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Graphene Device for High-Temperature Crystallography Visualizes How Enzymatic Reactivity is Rooted in Ensemble Probability

Padua, R.; Saha, S.; Ntangka, C.; Bachega, J. F.; Chen, L.; Cohen, A. E.; Perry, S. L.; Kern, D.

2026-08-21 biophysics
10.64898/2026.08.18.745618 bioRxiv
Show abstract

Life exists at temperatures ranging from -20 to 122 {degrees}C. However, the majority of high-resolution structural data in the Protein Data Bank (PDB) were obtained at cryogenic temperatures, where biological function is halted due to the lack of thermal fluctuations. To overcome this fundamental problem and directly link structure to biological function, we have created a graphene-based device that significantly extends the temperature range for high-resolution macromolecular X-ray diffraction data collection. Using the new device, we obtained models of the transition state ensembles for a psychrophilic, a mesophilic, and a thermophilic homolog of the enzyme orotidine 5-monophosphate decarboxylase from -173 to 65 {degrees}C. The data reveal how the active site ensemble structure at the transition state of each homolog changes with temperature, directly visualizing how the measured catalytic rates are rooted in the ensemble probabilities of reactive distances. The multi-temperature transition state ensembles further illuminate why cryogenic data, although useful, are inaccurate for describing biological processes.

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