Routine Determination of Ice Thickness for Cryo-EM Grids
| Autor: | Bridget Carragher, William J. Rice, Clinton S. Potter, Edward T. Eng, Anchi Cheng, Alex J. Noble, Laura Y. Kim |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Electron Microscope Tomography Aperture Cryo-electron microscopy Computer science 02 engineering and technology Article Specimen Handling Computational science 03 medical and health sciences Glutamate Dehydrogenase Structural Biology Fructose-Bisphosphate Aldolase Animals Sample preparation Instrumentation (computer programming) 030304 developmental biology Data processing 0303 health sciences Measure (data warehouse) Data collection Cryoelectron Microscopy Filter (signal processing) 021001 nanoscience & nanotechnology 030104 developmental biology Workflow Data quality Particle Rabbits 0210 nano-technology |
| Popis: | Recent advances in instrumentation and automation have made cryo-EM a popular method for producing near-atomic resolution structures of a variety of proteins and complexes. Sample preparation is still a limiting factor in collecting high quality data. Thickness of the vitreous ice in which the particles are embedded is one of the many variables that need to be optimized for collection of the highest quality data. Here we present two methods, using either an energy filter or scattering outside the objective aperture, to measure ice thickness for potentially every image collected. Unlike geometrical or tomographic methods, these can be implemented directly in the single particle collection workflow without interrupting or significantly slowing down data collection. We describe the methods as implemented into the Leginon/Appion data collection workflow, along with some examples from test cases. Routine monitoring of ice thickness should prove helpful for optimizing sample preparation, data collection, and data processing. |
| Databáze: | OpenAIRE |
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