1C and D) evidenced by 31%, 13% and 44% of all structures being obtained with each additive, respectively. Another common component of successful conditions were various salts, with concentrations ranging from 0–1 M, the absence of salt (38%) and 0.2 M (31%) being most popular ( Fig. 1E). Based on these findings we developed a crystallization screen for TCR/pMHC complexes (Tables 1A and 1B). Our screen consisted of two 48 well PEG/pH screens. Each PEG/pH screen consisted of four buffer systems (C2H6AsO2Na, MES, HEPES and TRIS) at a concentration of 0.1 M in combination with PEG 4000, or PEG 8000 at 15, 20 and 25%. These buffers allowed scanning the pH range from 6.0–8.5. 15% glycerol

was added to the first subscreen (Table 1A), whereas 0.2 M ammonium sulfate was added to the second subscreen (Table 1B). In some cases, TOPS generated several crystal selleckchem buy Olaparib hits that were of lower quality, i.e. the crystals were very small, contained cracks or impurities, or did not diffract to high resolution. In these cases, we extended the conditions that yielded crystals to generate a number of other fine screens that proved useful for specific TCR/pMHC complexes. TOPS1 (Supplementary Table 1) was designed by extending the lower range

of pH with C2H6AsO2Na pH 5.0 and 5.5 of the A07 condition of the TOPS screen. In addition, PEG 3350 was compared versus PEG 4000 in this screen. TOPS2 (Supplementary Table 2) was designed by extending the lower range of PEG concentration (10, 12.5, 15, 17.5, 20 and 22.5%) of the

second subscreen of the TOPS screen. In addition, one of the buffer systems (C2H6AsO2Na pH 6.0) was replaced by a non-buffered condition and supplemented by another precipitant (0.2 M sodium sulfate) as some good hits were obtained using a commercially available screen (PACTPremier, condition E08; 0.2 M sodium sulfate and 20% PEG 4000). TOPS3 (Supplementary Table 3) was designed by reducing the range of pH (from 6.5–7.5) and increasing the number of buffer system (MES pH 6.5, BIS TRIS propane pH 7.0 and TRIS pH 7.0) as well as the range of glycerol concentrations (0, 4.4, 8.7 and 17.4%). PEG 4000 was the PEG of choice in this screen. The only difference between TOPS3 and TOPS4 (Supplementary Table 4) was that TOPS4 contained 0.2 M ammonium sulfate. These screens generated IKBKE 5 TCR/pMHC complexes as detailed in Table 2. High-throughput crystallization trials were performed using 3 commercially available screens (PACT Premier, JBScreen and JCSG-plus (Molecular Dimensions Ltd, Suffolk, U.K.)) and/or 5 different “homemade” screens (TOPS, TOPS1, TOPS2, TOPS3 and TOPS4) (Tables 1A and 1B, Supplementary Tables 1–4), the last four screens being derivatives of the TOPS screen. Crystallization conditions were successfully identified for 25 TCR/pMHC complexes, 14 of which were derivatives from a common parent complex.