Proteins (TCR or TCR–pHLA complexes) were centrifuged before setting up crystallization trials. The final concentrations ranged between 10 and 20 mg/mL. All proteins were mixed in 96-well Greiner plates either as 2:1, 1:1, or 1:2 ratios, with the screen solution (mother liquor) from commercial primary sparse-matrix screens using a Mosquito liquid handler (SPT Labtech, UK). Final drop volumes were 200 nL; plates were incubated at 20°C in a Rock Imager (Formulatrix, USA). c728 TCR at 19 mg/mL crystallized when mixed 1:2 with 0.2 M lithium sulfate, 0.1 M MES pH 6.0, 20 % w/v PEG 4000 (ProPlex screen, Molecular Dimensions, UK). The following relate to trials using the PACT Premier screen (Molecular Dimensions, UK): c728 TCR–pHLA (at 10.7 mg/mL) crystals grew from 0.02 M sodium/potassium phosphate, 0.1 M bis-Tris propane pH 6.5, 20% w/v PEG 3350; c756 TCR–pHLA (at 10.3 mg/mL) crystals grew from 0.2 M sodium chloride, 0.1 M MES pH 6.0, 20% w/v PEG 6000; c796 TCR–pHLA (at 10.2 mg/mL) crystals grew in 0.2 M ammonium chloride, 0.1 M HEPES pH 7.0, 20% w/v PEG 6000. All crystals were harvested by exchanging first into mother liquor supplemented with 20% glycerol before cryopreservation in liquid nitrogen.
Synchrotron X-ray data were collected with an X-ray wavelength of 0.9763 Å at beamline I03 by Diamond Light Source Industrial Liaison Unit and beamline staff.
Synchrotron X-ray data were collected with an X-ray wavelength of 0.9763 Å at beamline I03 by Diamond Light Source Industrial Liaison Unit and beamline staff.