A Script-Assisted Microscopy (SAM) package to improve data acquisition rates on FEI Tecnai electron microscopes equipped with Gatan CCD cameras.
High throughput methods of data acquisition are advantageous for cryoelectron microscopy and single particle reconstruction as high-resolution structure determination requires thousands of particle images. We have developed a semi-automated data collection method that utilizes the scripting languages provided by FEI for their Tecnai User Interface (TUI) and by Gatan for their Digital Micrograph package. Our Script-Assisted Microscopy (SAM) method allows for the selection of multiple locations within a low magnification, search mode, micrograph and for subsequent automated imaging of these locations at a higher exposure magnification. The SAM approach permits the user to retain control over the microscope, while streamlining the most repetitive steps of collecting and evaluating micrographs. With SAM, we have found an average of 1000 micrographs can be collected per day on any grid type, either irregular homemade grids or prefabricated grids with regularly spaced holes. This rate of data collection represents a fivefold improvement over our manual collection rates. SAM provides an example of an individually tailored approach to data acquisition utilizing the scripting interfaces provided by the equipment manufacturers. The SAM method has proven valuable for determination of a subnanometer resolution cryoEM structure of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a 469kDa protein.
Shi
Jian
J
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232-0615, USA.
Williams
Dewight R
DR
Stewart
Phoebe L
PL
D020285:Cryoelectron Microscopy / Q000295:instrumentation*; D051747:DNA-Activated Protein Kinase / Q000737:chemistry; D036103:Nanotechnology; D010784:Photomicrography / Q000295:instrumentation*; D012984:Software
D051747:DNA-Activated Protein Kinase