particles to prevent host cell shutoff.