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Cryo-EM of soft-landed β-galactosidase: Gas-phase and native structures are remarkably similar

Esser, T. K.; Boehning, J.; Onur, A.; Chinthapalli, D. K.; Eriksson, L.; Grabarics, M.; Fremdling, P.; Konijnenberg, A.; Makarov, A. A.; Botman, A.; Peter, C.; Benesch, J. L. P.; Robinson, C. V.; Gault, J.; Baker, L.; Bharat, T. A. M.; Rauschenbach, S.

2023-08-17 biochemistry
10.1101/2023.08.17.553673 bioRxiv
Show abstract

Native mass spectrometry (native MS) is a powerful technique that provides information on stoichiometry, interactions, homogeneity and shape of protein complexes. However, the extent of deviation between protein structures in the mass spectrometer and in solution remains a matter of debate. Here, we uncover the gas-phase structure of {beta}-galactosidase using single particle electron cryomicroscopy (cryo-EM) down to 2.6 [A] resolution, enabled by soft-landing of mass-selected protein complexes onto cold TEM grids and in-situ ice coating. We find that large parts of the secondary and tertiary structure are retained from solution, with dehydration-driven subunit reorientation leading to consistent compaction in the gas phase. Our work enables visualizing the structure of gas-phase protein com-plexes from numerous experimental scenarios at side-chain resolution and demonstrates the possibility of more controlled cryo-EM sample preparation. One Sentence SummaryElectrospray ion-beam deposition on cold grids and in-vacuum ice growth enable cryo-EM of mass-selected proteins at 2.6 [A].

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