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(-) Å(-2)), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electron-diffraction data from a single ∼100 nm thick protein crystal. A highly parallel 200 keV electron beam (λ = 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.
Highlights
Protein crystallography is a major justification for large-scale X-ray facilities such as synchrotrons and free-electron lasers
Three-dimensional protein crystals that are smaller than about 0.5 mm are too small for standard X-ray crystallography, XFEL sources are expanding the method towards smaller crystals (Chapman et al, 2011)
Recent data demonstrate that useful high-resolution electron diffraction data can be obtained from nanosized three-dimensional protein crystals, where synchrotron X-rays fail (Jiang et al, 2009)
Summary
Protein crystallography is a major justification for large-scale X-ray facilities such as synchrotrons and free-electron lasers. Electrons are less damaging to proteins than X-rays by several orders of magnitude per elastically diffracted quantum (Henderson, 1995). This property of electrons explains the successes of two-dimensional electron crystallography. Recent data demonstrate that useful high-resolution electron diffraction data (up to 2.5 Aresolution) can be obtained from nanosized three-dimensional protein crystals, where synchrotron X-rays fail (Jiang et al, 2009). The Medipix chip only counts 200 keV electrons and, unlike many other detectors, is blind to soft X-rays of lower energy that are produced in great abundance inside any electron microscope In this fashion its noise is almost exclusively determined by the counting statistics of the electrons.
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