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.
Using three-dimensional particle-in-cell simulations, we demonstrate that femtosecond few-hundred keV electron pulses can be produced at a high repetition rate by tightly focusing few mJ few-cycle radially polarized laser pulses in a low density gas. In particular, we show that the laser pulse parameters and gas density can be optimized to cover the full 100–300 keV energy window that characterizes ultrafast electron diffraction imaging experiments. The active development of high-power laser sources promises routine operation at 1 kHz and above, allowing time-resolved electron diffraction on the femtosecond time scale.
1 Sentence summary: Serial diffraction (SerialED) enables high-throughput, low dose protein crystallography from sub-micron crystals using conventional S/TEM microscopes. AbstractWe describe a new method for serial electron diffraction of protein nanocrystals using a conventional scanning/transmission electron microscope (S/TEM). Randomly dispersed crystals are mapped, and dose-efficient diffraction patterns measured at each identified crystal position for structure determination. Each crystal is measured in a single orientation without rotation. The automated workflow is suitable for high-throughput applications with acquisition rates of up to 1 kHz at a high hit fraction. A dose fractionation scheme allows for minimization of radiation damage effects and inherently optimal acquisition settings. We demonstrate this method by solving the structure of crystalline granulovirus occlusion bodies and lysozyme to a resolution of 1.55 Å and 1.80 Å, respectively. Our method promises to provide rapid high-quality structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation.We thank Djordje Gitaric for mechanical design work, Fabian Westermeier, David Pennicard, and Heinz Graafsma for adapting the Lambda detector for electron imaging, Anton Barty and Henry Chapman for many helpful discussions and critical reading of the manuscript, Thomas A. White for help with modifying CrystFEL for electron diffraction, and Michiel de Kock for help with image processing. We are indebted to Kay Grünewald and his research group for lending to us their cryo-transfer holder.
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