Browsing by Author "Knoska, Juraj"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography
    (Springer Nature, 2018) Olmos, Jose Luis Jr.; Pandey, Suraj; Martin-Garcia, Jose M.; Calvey, George; Katz, Andrea; Knoska, Juraj; Kupitz, Christopher; Hunter, Mark S.; Liang, Mengning; Oberthuer, Dominik; Yefanov, Oleksandr; Wiedorn, Max; Heyman, Michael; Holl, Mark; Pande, Kanupriya; Barty, Anton; Miller, Mitchell D.; Stern, Stephan; Roy-Chowdhury, Shatabdi; Coe, Jesse; Nagaratnam, Nirupa; Zook, James; Verburgt, Jacob; Norwood, Tyler; Poudyal, Ishwor; Xu, David; Koglin, Jason E.; Seaberg, Matthew H.; Zhao, Yun; Bajt, Saša; Grant, Thomas; Mariani, Valerio; Nelson, Garrett; Subramanian, Ganesh; Bae, Euiyoung; Fromme, Raimund; Fung, Russell; Schwander, Peter; Frank, Matthias; White, Thomas A.; Weierstall, Uwe; Zatsepin, Nadia; Spence, John; Fromme, Petra; Chapman, Henry N.; Pollack, Lois; Tremblay, Lee; Ourmazd, Abbas; Phillips, George N.Jr.; Schmidt, Marius
    BACKGROUND: Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. RESULTS: Here, we demonstrate a general method for capturing enzyme catalysis "in action" by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. CONCLUSIONS: MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.
  • Thumbnail Image
    Item
    Heterogeneity in M. tuberculosis β-lactamase inhibition by Sulbactam
    (Springer Nature, 2023) Malla, Tek Narsingh; Zielinski, Kara; Aldama, Luis; Bajt, Sasa; Feliz, Denisse; Hayes, Brendon; Hunter, Mark; Kupitz, Christopher; Lisova, Stella; Knoska, Juraj; Martin-Garcia, Jose Manuel; Mariani, Valerio; Pandey, Suraj; Poudyal, Ishwor; Sierra, Raymond G.; Tolstikova, Alexandra; Yefanov, Oleksandr; Yoon, Chung Hong; Ourmazd, Abbas; Fromme, Petra; Schwander, Peter; Barty, Anton; Chapman, Henry N.; Stojkovic, Emina A.; Batyuk, Alexander; Boutet, Sébastien; Phillips, George N.; Pollack, Lois; Schmidt, Marius
    For decades, researchers have elucidated essential enzymatic functions on the atomic length scale by tracing atomic positions in real-time. Our work builds on possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals. Here, we report in atomic detail (between 2.2 and 2.7 Å resolution) by room-temperature, time-resolved crystallography with millisecond time-resolution (with timepoints between 3 ms and 700 ms) how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating, cooperativity, induced fit, and conformational selection all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme noncovalently before reacting to a trans-enamine. This was made possible in part by the application of singular value decomposition to the MISC data using a program that remains functional even if unit cell parameters change up to 3 Å during the reaction.