MicroED Structures from Micrometer Thick Protein Crystals

Abstract

SummaryAtomic resolution protein structures can be determined by MicroED from crystals that surpass the theoretical maximum thickness limit by an order of magnitude. AbstractTheoretical calculations suggest that crystals exceeding 100 nm thickness are excluded by dynamical scattering from successful structure determination using microcrystal electron diffraction (MicroED). These calculations are at odds with experimental results where MicroED structures have been determined from significantly thicker crystals.Here we systematically evaluate the influence of thickness on the accuracy of MicroED intensities and the ability to determine structures from protein crystals one micrometer thick. To do so, we compare ab initio structures of a human prion protein segment determined from thin crystals to those determined from crystals up to one micrometer thick. We also compare molecular replacement solutions from crystals of varying thickness for a larger globular protein, proteinase K. Our results indicate that structures can be reliably determined from crystals at least an order of magnitude thicker than previously suggested by simulation, opening the possibility for an even broader range of MicroED experiments.peer-reviewed)

Analysis of global and site-specific radiation damage in cryo-EM

“…Frozen-hydrated crystals of proteinase K and a 7-residue peptide were prepared as previously described Martynowycz et al, 2017), except the blotting force and time of the FEI (now Thermal Fischer) Vitrobot Mark IV were optimized to position 24 and 12 s, respectively, at an environment humidity of 30% for proteinase K and 20 and 20 s for GSNQNNF. Electron diffraction datasets from separate crystals were collected using an FEI Tecnai F20 transmission electron microscope operated at 200 kV, with the objective aperture fully open to evenly illuminate an area extending beyond the sample and setting the selected area aperture to closely match the size of the crystal.…”

“…Recent MicroED experiments demonstrated that complete data sets could be collected from a single nanocrystal using a total exposure of less than ~1-2 e -Å -2 (Nannenga et al, 2014a;de la Cruz et al, 2017), making it possible to design new and improved experiments to test for beam induced damage to the specimen with increasing exposure to the electron beam.…”

Efficient, high-throughput ligand incorporation into protein microcrystals by on-grid soaking

“…Milling protein crystals has recently seen a high amount of interest for preparing crystals that are too small for synchrotron X-ray experiments, but still too large for MicroED. By milling the ligand soaked crystals we are no longer limited to crystals that happen to be in thin ice and happen to be smaller than 1μm thick, though this method is still tractable when a FIB/SEM is unavailable by means of crystal fragmentation (de la Cruz et al, 2017; Martynowycz et al, 2017). Using the FIB/SEM, even crystals large enough for synchrotron or home source X-ray experiments become amenable to MicroED.…”

MicroED structure of lipid-embedded mammalian mitochondrial voltage dependent anion channel

“…Briefly, a small amount of liquid is added to a TEM grid, the grid is gently blotted to remove the excess solvent, vitrified in liquid ethane, and stored in liquid nitrogen for future investigation. However, only crystals that happened to be very thin (<500nm) or could be fragmented by sonication or vortexing were amenable to MicroED data collection strategies (de la Cruz et al, 2017;Martynowycz et al, 2017). Unfortunately, membrane protein crystals rarely form crystals thin enough for MicroED.…”