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Abstract
Electron diffraction allows protein structure determination when only nanosized crystals are available. Nevertheless, multiple elastic (or dynamical) scattering, which is prominent in electron diffraction, is a concern. Current methods for modeling dynamical scattering by multi-slice or Bloch wave approaches are not suitable for protein crystals because they are not designed to cope with large molecules. Here, dynamical scattering of nanocrystals of insulin, thermolysin and thaumatin was limited by collecting data from thin crystals. To accurately measure the weak diffraction signal from the few unit cells in the thin crystals, alow-noise hybrid pixel Timepix electron-counting detector was used. The remaining dynamical component was further reduced in refinement using a likelihood-based correction, which was introduced previously for analyzing electron diffraction data of small-molecule nanocrystals and was adapted here for protein crystals. The procedure is shown to notably improve the structural refinement, in one case allowing the location of solvent molecules. It also allowed refinement of the charge states of bound metal atoms, an important element in protein function, through B-factor analysis of the metal atoms and their ligands. These results clearly increase the value of macromolecular electron crystallography as a complementary structural biology technique.
Highlights
The strong interaction of electrons with matter favors electron crystallography for solving three-dimensional structures from beam-sensitive sub-micrometre-sized crystals such as protein nanocrystals (Clabbers & Abrahams, 2018; Henderson, 1995)
Our results confirm that a statistical correction for the overestimation of the intensity of weak reflections arising from dynamical scattering can be generally extended to protein crystals
The correction procedure was originally developed for organic molecule electron diffraction data and to date had only been demonstrated for protein data using a single lysozyme test case
Summary
The strong interaction of electrons with matter favors electron crystallography for solving three-dimensional structures from beam-sensitive sub-micrometre-sized crystals such as protein nanocrystals (Clabbers & Abrahams, 2018; Henderson, 1995). The major advantage of image-based methods over diffraction is that they provide experimental data with crystallographic phases. Various protein structures have recently been solved using diffraction data collected with continuous rotation of 3D crystals (Clabbers et al, 2017; de la Cruz et al, 2017; Nannenga et al, 2014; Xu et al, 2018; Yonekura et al, 2015)
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Schedule a callMore From: Acta Crystallographica Section D Structural Biology
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