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- EMDB-4261: Volta phase plate data collection facilitates image processing an... -

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Basic information

Entry
Database: EMDB / ID: EMD-4261
TitleVolta phase plate data collection facilitates image processing and cryo-EM structure determination
Map data
Sample
  • Complex: ribosome
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
Authorsvon Loeffelholz O / Klaholz BP / Natchiar SK
CitationJournal: J Struct Biol / Year: 2018
Title: Volta phase plate data collection facilitates image processing and cryo-EM structure determination.
Authors: Ottilie von Loeffelholz / Gabor Papai / Radostin Danev / Alexander G Myasnikov / S Kundhavai Natchiar / Isabelle Hazemann / Jean-François Ménétret / Bruno P Klaholz /
Abstract: A current bottleneck in structure determination of macromolecular complexes by cryo electron microscopy (cryo-EM) is the large amount of data needed to obtain high-resolution 3D reconstructions, ...A current bottleneck in structure determination of macromolecular complexes by cryo electron microscopy (cryo-EM) is the large amount of data needed to obtain high-resolution 3D reconstructions, including through sorting into different conformations and compositions with advanced image processing. Additionally, it may be difficult to visualize small ligands that bind in sub-stoichiometric levels. Volta phase plates (VPP) introduce a phase shift in the contrast transfer and drastically increase the contrast of the recorded low-dose cryo-EM images while preserving high frequency information. Here we present a comparative study to address the behavior of different data sets during image processing and quantify important parameters during structure refinement. The automated data collection was done from the same human ribosome sample either as a conventional defocus range dataset or with a Volta phase plate close to focus (cfVPP) or with a small defocus (dfVPP). The analysis of image processing parameters shows that dfVPP data behave more robustly during cryo-EM structure refinement because particle alignments, Euler angle assignments and 2D & 3D classifications behave more stably and converge faster. In particular, less particle images are required to reach the same resolution in the 3D reconstructions. Finally, we find that defocus range data collection is also applicable to VPP. This study shows that data processing and cryo-EM map interpretation, including atomic model refinement, are facilitated significantly by performing VPP cryo-EM, which will have an important impact on structural biology.
History
DepositionJan 15, 2018-
Header (metadata) releaseJan 24, 2018-
Map releaseJan 31, 2018-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0299
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0299
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4261.png

Downloads & links

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Map

FileDownload / File: emd_4261.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 0.0299 / Movie #1: 0.0299
Minimum - Maximum-0.095998526 - 0.15565197
Average (Standard dev.)0.00075711764 (±0.0071800277)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 457.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z420420420
origin x/y/z0.0000.0000.000
length x/y/z457.800457.800457.800
α/β/γ90.00090.00090.000
start NX/NY/NZ434333
NX/NY/NZ116118137
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS420420420
D min/max/mean-0.0960.1560.001

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Supplemental data

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Sample components

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Entire : ribosome

EntireName: ribosome
Components
  • Complex: ribosome

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Supramolecule #1: ribosome

SupramoleculeName: ribosome / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Strain: HeLa

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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: 7.6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 45871 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 45871
Specialist opticsPhase plate: VOLTA PHASE PLATE
Spherical aberration corrector: CS corrector used, opening up to 13 mrad at 26 mrad tilt
Energy filter - Name: GIF Quantum LS
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 / Digitization - Dimensions - Width: 7420 pixel / Digitization - Dimensions - Height: 7676 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-29 / Number grids imaged: 1 / Number real images: 316 / Average exposure time: 4.5 sec. / Average electron dose: 1.03 e/Å2

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Image processing

Startup modelType of model: OTHER
Details: e2initialmodel.py output from 2D classes calculated with Relion
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 15266
FSC plot (resolution estimation)

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