Two crystal structures of the double Cys mutant CtUGGT-N aka CtUGGT S180C:T742C have been determined by X-ray crystallography. The trigonal one, in space group P3212 was determined at 4.7 Å resolution by molecular replacement using PDB ID 5NV4 as a search model, against the data collected by Mario Hensen and Roberta Ibba on I24@Diamond on 08.08.2018. The orthorhombic one, in space group P212121, was determined to 4.6 Å resolution by molecular replacement using the P3212 CtUGGT-N model as a search model, against the data collected by Pietro Roversi and Roberta Ibba on I03@Diamond on 05.05.2018. Both structures show the disulphide bridge across the engineered double mutation S180C:T742C, and the TRXL1 and TRXL3 domains clamped shut. In the P3212 form the orientation of TRXL2 with respect to the rest of the protein is the most opened ever observed so far (a rotation of 60 degrees away from the TRXL2 in PDB ID). The orthorhombic form proves that the clamped shut structure (CtUGGT-N P212121) is compatible with the bent shut structure (CtUGGT-H, P212121, PDB ID 5NV) i.e. both clamping and bending movements can close at the same time. A quadruple CtUGGT mutant, containing both D611C:G1050C and S180C:T742C, should be feasible and its human UGGT1 equivalent could provide the best rigidification of the human structure conceivable so far.

Cloning, protein expression, purification and crystallisation took place at the Department of Biochemistry, Oxford University and were supported and funded by Prof. Nicole Zitzmann through the Oxford Glycobiology Institute Endowment. Thanks to Ed Lowe for the management of the Crystallography Facility and the coordination of the diffraction experiments. PR is funded by UK Wellcome Trust ISSF Grant 204801/Z/16/Z. JCH is funded by UK Wellcome Trust ISSF Grant 106272/Z/14/Z. RI is funded by a PhD Studentship by the Sardinian Regional Authority (Regione Sardegna), Italy.