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- EMDB-20225: Dataset I: Sub-3 Angstrom Apoferritin Structure Determined With F... -

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Entry
Database: EMDB / ID: EMD-20225
TitleDataset I: Sub-3 Angstrom Apoferritin Structure Determined With Full Range of Phase Shifts Using A Single Position Of Volta Phase Plate
Map datasharpened full map
Sample
  • Organelle or cellular component: Human Apoferritin light chainFerritin
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsLi K / Sun C / Klose T / Irimia-Dominguez J / Vago FS / Vidal R / Jiang W
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesU24 GM116789 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke1U01NS110437 United States
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesR01 AI111095 United States
National Institutes of Health/National Institute of Neurological Disorders and StrokeR01 NS050227 United States
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.
History
DepositionMay 16, 2019-
Header (metadata) releaseMay 29, 2019-
Map releaseMay 29, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 17
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 17
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20225.png

Downloads & links

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Map

FileDownload / File: emd_20225.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened full map
Voxel sizeX=Y=Z: 0.658 Å
Density
Contour LevelBy AUTHOR: 17 / Movie #1: 17
Minimum - Maximum-33.847168 - 67.73772
Average (Standard dev.)0.23494624 (±3.5711124)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-180-180-180
Dimensions360360360
Spacing360360360
CellA=B=C: 236.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6580.6580.658
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z236.880236.880236.880
α/β/γ90.00090.00090.000
start NX/NY/NZ79740
NX/NY/NZ93103213
MAP C/R/S123
start NC/NR/NS-180-180-180
NC/NR/NS360360360
D min/max/mean-33.84767.7380.235

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Supplemental data

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Mask #1

Fileemd_20225_msk_1.map
Projections & Slices
AxesZYX

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Mask #2

Fileemd_20225_msk_2.map
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Half map: #2

Fileemd_20225_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_20225_half_map_2.map
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Sample components

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Entire : Human Apoferritin light chain

EntireName: Human Apoferritin light chainFerritin
Components
  • Organelle or cellular component: Human Apoferritin light chainFerritin

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Supramolecule #1: Human Apoferritin light chain

SupramoleculeName: Human Apoferritin light chain / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 440 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Specialist opticsPhase plate: VOLTA PHASE PLATE
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 628 / Average electron dose: 35.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: O (octahedral) / Resolution.type: BY AUTHOR / Resolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 72521
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2ffx

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