Speaker
Description
Macromolecular crystallography changed substantially with the advent of the brightest X-ray sources the world has ever seen, the free electron lasers for hard X-rays (XFELs). The immense brilliance and the ultrashort pulses available at these machines triggered the development of serial crystallography, where a very large number of very small crystals are exposed to the X-ray pulses, one by one and in random orientation. We established time-resolved serial femtosecond crystallography (TR-SFX) at the Linac Coherent Light Source1. We followed the trans to cis isomerization of the central p-coumaric acid chromophore in the photoactive yellow protein in real time from 100 femtoseconds to 3 picoseconds2. With X-ray structures determined on the femtosecond time scale we structurally characterize protein structural changes on the excited state energy surface, as well as the isomerization reaction through a conical intersection, for the first time. Catalysis by bio-medically important enzymes is observed on different, slower time scales. The holy grail of time-resolved crystallography is a practical and general method to investigate enzyme catalysis. We developed such an approach called “mix-and-inject serial crystallography” (MISC) to trigger reactions by diffusion of substrate3, 4. We demonstrate that the approach is feasible using the M. tuberculosis β-lactamase (BlaC). BlaC is an enzyme that promotes broad-scale antibiotic resistance by chemically inactivating β-lactam antibiotics. We characterize the structure of the enzyme-substrate complex and that of a reaction intermediate along the catalytic pathway of the BlaC reaction with a third generation antibiotic (Ceftriaxone)5. This talk aims of addressing how MISC may be feasible at new-generation synchrotron X-ray sources.
J. Tenboer, S. Basu, N. Zatsepin, K. Pande, D. Milathianaki, M. Frank, M. Hunter, S. Boutet, G. J. Williams, J. E. Koglin, D. Oberthuer, M. Heymann, C. Kupitz, C. Conrad, J. Coe, S. Roy-Chowdhury, U. Weierstall, D. James, D. Wang, T. Grant, A. Barty, O. Yefanov, J. Scales, C. Gati, C. Seuring, V. Srajer, R. Henning, P. Schwander, R. Fromme, A. Ourmazd, K. Moffat, J. J. Van Thor, J. C. Spence, P. Fromme, H. N. Chapman and M. Schmidt, Science 346 (6214), 1242-1246 (2014).
K. Pande, C. D. M. Hutchison, G. Groenhof, A. Aquila, J. S. Robinson, J. Tenboer, S. Basu, S. Boutet, D. Deponte, M. Liang, T. White, N. Zatsepin, O. Yefanov, D. Morozov, D. Oberthuer, C. Gati, G. Subramanian, D. James, Y. Zhao, J. Koralek, J. Brayshaw, C. Kupitz, C. Conrad, S. Roy-Chowdhury, J. D. Coe, M. Metz, P. Lourdu Xavier, T. D. Grant, J. Koglin, K. G., R. Fromme, V. Srajer, R. Henning, J. H. C. Spence, A. Ourmazd, P. Schwander, U. Weierstall, M. Frank, P. Fromme, A. Barty, H. N. Chapman, K. Moffat, J. J. Van Thor and M. Schmidt, Science 352 (6286), 725-729 (2016).
C. Kupitz, J. L. Olmos, Jr., M. Holl, L. Tremblay, K. Pande, S. Pandey, D. Oberthur, M. Hunter, M. Liang, A. Aquila, J. Tenboer, G. Calvey, A. Katz, Y. Chen, M. O. Wiedorn, J. Knoska, A. Meents, V. Majriani, T. Norwood, I. Poudyal, T. Grant, M. D. Miller, W. Xu, A. Tolstikova, A. Morgan, M. Metz, J. M. Martin-Garcia, J. D. Zook, S. Roy-Chowdhury, J. Coe, N. Nagaratnam, D. Meza, R. Fromme, S. Basu, M. Frank, T. White, A. Barty, S. Bajt, O. Yefanov, H. N. Chapman, N. Zatsepin, G. Nelson, U. Weierstall, J. Spence, P. Schwander, L. Pollack, P. Fromme, A. Ourmazd, G. N. Phillips, Jr. and M. Schmidt, Struct Dyn 4 (4), 044003 (2017).
M. Schmidt, Advances on Condensed Matter Physics (2013), 1-10 (2013).
J. L. Olmos, Jr., S. Pandey, J. M. Martin-Garcia, G. Calvey, A. Katz, J. Knoska, C. Kupitz, M. S. Hunter, M. Liang, D. Oberthuer, O. Yefanov, M. Wiedorn, M. Heyman, M. Holl, K. Pande, A. Barty, M. D. Miller, S. Stern, S. Roy-Chowdhury, J. Coe, N. Nagaratnam, J. Zook, J. Verburgt, T. Norwood, I. Poudyal, D. Xu, J. Koglin, M. H. Seaberg, Y. Zhao, S. Bajt, T. Grant, V. Mariani, G. Nelson, G. Subramanian, E. Bae, R. Fromme, R. Fung, P. Schwander, M. Frank, T. A. White, U. Weierstall, N. Zatsepin, J. Spence, P. Fromme, H. N. Chapman, L. Pollack, L. Tremblay, A. Ourmazd, G. N. Phillips, Jr. and M. Schmidt, BMC Biol 16 (1), 59 (2018).
