Summary of key activities carried out by the Programme

A summary of the structural analysis performed with full-length and domain centrosomal proteins follows:

SNCG (full-length)

SNCG was expressed in E. coli and purified to homogeneity. Despite numerous crystallization experiments no crystals of full-length SNCG have been thus far obtained. As evidenced by NMR analysis (in collaboration with consortium partner M. Bruix) the protein seems to be largely unfolded. In order to elucidate its structure by NMR, micelles of SNCG will be prepared.

TXNDC9 (full-length)

The protein was expressed and purified to homogeneity. Crystallization screening resulted in micro-needles which diffracted to 9-10 Å using synchrotron radiation. Optimization of these preliminary crystals is underway. In parallel, we cloned a shorter version of TXNDC9 (residues 6-181) lacking predicted disordered regions at both the N and C termini. Furthermore, we showed by yeast two-hybrid analysis that TXNDC9 interacts with FEZ1 and we are now attempting to confirm this interaction in vitro.

RABGAP1 (domain 536-849)

RABGAP1 is a RAB GTPase activator whose target is likely RAB6A. It was reported to interact with ϒ-tubulin as well. We purified and crystallized a domain of RABGAP1 spanning residues 536-849 in three crystal forms (space groups P1, P21, and P212121). Data were collected to 1.9 Å at the ESRF synchrotron and the structure was solved (Fig. 1).

Fig. 1. Crystal structure of RABGAP1 TBC domain

TUBULIN FOLDING COFACTORS (In collaboration with partner JC Zabala)

TBCB

We performed crystallization screenings for human and murine TBCB, obtaining needle-like crystals.

TBCE

Human TBCE Δ52-55 were subjected to a crystallization screening. Full-length TBCE appears to undergo some proteolysis during the experiments and new preparation conditions are being investigated in order to prevent it.

Cloning and expression facility

Cloning of 59 full-length centrosomal target proteins in suitable GST-tagged expression constructs was completed. Of these, 23 clones were distributed among other partners of the consortium. Cloning of further 19 full-length synthetic genes has been started. A high-throughput expression and purification screening of the 59 full-length GST-fused targets was completed resulting in 18 small-scale purified proteins. In order to facilitate the structural analysis of the targets, either by crystallography or NMR, we designed domain constructs of the target proteins based on a combined bioinformatics analysis (e.g. literature mining, structural homology to available PDB structures, fold prediction, etc). In total 37 domain constructs, out of 22 target proteins, were designed. All of them were cloned in the pOPINJ expression vector. We also performed both automated and manual expression and solubility screening in E. coli with all cloned domains. As a result, 4 domains of unknown structure were found to be soluble and amenable for crystallization or NMR studies. <
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