Materials and methods

MATERIAL S AND METHODS

Protein production and purification: Soluble TCR (MS2-3C8) and MBP-bound MHC (HLA-DR4) were prepared in vitro folding from inclusion bodies produced in E.Coli. For affinity maturation of CD4, overlap PCR with degenerate primers was used to mutate library of CD4 D1-D2 mutant clones. They were then sorted by flow cytometry using MHC to isolate out the CD4 mutants with increased affinity. Then the mutant was inserted into expression vector with a C-terminal Flag tag and finally expressed in insect cells.

SPR Analysis: The interaction of the recombinant (affinity-matured) CD4 with MHC (HLA-DR4) was assessed by SPR. Solutions of different concentrations  of CD4 were injected sequentially over flow cells immobilized with MHC and the dissociation constant was then calculated.

Crystallisation and Data Collection: Crystallisation occured spontaneously upon equimolar mixing of purified TCR, MBP-HLA-DR4, and CD4 under specific conditions (supplied in the paper). Crystals up to 0.2x0.1x0.1 mm were transferred to a cryoprotectant and flash-cooled in liquid nitrogen. X-Ray data was collected, up to a resolution of 4.0Å, and manipulated with the program CrystalClear (Rigaku).
Structure Determination and Refinement:
The structure was solved by molecular replacement with the program Phaser. The molecule templates used were a previously published structure of MS2-3C8-MBP-DR4, as well as a structure of CD4 D1-D3. The D4 domain was positioned into the model afterwards. The model was refined, manually built, and further refined.
The R-factors of the finalized model are 0.238 (Rfree) and 0.305 (Rwork). Given the low resolution, these compare favourable to other published models, indicating that the model is likely to be accurate.