Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography

Pandey, S.; Calvey, G.; Katz, A.M.; Malla, T.N.; Koua, F.H.M.; Martin-Garcia, J.M.; Poudyal, I.; Yang, J.-H.; Vakili, M.; Yefanov, O.; Zielinski, K.A.; Bajt, S.; Awel, S.; Doerner, K.; Frank, M.; Gelisio, L.; Jernigan, R.; Kirkwood, H.; Kloos, M.; Koliyadu, J.; Mariani, V.; Miller, M.D.; Mills, G.; Nelson, G.; Olmos Jr., J.L.; Sadri, A.; Sato, T.; Tolstikova, A.; Xu, W.; Ourmazd, A.; Spence, J.H.C.; Schwander, P.; Barty, A.; Chapman, H.N.; Fromme, P.; Mancuso, A.P.; Phillips Jr, G.N.; Bean, R.; Pollack, L.; Schmidt, M.

doi:10.1107/S2052252521008125

Figure 6
BlaC–CEF complex formation as a function of time. (a) Occupancies of CEF in the active site of BlaC at 5, 10, 30 and 50 ms in subunits B (spheres) and D (squares) as well as those of P_i (green triangles and blue diamonds) are plotted as a function of delay (the 30 ms data are from Olmos et al., 2018

). The data are fitted with saturation curves [equations (1

) and (2

), black and blue dashed lines]. The two curves intersect at around 6 ms. Inset: the corresponding change in the unit-cell axis c. (b) Concentrations (in %) as calculated from diffusion and binding [equations (1)–(5)]; green dashed line and green triangles, increase of the calculated BlaC–CEF complex concentration averaged over all voxels in the crystal; red dashed line and squares, decrease of the free enzyme (BlaC); blue dashed line and diamonds, increase of the BlaC–CEF complex in the center of the platelet-shaped crystals. For comparison, the observed (refined) occupancies of the BlaC–CEF complex (normalized to 100% at 50 ms) are also shown (black spheres).

IUCrJ

Volume 8| Part 6| November 2021| Pages 878-895

ISSN: 2052-2525

https://doi.org/10.1107/S2052252521008125

BIOLOGY | MEDICINE

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