Speaker
Description
Cryogenic temperatures may introduce artefacts that limit the understanding of protein dynamics, crucial to their biological functions. To address this, we developed a room-temperature (RT) X-ray crystallographic method that captures movie-like structural snapshots triggered by temperature [1]. This method revealed binding-mode changes of TL00150, a 175.15 Da fragment, in endothiapepsin. Building on this, we further developed Cryo2RT, a high-throughput RT data-collection method using cryo-cooled crystals, which leverages the cryo-crystallography workflow [2]. This method has been applied to endothiapepsin with four soaked fragments, thaumatin, and SARS-CoV-2 3CLpro, Cryo2RT uncovered distinct ligand-binding modes at RT, not seen at cryogenic temperatures. To minimize radiation damage, X-ray doses were controlled below 500 kGy, a threshold considered safe for both cryo and RT crystallography. Despite similar doses, RT datasets showed slightly lower resolution and higher B-factors (30–40 Ų vs. ~20 Ų at cryo), likely due to increased atomic motion at RT. These findings provide insights into structural interpretation at RT and highlight Cryo2RT's potential for fragment-based screening and studying temperature-dependent dynamics.
References
[1] Huang, C.-Y., Aumonier, S., Engilberge, S., Eris, D., Smith, K. M. L., Leonarski, F., Wojdyla, J. A., Beale, J. H., Buntschu, D., Pauluhn, A., Sharpe, M. E., Metz, A., Olieric, V., and Wang, M. (2022) Acta Cryst. D78: 964-974. https://doi.org/10.1107/S205979832200612X
[2]: Huang, C.-Y., Aumonier, S., Olieric, V., and Wang, M. (2024) Acta Cryst. D80: 620-628. https://doi.org/10.1107/S2059798324006697
