October 2006 issue
Structural Proteomics IN Europe
The technology output from the Structural Proteomics In Europe (SPINE) consortium for cloning and expression in E. coli is reviewed.
Strategies and experience resulting from the use of eukaryotic expression systems in SPINE are described.
Characteristics such as the purity of target proteins are key factors that can influence the success of structure determination. Here, the application and efficacy of various quality-assessment methodologies are discussed.
The introduction of automation and minimization into the crystallization pipeline has improved the yield of high-quality crystals for structural determination using more reproducible experimental protocols.
Automated data collection for macromolecular crystallography at beamlines with protocols for automatic instrumentation improves the efficient use of available beamtime. These developments were a result of a collaboration between the main partners of SPINE and members of other similar European and national initiatives.
The results of a workshop on the automation of protein crystal structure solution organized by the Structural Proteomics In Europe (SPINE) consortium are reported.
This article reviews the infrastructure of software that has been developed under the SPINE initiative for high-throughput protein-structure analysis covering target selection and prototype LIMS for protein production through to structure annotation, with contributions to the management of a large-scale crystallization set-up and structure determination.
Progress by the SPINE consortium on the determination of structures of proteins of bacterial and viral pathogens is outlined, with particular reference to the impact of high-throughput technologies.
Progress in the determination of structures of human proteins by the Structural Proteomics In Europe (SPINE) consortium is outlined with highlights indicating the impact of high-throughput technologies.
This article presents a comparative analysis of the standard methods for small-scale screening of recombinant protein expression/solubility implemented in laboratories of the Structural Proteomics In Europe (SPINE) consortium.
A multifermentation system, GRETA, has been developed which specifically accommodates features that respond to the needs of future structural genomics programmes. The GRETA multifermenter provides a robust platform for streamlined parallel fermentation, requiring minimal manual intervention, while at the same time providing a versatile fermentation platform for process optimization.
The design of a new system for multi-expression in E. coli and a database for co-expression data are described. Lessons are drawn from case studies using these and other strategies for co-expression and analysis of protein complexes.
A simple, fast and affordable method for transient protein expression in mammalian cells is presented. It combines several features of interest for X-ray crystallography such as high protein yield, straightforward purification, selenomethinonine incorporation and control of N-linked glycosylation.
A sample holder standard for use with robotic sample changers is defined. The standard includes a system for sample identification, tracking and management of data flow in a macromolecular structure-determination pipeline. A robotic sample changer designed for use with the sample standard is described.
The current state of methods for the in silico prediction of natively disordered regions of proteins is discussed in the context of the potential for improvement and application of these predictions in structural proteomics.
The current state of high-throughput technologies for protein production and structure determination is assessed in relation to progress on the SPINE structural proteomics project on proteins from B. anthracis.