Changes in an enzyme ensemble during catalysis observed by high-resolution XFEL crystallography

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Acknowledgement

Funding

R.G.S. is supported by the Office of Basic Energy Sciences through the Atomic, Molecular, and Optical Sciences Program within the Chemical Sciences, Geosciences, and Biosciences Division and of the US Department of Energy (DOE) through the SLAC Laboratory Directed Research and Development Program. A.S.B., I.D.Y., and N.K.S. are supported by NIH grant R01GM117126 to N.K.S. for data processing methods. M.C.T. and A.M.W. were funded by a discretionary award from the BioXFEL Science and Technology Center (NSF STC-1231306). A.H.F. acknowledges support from R01GM120349 (principal investigator: A. S. Borovik). This work was supported in part by NIH grant P41GM139687. R.G.H. is supported by NIH grant R35GM142595. M.E.W. and D.C.W. are supported by the Exascale Computing Project (17-SC-20-SC), a collaborative effort of the DOE, Office of Science, and the National Nuclear Security Administration, and the UC Office of the President Laboratory Fees Research Program (LFR-17-476732). These studies were facilitated by the IR/D (Individual Research and Development) program associated with D.B.B.’s appointment at the National Science Foundation. D.B.B., N.K., and V.K.T. acknowledge NIH (SIG-1-510-RR-06307) and NSF (CHE-0091975, MRI-0079750) support for NMR instrumentation support and the NIH (RR016544) for research facilities. A helium recovery system supporting the NMR instruments was purchased with support from the NCIBC Systems Biology Core (NIH NIGMS P20 GM113126). N.S., C.D., and M.A.W. are supported by NIH grant R01GM139978 to M.A.W.

Contributions

M.E.W., R.G.H., M.C.T., A.H.F., and M.A.W. planned the experiments. V.K.T., N.K., and D.B.B. generated key reagents. N.S., M.D., C.D., R.G.S., S.L., D.M.-R., A.E.C., S.B., M.S.H., C.K., F.P., F.R.M., A.S.B., N.K.S., I.D.Y., A.M.W., M.C.T., A.H.F., M.E.W., and M.A.W. performed the experiments. N.S., M.D., D.C.W., D.W.M.-M., A.S.B., N.K.S., I.D.Y., A.M.W., A.H.F., M.E.W., and M.A.W. analyzed and interpreted the data. N.S., D.C.W., M.E.W., R.G.S., and M.A.W. wrote the manuscript. N.S., M.D., D.C.W., R.G.S., S.B., M.S.H., C.K., D.M.-R., N.K.S., I.D.Y., D.B.B., A.M.W., M.C.T., A.H.F., M.E.W., M.A.W., and R.G.H. edited the manuscript.

Contributions

All refined structural models and structure factor data are deposited with the PDB with the following accession codes: 8VPW (G150T ICH XFEL free enzyme), 8VQ1 (G150T ICH XFEL thioimidate intermediate, 30-s mixing), 8TSX (G150T ICH 100 K), 8TSU (G150T ICH 274 K synchrotron dataset 1), 8TSY (G150T ICH 274 K synchrotron dataset 2), 8TSZ (G150T ICH 274 K synchrotron dataset 3), 8TT0 (wild-type ICH, pH 4.2), 8TT1 (wild-type ICH, pH 5.0), 8TT2 (wild-type ICH, pH 5.4), 8TT4 (wild-type ICH, pH 6.0), and 8TT5 (wild-type ICH, pH 8.3). All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

Conflict of Interest

M.E.W. is a consultant for Eli Lilly and Company. The other authors declare that they have no competing interests.