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 and might be used to further develop processing routines.

Fig. 1. Visualization of the acquisition schemes.

a Overview of different angular acquisition schemes used. b Zero-degree projections from representative tilt-series and their corresponding periodograms with fitted CTF model estimated by CTFFind4. For DS VPP foc scheme a conventional Fourier power spectrum is shown.

Fig. 2. Resolution estimates of structures obtained using different acquisitions schemes.

a Resolution between the half maps with different symmetries obtained by FSC at 0.5 criterion. b Same as a, but the resolution was estimated using the 0.143 criterion. c Resolution estimate by FSC (0.5 criterion) between the EMD-3782 map and the respective maps with C6 symmetry applied. The respective FSC curves are shown in Supplementary Fig. 1. d B-factor analysis. Plot of resolution of C6-symmetrized structures at 0.5 criterion as a function of number of particles (x-axis scaled logarithmically). e Same as d, but the resolution was estimated at 0.143 criterion.

Fig. 3. Structural details of averages obtained using different acquisition schemes.

An individual helix of HIV-1 CA-SP1 as structurally determined previously is shown in comparison to the dose-symmetric, bidirectional and continuous scheme used in this study. PDB 5L93 indicating the position of the helix is shown left.

Fig. 4. Cryo-EM maps of HIV-1 CA-SP1.

a Structure obtained by the dose-symmetric scheme (after CTF-reweighting and sharpening). b A raw structure obtained from DS VPP foc scheme and its sharpened version (right). c Raw structure obtained from DS VPP def scheme, corresponding CTF-reweighted structure (middle) and final structure after CTF-reweighting and sharpening (right). All images are color coded according to a single chain from PDB 5L93 of HIV-1 CA-SP1 (see Fig. 3 for comparison).