![]() The method consists in reconstructing the electron scattering map of a macromolecule of interest from projection images of cryopreserved specimens recorded in a transmission electron microscope. Three-dimensional cryo-electron microscopy (cryo-EM) is now routinely used to determine the structure of macromolecular complexes at near-atomic resolution. We validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300 kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved. We achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. Here, we introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by up to an order of magnitude and improve map resolution compared to existing approaches. ![]() However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment.
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