A Medipix quantum area detector allows rotation electron diffraction data collection from submicrometre three-dimensional protein crystals

Abstract

When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e À Å À2 ), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electrondiffraction data from a single $100 nm thick protein crystal. A highly parallel 200 keV electron beam ( = 0.025 Å ) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4 . This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLM and SCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins.

Protein structure determination by electron diffraction using a single three-dimensional nanocrystal

“…Hen egg-white lysozyme nanocrystals were prepared as described previously (Nederlof et al, 2013). The microscope was operated at 200 kV and aligned for diffraction with a parallel beam that had a diameter of 2.0 and 1.7 mm in microprobe mode for the Talos Arctica and Titan Krios TEMs, respectively.…”

“…Thus, hybrid pixel detectors sacrifice pixel size to achieve radiation hardness, a high dynamic range and megahertz counting modes. Pixel size is less important in diffraction data acquisition than in imaging, since the resolution of the data is not determined by the level of detail on the detector but by the number of diffraction orders that can be resolved (Nederlof et al, 2013).…”

“…In electron crystallography, alignment of the crystal with the rotation axis is not always straightforward and the rotation stages are not always as accurate as desired, which prompted the enhancement of the method using either conical beam precession (Vincent & Midgley, 1994;Kolb et al, 2007Kolb et al, , 2008Gemmi et al, 2013) or beam tilt (Zhang et al, 2010;Wan et al, 2013;Yun et al, 2015). Recently, continuous three-dimensional data collection from ISSN 2059-7983 protein nanocrystals was accomplished (Nederlof et al, 2013). The first protein structure of a micrometre-sized crystal was determined soon after, using discrete rotation steps (Shi et al, 2013).…”

“…Detector features that are of particular interest for electron diffraction are the absence of readout noise, a high dynamic range and the ability to distinguish between the signal from diffracted electrons and that from the high X-ray background that is inherently present in any TEM (Georgieva et al, 2011;Nederlof et al, 2013;van Genderen et al, 2016). These features require a counting detector, a concept that has recently also been introduced in monolithic and CMOS detectors.…”

“…Previously, we used a single quad Medipix detector (Georgieva et al, 2011;Nederlof et al, 2013) and a Timepix detector (van Genderen et al, 2016) of 512 Â 512 pixels (55 Â 55 mm pixel size). For very well ordered crystals this detector size is sufficient for resolving up to 50 orders of diffraction.…”