Application of In-Fusion™ cloning for the parallel construction of E. coli expression vectors.

In-Fusion™ cloning is a flexible DNA ligase-independent cloning technology that has wide-ranging uses in molecular biology. In this chapter we describe the protocols used in the OPPF-UK to design and construct expression vectors using In-Fusion™. Our method for small scale expression screening in Escherichia coli of constructs generated by In-Fusion™ is also outlined.

xtalPiMS: a PiMS-based web application for the management and monitoring of crystallization trials.

A major advance in protein structure determination has been the advent of nanolitre-scale crystallization and (in a high-throughput environment) the development of robotic systems for storing and imaging crystallization trials. Most of these trials are carried out in 96-well (or higher density) plates and managing them is a significant information management challenge. We describe xtalPiMS, a web-based application for the management and monitoring of crystallization trials.

Recording information on protein complexes in an information management system.

The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes.

The Protein Information Management System (PiMS): a generic tool for any structural biology research laboratory.

The techniques used in protein production and structural biology have been developing rapidly, but techniques for recording the laboratory information produced have not kept pace. One approach is the development of laboratory information-management systems (LIMS), which typically use a relational database schema to model and store results from a laboratory workflow. The underlying philosophy and implementation of the Protein Information Management System (PiMS), a LIMS development specifically targeted at the flexible and unpredictable workflows of protein-production research laboratories of all scales, is described.

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.

Crystal lattice as biological phenotype for insect viruses.

Many insect viruses survive for long periods by occlusion within robust crystalline polyhedra composed primarily of a single polyhedrin protein. We show that two different virus families form polyhedra which, despite lack of sequence similarity in the virally encoded polyhedrin protein, have identical cell constants and a body-centered cubic lattice. It is almost inconceivable that this could have arisen by chance, suggesting that the crystal lattice has been preserved because it is particularly well-suited to its function of packaging and protecting 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.

Benefits of automated crystallization plate tracking, imaging, and analysis.

We describe the design of a database and software for managing and organizing protein crystallization data. We also outline the considerations behind the design of a fast web interface linking protein production data, crystallization images, and automated image analysis. The database and associated interfaces underpin the Oxford Protein Production Facility (OPPF) crystallization laboratory, collecting, in a routine and automatic manner, up to 100,000 images per day.

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 A resolution by cryo-electron microscopy and image reconstruction, no detailed crystallographic structure of a membrane system has been described.

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 A 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 bluetongue virus core: a nano-scale transcription machine.

The replication phase of the bluetongue virus (BTV) infection cycle is initiated when the virus core is delivered into the cytoplasm of a susceptible host cell. The 10 segments of the viral genome remain packaged within the core throughout the replication cycle, helping to prevent the activation of host defence mechanisms that would be caused by direct contact between the dsRNA and the host cell cytoplasm. However, the BTV core is a biochemically active 'nano-scale' machine, which can simultaneously and repeatedly transcribe mRNA from each of the 10 genome segments, which are packaged as a liquid crystal array within a central cavity.

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 beta-barrel with a fold previously unseen in single domain proteins.

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.