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Author(s): Nicola G. A. Abrescia [1, 5]; Joseph J. B. Cockburn [1, 2, 5]; Jonathan M. Grimes [1]; Geoffrey C. Sutton [1]; Jonathan M. Diprose [1]; Sarah J. Butcher [3]; [...]

Author(s): Joseph J. B. Cockburn [1, 2]; Nicola G. A. Abrescia [1]; Jonathan M. Grimes [1]; Geoffrey C. Sutton [1]; Jonathan M. Diprose [1]; James M. Benevides [3]; George J. [...]

Abstract: 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. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. [Copyright &y& Elsevier]

The article describes the philosophy and implementation of the Protein Information Management System (PiMS), which is targeted at the workflows of protein-production laboratories. It refers to a Web-based Java application underpinned by a relational database management system. It does not use tightly predefined workflows to make it easier to record experimental information and other data items. A list of the protocols supplied as part of the standard PiMS installation is presented.

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. [ABSTRACT FROM AUTHOR]

Summary: 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. Over 17 million separate images are currently held in this database. We discuss the substantial scientific benefits automated tracking, imaging, and analysis of crystallizations offers to the structural biologist: analysis of the time course of the trial and easy analysis of trials with related crystallization conditions. Features of this system address requirements common to many crystallographic laboratories that are currently setting up (semi-)automated crystallization imaging systems. [Copyright &y& Elsevier]

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. These mRNAs, which are also capped and methylated within the core, are extruded into the cytoplasm through pores at the vertices of the icosahedral structure, where they are translated into viral proteins. One copy of each of the viral mRNAs is also assembled with these newly synthesised proteins to form nascent virus particles, which mature by a process that involves −ve RNA strand synthesis on the +ve stand template, thereby reforming dsRNA genome segments within progeny virus cores. The structure of the BTV core particle has been determined to atomic resolution by X-ray crystallography, revealing the organisation and interactions of its major protein components (VP3(T2)-subcore shell and VP7(T13) outer core layer) and important features of the packaged dsRNA. By soaking crystals of BTV cores with metal ions and substrates/products of the transcription reactions prior to analysis by X-ray crystallography, then constructing difference maps, it has been possible to identify binding sites and entry/exit routes for these ions, substrates and products. This has revealed how BTV solves the many logistical problems of multiple and simultaneous transcription from the 10 genome segments within the confined space of the core particle. The crystal structure of the BTV core has also revealed an outer surface festooned with dsRNA. This may represent a further protective strategy adopted by the virus to prevent host cell shut-off, by sequestering any dsRNA that may be released from damaged particles. [Copyright &y& Elsevier]

Focuses on the highly ordered double-stranded RNA genome of bluetongue virus (BTV). Description of the outer layer of RNA; Aspects of replication strategy; Powder rings in diffraction patterns; Interpretation of the consistent density within the cores BTV-1 and BTV-10.

Abstract: 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. xtalPiMS has a user-interface layer based on the standards of the Protein Information Management System (PiMS) and a database layer which links the crystallization trial images to the meta-data associated with a particular crystallization trial. The user interface has been optimized for the efficient monitoring of high-throughput environments with three different automated imagers and work to support a fourth imager is in progress, but it can even be of use without robotics. The database can either be a PiMS database or a legacy database for which a suitable mapping layer has been developed. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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 2000 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 4 A˚. 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. [Copyright &y& Elsevier]

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). [Copyright &y& Elsevier]

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.