However, further analysis revealed that, although the vast majority of TCR/pMHC complexes crystallized within the remit of these conditions, a number of structures crystallized in conditions outside of this range (Fig. 4). Thus, although it could be tempting to limit the number of conditions in a protein crystal screen to improve efficiency and reduce protein consumption, selleck broader screens are required to ensure that crystallization conditions are not missed for important proteins. The ability of T cells to respond to antigen depends on the productive

interaction between the TCR and pMHC. The crystal structures of a number of TCR/pMHC complexes have been solved and show that the TCR has a relatively conserved mode of binding to pMHC in which the selleck chemicals llc TCR lines up approximately diagonally to the MHC peptide binding groove, with the TCR α

chain contacting the MHC α2 domain and the TCR β chain contacting the MHC α1 domain. The antigen specific portion of the TCR/pMHC interaction occurs between the pMHC surface and the TCR complementarity determining region loops (CDR-loops) (Rudolph et al., 2006). These CDR-loops serve different roles during TCR binding to pMHC: the variable (V)-gene encoded CDR2-loops contact mainly the conserved helical region of the MHC surface, the V-gene encoded CDR1-loops can contact both the MHC and the peptide and the more variable somatically rearranged CDR3-loops contact mainly the antigenic peptide. Although the general features of TCR/pMHC binding have been defined, there remains a number of conflicting models that describe the structural basis of T cell MHC-restriction, cross-reactivity, autoimmunity and alloreactivity. Furthermore, each previous TCR/pMHC complex has been governed by a unique set of contacts that enable T cell antigen recognition. Thus, there is still a pressing need to increase the number of TCR/pMHC complex structures in the literature in order to: (1) determine an accepted set of rules

Cediranib (AZD2171) that describe the generalities of T cell specificity, and (2) understand the unique features of individual TCR/pMHC interactions that allow T cells to target different disease epitopes. The study of TCR/pMHC complexes has been limited by the challenges in expression, purification and successful crystallization of these soluble proteins. Here, we report a new systematic and directed approach for the design of a TCR/pMHC Optimized Protein crystallization Screen (TOPS) that has proved to be useful for the crystallization of this family of immuno-proteins. With this novel crystallization screen, we have successfully generated the majority of our current portfolio of structures that includes 21 TCR/pMHC complexes (13 derived from a common parent complex), 3 TCRs and 8 pMHCs. We found that TCR/pMHC complex crystals most commonly formed at a neutral pH, with 15%–20% of PEG 4000 and 0.2 M ammonium sulfate.