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TitleA new cryo-EM system for electron 3D crystallography by eEFD.
Journal, issue, pagesJ Struct Biol, Vol. 206, Issue 2, Page 243-253, Year 2019
Publish dateMay 1, 2019
AuthorsKoji Yonekura / Tetsuya Ishikawa / Saori Maki-Yonekura /
PubMed AbstractA new cryo-EM system has been developed and investigated for use in protein electron 3D crystallography. The system provides parallel illumination of a coherent 300 kV electron beam to a sample, ...A new cryo-EM system has been developed and investigated for use in protein electron 3D crystallography. The system provides parallel illumination of a coherent 300 kV electron beam to a sample, filters out energy-loss electrons through the sample with an in-column energy filter, and allows rotational data collection on a fast camera. It also possesses motorized cryo-sample loading and automated liquid-nitrogen filling for cooling of multiple samples. To facilitate its use, we developed GUI programs for efficient operation and accurate structure analysis. Here we report on the performance of the system and first results for thin 3D crystals of the protein complexes, catalase and membrane protein complex ExbBD. Data quality is remarkably improved with this approach, which we name eEFD (electron energy-filtered diffraction of 3D crystals), compared with those collected at 200 kV without energy filtration. Key advances include precise control of the microscope and recordings of lens fluctuations, which the programs process and respond to. We also discuss the merits of higher-energy electrons and filtration of energy-loss electrons in electron 3D crystallography.
External linksJ Struct Biol / PubMed:30928615
MethodsEM (electron crystallography)
Resolution3 Å
Structure data

PDB-6jnt:
Catalase structure determined by eEFD (dataset 1)
Method: ELECTRON CRYSTALLOGRAPHY / Resolution: 3.0 Å

PDB-6jnu:
Catalase structure determined by eEFD (dataset 2)
Method: ELECTRON CRYSTALLOGRAPHY / Resolution: 3.0 Å

Chemicals

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

ChemComp-NDP:
NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

ChemComp-HOH:
WATER

Source
  • bos taurus (cattle)
KeywordsOXIDOREDUCTASE / Electron 3D crystallography / eEFD / energy filter / ParallEM / CRYO ARM

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