The Scottish Structural Proteomics Facility: targets, methods and outputs

Abstract

The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.Electronic supplementary materialThe online version of this article (doi:10.1007/s10969-010-9090-y) contains supplementary material, which is available to authorized users.

Functional and Structural Analysis of the Siderophore Synthetase AsbB through Reconstitution of the Petrobactin Biosynthetic Pathway from Bacillus anthracis

“…Additionally, in the pocket, partial electron density corresponding to an ADP molecule (although not fully ordered) is found in chain B (supplemental Fig. 3) that is consistent in conformation with the ATP molecule of the AlcC structure (PDB ID 2X0Q) (48).…”

Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase

“…35 The expression vector pJexpress411 harbouring the coding sequence for an N-terminal His-tagged MtPPase (a kind gift from Dr Luiz Pedro S. de Carvalho, The Francis Crick Institute) was transformed into E. coli BL21(DE3) and expressed as previously described. 3 Purification of PaHisGS and PaHisZ.…”

“…All purification steps were carried out at 4 °C, and all chromatographic steps employed an AKTA Start FPLC system. TEVP was prepared as previously published 35 X-ray data collection and processing. Crystals were cross-linked by vapour diffusion with 25% (v/v) aqueous solution of glutaraldehyde for 1 h prior to being cryoprotected in 15% 2,4-methylpentanediol freshly prepared in reservoir solution, then flash-cooled in liquid nitrogen.…”

NMR Spectroscopic and Theoretical Analysis of a Spontaneously Formed Lys–Asp Isopeptide Bond

“…The structure of CnaB2 and the presence of the isopeptide were identified by X-ray crystallography. [13] While many proteins have been found or predicted to contain Asn-Lys isopeptides, [3] several potential Asp-Lys CnaB domains can now be added to the rapidly growing list of isopeptide proteins ( Figure S1 in the Supporting Information). [14] CnaB2 is unusual as it occurs isolated in sequence and represents an independently folded domain ideally suited to address questions relating to the structural role of isopeptide bonds and the mechanism of their formation.…”

“…[3,5] The isopeptide cross-links the parallel N-terminal and C-terminal β-strands and is surrounded by hydrophobic groups (Figure 1b,c). CnaB2 and mutants K470A, E516Q and D556A, expected to lack the isopeptide bond, were expressed in unlabeled, 15 Nor 13 C, 15 N-(CnaB2, E516Q) labeled forms. As all previously found bacterial isopeptide bonds are formed by asparagine, a mutant D556N was also generated.…”