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- EMDB-20229: Dataset IV: Sub-3 Angstrom Apoferritin Structure Determined With ... -

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Entry
Database: EMDB / ID: EMD-20229
TitleDataset IV: Sub-3 Angstrom Apoferritin Structure Determined With Full Range of Phase Shifts Using A Single Position Of Volta Phase Plate
Map data
SampleHuman Apoferritin light chainFerritin
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsLi K / Sun C / Klose T / Irimia-Dominguez J / Vago FS / Vidal R / Jiang W
CitationJournal: 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.
DateDeposition: May 16, 2019 / Header (metadata) release: May 29, 2019 / Map release: May 29, 2019 / Update: Jun 5, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 18
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 18
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20229.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 273.92 Å
1.07 Å/pix.
x 256 pix.
= 273.92 Å
1.07 Å/pix.
x 256 pix.
= 273.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 18.0 / Movie #1: 18
Minimum - Maximum-48.728622000000001 - 69.919659999999993
Average (Standard dev.)-0.0075682807 (±3.0229187)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z273.920273.920273.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-128-128-128
NC/NR/NS256256256
D min/max/mean-48.72969.920-0.008

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Supplemental data

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Mask #1

Fileemd_20229_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Mask #2

Fileemd_20229_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Human Apoferritin light chain

EntireName: Human Apoferritin light chainFerritin / Number of components: 1

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Component #1: cellular-component, Human Apoferritin light chain

Cellular-componentName: Human Apoferritin light chainFerritin / Recombinant expression: No
MassTheoretical: 440 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 35 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Raw dataEMPIAR-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])

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: O (octahedral) / Number of projections: 73314
3D reconstructionResolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Input PDB model: 2FFX

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