Benchmarking tomographic acquisition schemes for high-resolution structural biology

Abstract

Cryo electron tomography with subsequent subtomogram averaging is a powerful technique to structurally analyze macromolecular complexes in their native context. Although close to atomic resolution, in principle, can be obtained, it is not clear how individual experimental parameters contribute to the attainable resolution. Here, we have used immature HIV-1 lattice as a benchmarking sample to optimize the attainable resolution for subtomogram averaging. We systematically tested various experimental parameters such as the order of projections, different angular increments and the use of the Volta phase plate. We find that although any of the prominently used acquisition schemes is sufficient to obtain subnanometer resolution, dose-symmetric acquisition provides considerably better outcome. We discuss our findings in order to provide guidance for data acquisition.Our data is publicly available at EMPIAR-10277 as well as EMD-10207 and might be used to further develop processing routines.

A guided approach for subtomogram averaging of challenging macromolecular assemblies

“…While single particle EM gained widespread notoriety for its utility in solving high resolution structures of purified proteins, cryo-electron tomography (cryo-ET) has emerged as the leading technique for visualizing the structures of large, transient, dynamic, flexible, and/or heterogeneous samples in native or near-native reconstituted cellular environments ( Baumeister, 2013 , Oikonomou and Jensen, 2017 ). The implementation of automated data collection ( Blocker et al, 1997 ) packages ( Mastronarde, 2005 , Suloway et al, 2009 ) and optimized tomographic acquisition schemes ( Chreifi et al, 2019 , Eisenstein et al, 2019 , Hagen et al, 2017 , Turoňová et al, 2019 ), combined with direct electron detectors, energy filters, and phase plates ( Khoshouei et al, 2017 ) has revolutionized the feasibility of visualizing cellular machinery for functional and physiological interpretation. Multiple copies of the biological complex of interest can be identified within reconstructed tomograms, and 3D “subvolumes” or “subtomograms” can be extracted and averaged together in a process called subtomogram averaging (STA) to obtain better-resolved 3D reconstructions of the complex of interest.…”

A guided approach for subtomogram averaging of challenging macromolecular assemblies

“…While single particle EM gained widespread notoriety for its utility in solving high resolution structures of purified proteins, cryo-electron tomography (cryo-ET) has emerged as the leading technique for visualizing the structures of large, transient, dynamic, flexible, and/or heterogeneous samples in native or near-native reconstituted cellular environments 1,2 . The implementation of automated data collection 3 packages 4,5 and optimized tomographic acquisition schemes [6][7][8][9] , combined with direct electron detectors, energy filters, and phase plates 10 has revolutionized the feasibility of visualizing cellular machinery for functional and physiological interpretation 11 . Multiple copies of the biological complex of interest can be identified within reconstructed tomograms, and 3D "subvolumes" can be extracted and averaged together in a process called subtomogram averaging (STA) [12][13][14][15][16][17][18][19][20] to obtain better resolved 3D reconstructions of the complex of interest.…”