![]() ![]() realized that it was possible to obtain diffraction patterns without severely damaging the crystal if they dramatically reduced the normal low-dose electron beam. The normal ‘low-dose’ electron beam in one of these microscopes would normally damage the crystal after a single diffraction pattern had been collected. Called MicroED, this technique involves placing the crystal in a transmission electron cryo-microscope, which is a fairly standard piece of equipment in many laboratories. report a new approach to electron crystallography that works with very small three-dimensional crystals. Researchers have developed methods to merge the diffraction patterns produced by hundreds of small crystals, but to date these techniques have only worked with very thin two-dimensional crystals that contain only one layer of the protein of interest. However, it is normally only possible to collect one diffraction pattern from each crystal because of beam induced damage. This means that meaningful amounts of data can be collected from much smaller crystals. However, the interactions between the electrons in the beam and the crystal are much stronger than those between the X-ray photons and the crystal. There is, therefore, a need for other approaches that can determine the structure of proteins that only form small crystals.Įlectron crystallography is similar to X-ray crystallography in that a protein crystal scatters a beam to produce a diffraction pattern. It is possible to overcome this problem by using extremely short pulses of X-rays, but this requires a very large number of small crystals and ultrashort X-ray pulses are only available at a handful of research centers around the world. However, some proteins do not form crystals at all, and others only form small crystals. The crystals used for X-ray crystallography must be large to withstand the damage caused by repeated exposure to the X-ray beam. Finally, after this process has been repeated enough times, it is possible to work backwards from the diffraction patterns to figure out the structure of the protein. The crystal is then rotated through a small angle and another diffraction pattern is recorded. In a typical X-ray crystallography experiment, a beam of X-rays is directed at a protein crystal, which scatters some of the X-ray photons to produce a diffraction pattern. X-ray crystallography has been used to work out the atomic structure of a large number of proteins. This proof of principle paves the way for the implementation of a new technique, which we name ‘MicroED’, that may have wide applicability in structural biology. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1–1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). ![]()
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