K.). Protein quality, either under non-reducing or reducing conditions, was analyzed by Coomassie-stained SDS-PAGE. Crystals were grown at 18 °C by vapor diffusion via the sitting drop technique. All crystallization screening and optimization experiments were completed with an Art-Robbins Phoenix dispensing robot (Alpha Biotech Ltd, U.K.). 200 nL of 10–20 mg/ml TCR, pMHC, or TCR and pMHC complex mixed at a 1:1 molar ratio,

was added to 200 nL of reservoir solution. Intelli-plates were then sealed and incubated in a crystallization incubator (18 °C) (Molecular Dimensions) and analyzed for crystal formation. Crystals selected for further analysis were cryoprotected with 25% ethylene glycol and then flash cooled in liquid nitrogen in Litho loops (Molecular

Dimensions). Diffraction data was collected at a number of different beamlines at the Diamond Light Source, Oxford, using a Pilatus 2M, or a QADSC, selleck chemical detector. click here Using a rotation method, 400 frames were recorded each covering 0.5° of rotation. Reflection intensities were estimated with the XIA2 package (Winter, 2010) and the data were scaled, reduced and analyzed with SCALA and the CCP4 package (Collaborative Computational Project, N, 1994). The TCR, pMHC, or TCR/pMHC complex structures were solved with molecular replacement using PHASER (McCoy et al., 2005), or AMORE (Trapani and Navaza, 2008). The model sequences were adjusted with COOT (Emsley and Cowtan, 2004) and the models refined with

REFMAC5. TCR/pMHC complex structures have previously been solved by a number of different groups using individually determined crystallization conditions. In order to combine these data to generate a comprehensive TCR/pMHC Optimized Protein crystallization Screen (TOPS), we investigated the crystallization conditions of 16 previously published TCR/pMHC complexes ( Garboczi et al., 1996, Garcia et al., 1996, Ding et al., 1998, Ding et al., 1999, Hennecke et al., 2000, Reiser et al., 2000, Reiser et al., 2003, Hennecke and Wiley, 2002, Kjer-Nielsen et al., 2003, Stewart-Jones et al., 2003, Chen et al., 2005, Li et al., 2005, Maynard et al., 2005, Tynan et al., 2005, Tynan et al., 2007, Sami et al., 2007 and Cole ADP ribosylation factor et al., 2009) ( Fig. 1). Although there was a substantial variation in the crystallization conditions identified for different TCR/pMHC complexes, we noticed certain trends. The pH lay between 5.6–8.5 in all cases, with the TCR/pMHC complexes tending to crystallize at the higher end of this pH range ( Fig. 1A); with 25%, 19% and 19% of complexes crystallizing in the pH range of 7.0–7.5, 7.5–8.0 and 8.0–8.5, respectively. Six conditions (38%) contained glycerol as cryoprotectant ( Fig. 1B). All conditions contained PEG (polyethylene glycol), although the weight (550–8000 g/mol) and percentage (10–25%) were very variable. The best PEG concentration, representing 31% of the previous structures reported, was between 15%–17.5%.