|Entry||Database: EMDB / ID: EMD-20225|
|Title||Dataset I: Sub-3 Angstrom Apoferritin Structure Determined With Full Range of Phase Shifts Using A Single Position Of Volta Phase Plate|
|Sample||Human Apoferritin light chainFerritin|
|Biological species||Homo sapiens (human)|
|Method||single particle reconstruction / cryo EM / Resolution: 2.94 Å|
|Authors||Li K / Sun C / Klose T / Irimia-Dominguez J / Vago FS / Vidal R / Jiang W|
|Citation||Journal: J. Struct. Biol. / Year: 2019|
Title: Sub-3 Å apoferritin structure determined with full range of phase shifts using a single position of volta phase plate.
Authors: Kunpeng Li / Chen Sun / Thomas Klose / Jose Irimia-Dominguez / Frank S Vago / Ruben Vidal / Wen Jiang /
Abstract: Volta Phase Plate (VPP) has become an invaluable tool for cryo-EM structural determination of small protein complexes by increasing image contrast. Currently, the standard protocol of VPP usage ...Volta Phase Plate (VPP) has become an invaluable tool for cryo-EM structural determination of small protein complexes by increasing image contrast. Currently, the standard protocol of VPP usage periodically changes the VPP position to a fresh spot during data collection. Such a protocol was to target the phase shifts to a relatively narrow range (around 90°) based on the observations of increased phase shifts and image blur associated with more images taken with a single VPP position. Here, we report a 2.87 Å resolution structure of apoferritin reconstructed from a dataset collected using only a single position of VPP. The reconstruction resolution and map density features are nearly identical to the reconstruction from the control dataset collected with periodic change of VPP positions. Further experiments have verified that similar results, including a 2.5 Å resolution structure, could be obtained with a full range of phase shifts, different spots of variable phase shift increasing rates, and at different ages of the VPP post-installation. Furthermore, we have found that the phase shifts at low resolutions, probably related to the finite size of the Volta spots, could not be correctly modeled by current CTF model using a constant phase shift at all frequencies. In dataset III, severe beam tilt issue was identified but could be computationally corrected with iterative refinements. The observations in this study may provide new insights into further improvement of both the efficiency and robustness of VPP, and to help turn VPP into a plug-and-play device for high-resolution cryo-EM.
|Date||Deposition: May 16, 2019 / Header (metadata) release: May 29, 2019 / Map release: May 29, 2019 / Update: Jun 5, 2019|
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_20225.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 0.658 Å|
|Symmetry||Space group: 1|
CCP4 map header:
|Projections & Slices|
-Entire Human Apoferritin light chain
|Entire||Name: Human Apoferritin light chainFerritin / Number of components: 1|
-Component #1: cellular-component, Human Apoferritin light chain
|Cellular-component||Name: Human Apoferritin light chainFerritin / Recombinant expression: No|
|Mass||Theoretical: 440 kDa|
|Source||Species: Homo sapiens (human)|
|Source (engineered)||Expression System: Escherichia coli (E. coli)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||pH: 7.4|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 35 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Cs: 2.7 mm / Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Image acquisition||Number of digital images: 628|
|Raw data||EMPIAR-10263 (Title: Sub-3 Å Apoferritin Structure Determined With Full Range of Phase Shifts Using A Single Position Of Volta Phase Plate|
Data size: 778.8
Data #1: Unaligned multi-frame movie of apoferritin Dataset I [micrographs - multiframe]
Data #2: Unaligned multi-frame movie of apoferritin Dataset II [micrographs - multiframe]
Data #3: Unaligned multi-frame movie of apoferritin Dataset III [micrographs - multiframe]
Data #4: Unaligned multi-frame movie of apoferritin Dataset IV [micrographs - multiframe])
|Processing||Method: single particle reconstruction / Applied symmetry: O (octahedral) / Number of projections: 72521|
|3D reconstruction||Resolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF|
|FSC plot (resolution estimation)|
-Atomic model buiding
|Modeling #1||Input PDB model: 2FFX|
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