Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography

Güven, Ömür; Gul, Mehmet; Ayan, Esra; Johnson, J Austin; Cakilkaya, Baris; Usta, Gozde; Ertem, Fatma Betül; Tokay, Nurettin; Yüksel, Büşra; Göcenler, Oktay; Büyükdağ, Cengizhan; Botha, Sabine; Ketawala, Gihan; Su, Zhen; Hayes, Brandon; Poitevin, Frédéric; Batyuk, A.; Yoon, Chun Hong; Kupitz, Christopher; Durdağı, Serdar; Sierra, Raymond G.; DeMi̇rci̇, Hasan

doi:10.1101/2021.11.28.468932

Cited by 2 publications

(2 citation statements)

References 54 publications

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“…We conclude that the large temperature difference between cryo and RT (>178 K) underlies the observed changes in binding. This is in accord with recent studies in which only 0 of 9 (Guven et al, 2021) and 5 of 30 (Gildea et al, 2021) cryo-hit ligands were seen to bind at RT, and in which lower occupancies were seen at RT than at cryo for <10 ligands (Bradford et al, 2021).…”

Section: Discussionsupporting

confidence: 92%

Room-temperature crystallography reveals altered binding of small-molecule fragments to PTP1B

Mehlman

Biel

Azeem

et al. 2022

Preprint

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Much of our current understanding of how small-molecule ligands interact with proteins stems from X-ray crystal structures determined at cryogenic (cryo) temperature. For proteins alone, room-temperature (RT) crystallography can reveal previously hidden, biologically relevant alternate conformations. However, less is understood about how RT crystallography may impact the conformational landscapes of protein-ligand complexes. Previously we showed that small-molecule fragments cluster in putative allosteric sites using a cryo crystallographic screen of the therapeutic target PTP1B (Keedy*, Hill*, 2018). Here we have performed two RT crystallographic screens of PTP1B using many of the same fragments, representing the largest RT crystallographic screens of a diverse library of ligands to date, and enabling a direct interrogation of the effect of data collection temperature on protein-ligand interactions. We show that at RT, fewer ligands bind, and often more weakly -- but with a variety of temperature-dependent differences, including unique binding poses, changes in solvation, new binding sites, and distinct protein allosteric conformational responses. Overall, this work suggests that the vast body of existing cryogenic-temperature protein-ligand structures may provide an incomplete picture, and highlights the potential of RT crystallography to help complete this picture by revealing distinct conformational modes of protein-ligand systems. Our results may inspire future use of RT crystallography to interrogate the roles of protein-ligand conformational ensembles in biological function.

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Section: Discussionsupporting

confidence: 92%

Room-temperature crystallography reveals altered binding of small-molecule fragments to PTP1B

Mehlman

Biel

Azeem

et al. 2022

Preprint

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“…Fragment screening experiments such as these are typically carried out using conventional cryogenic conditions to minimise the effects of radiation damage, with each structure obtained from a single crystal. Room-temperature data, however, can usefully identify or rule out structural artefacts induced by pushing the temperature far from the biologically relevant level (Durdagi et al ., 2021; Guven et al ., 2021).…”

Section: Applicationsmentioning

confidence: 99%

xia2.multiplex: a multi-crystal data analysis pipeline

Gildea¹,

Beilsten-Edmands²,

Axford³

et al. 2022

Preprint

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In macromolecular crystallography radiation damage limits the amount of data that can be collected from a single crystal. It is often necessary to merge data sets from multiple crystals, for example small-wedge data collections on micro-crystals, in situ room-temperature data collections, and collection from membrane proteins in lipidic mesophase. Whilst indexing and integration of individual data sets may be relatively straightforward with existing software, merging multiple data sets from small wedges presents new challenges. Identification of a consensus symmetry can be problematic, particularly in the presence of a potential indexing ambiguity. Furthermore, the presence of non-isomorphous or poor-quality data sets may reduce the overall quality of the final merged data set. To facilitate and help optimise the scaling and merging of multiple data sets, we developed a new program, xia2.multiplex, which takes data sets individually integrated with DIALS and performs symmetry analysis, scaling and merging of multi-crystal data sets. xia2.multiplex also performs analysis of various pathologies that typically affect multi-crystal data sets, including non-isomorphism, radiation damage and preferential orientation. After describing a number of use cases, we demonstrate the benefit of xia2.multiplex within a wider autoprocessing framework in facilitating a multi-crystal experiment collected as part of in situ room-temperature fragment screening experiments on the SARS-CoV-2 main protease.

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Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography

Cited by 2 publications

References 54 publications

Room-temperature crystallography reveals altered binding of small-molecule fragments to PTP1B

Room-temperature crystallography reveals altered binding of small-molecule fragments to PTP1B

xia2.multiplex: a multi-crystal data analysis pipeline

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