Challenges in solving structures from radiation-damaged tomograms of protein nanocrystals assessed by simulation

Abstract

Structure determination methods are needed to resolve the atomic details that underlie protein function. X-ray crystallography has provided most of our knowledge of protein structure but is constrained by the need for large, well-ordered crystals and the loss of phase information. The rapidly developing methods of serial femtosecond crystallography, micro-electron diffraction, and single-particle reconstruction circumvent the first of these limitations by enabling data collection from nanocrystals or purified proteins. However, the first two methods also suffer from the phase problem, while many proteins fall below the molecular weight threshold required by single-particle reconstruction. Cryo-electron tomography of protein nanocrystals has the potential to overcome these obstacles of mainstream structure determination methods. Here we present a data processing scheme that combines routines from X-ray crystallography and new algorithms we developed to solve structures from tomograms of nanocrystals. This pipeline handles image processing challenges specific to tomographic sampling of periodic specimens and is validated using simulated crystals. We also assess the tolerance of this workflow to the effects of radiation damage. Our simulations indicate a trade-off between a wider tilt-range to facilitate merging data from multiple tomograms and a smaller tilt increment to improve phase accuracy. Since phase errors but not merging errors can be overcome with additional datasets, these results recommend distributing the dose over a wide angular range rather than using a finer sampling interval to solve the protein structure.

Macromolecular crystallography using microcrystal electron diffraction

“…After obtaining initial atomic positions and crystallographic structure-factor phases, the reflection intensities extracted from electron diffraction patterns can be used for refinement of the atomic positions and extension to higher resolution (Weirich et al, 1996;Zandbergen, 1997;Jansen et al, 1998;Zou et al, 2003). These routines have not successfully been applied to phase macromolecular structures from 3D crystals thus far Peck et al, 2020). Phasing of 3D macromolecular crystals is complicated by crystal thickness, breaking the weak phase object approximation, and corrections for the contrast transfer function (CTF), as well as difficulty in finding a common origin.…”