Serial protein crystallography in an electron microscope

Abstract

Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nanocrystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 Å and 1.80 Å, respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation.

Fast Pixelated Detectors in Scanning Transmission Electron Microscopy. Part II: Post-Acquisition Data Processing, Visualization, and Structural Characterization

“…For diffraction data, this includes strain mapping, the number and spatial extent of different phases or grains (see Fig. 1), and the spot intensities for structure factor characterization (Midgley & Eggeman, 2015) and structure determination (Mugnaioli et al, 2009;Clabbers et al, 2017;Bücker et al, 2020), and for the analysis of noncentrosymmetric crystals. Intensity mapping may be done by generating a synthetic aperture based on the extracted lattice parameters and then applying it to the data, as discussed in the section "Virtual detector images", or, for point-like spots, fitting a 2D Gaussian to the peak regions using the functions provided in the fpd.utils module, which are also used in the sub-pixel peak finding described above, yielding details of the peak properties.…”

“…Alternatively, different DEDs with higher pixel counts may be used, or more pixels may be added to a Medipix3 detector by tiling the detector chips. The Medipix3 family of detectors are 3-side buttable (Ballabriga et al, 2013), and thus may be tiled in 2 × N arrays (Bücker et al, 2020), with larger pixels at the joints which must be accounted for. Furthermore, employment of the through-silicon via feature of the Medipix3 family can allow tiling on all four sides of the sensor in even larger arrays (Tick & Campbell, 2011; Ponchut et al, 2015) with minimal dead areas for the readout circuitry.…”

Demonstration of electron diffraction from membrane protein crystals grown in a lipidic mesophase after lamella preparation by focused ion beam milling at cryogenic temperatures

“…Since 2013, when the first protein structure was determined by the microcrystal electron diffraction technique (Shi et al, 2013), 3D macromolecular crystallography with electrons has undergone significant developments in data collection and data analysis (Nannenga et al, 2014;van Genderen et al, 2016;Clabbers et al, 2017;Gruene et al, 2018;Hattne et al, 2019;Bü cker et al, 2020), and a number of new protein and peptide structures have been solved (Rodriguez et al, 2015;de la Cruz et al, 2017;Xu et al, 2019). For a typical ED data collection experiment, crystals in solution are pipetted onto a transmission electron microscopy (TEM) grid, blotted to remove excess solution, and vitrified in liquid ethane or nitrogen.…”

Demonstration of electron diffraction from membrane protein crystals grown in a lipidic mesophase after lamella preparation by focused ion beam milling at cryogenic temperatures

“…Since 2013, when the first protein structure was determined by the microcrystal electron diffraction (MicroED) technique (Shi et al, 2013), 3D macromolecular crystallography with electrons has undergone significant developments in data collection and data analysis (Nannenga et al, 2014;van Genderen et al, 2016;Clabbers et al, 2017;Gruene et al, 2018;Hattne et al, 2019;Bücker et al, 2020), and a number of new protein and peptide structures have been solved (Rodriguez et al, 2015;de la Cruz et al, 2017;Xu et al, 2019). For a typical ED data collection experiment, crystals in solution are pipetted onto a transmission electron microscopy (TEM) grid, blotted to remove excess solution, and vitrified in liquid ethane or nitrogen.…”