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- EMDB-20111: Separating distinct macromolecular assemblies from cryo-EM images -

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Basic information

Entry
Database: EMDB / ID: EMD-20111
TitleSeparating distinct macromolecular assemblies from cryo-EM images
Map dataReconstruction of 80S from mixture
Sample
  • Complex: 80S ribosome
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.4 Å
AuthorsVerbeke EJ / Zhou Y / Horton AP / Mallam AL / Taylor DW / Marcotte EM
CitationJournal: J Struct Biol / Year: 2020
Title: Separating distinct structures of multiple macromolecular assemblies from cryo-EM projections.
Authors: Eric J Verbeke / Yi Zhou / Andrew P Horton / Anna L Mallam / David W Taylor / Edward M Marcotte /
Abstract: Single particle analysis for structure determination in cryo-electron microscopy is traditionally applied to samples purified to near homogeneity as current reconstruction algorithms are not designed ...Single particle analysis for structure determination in cryo-electron microscopy is traditionally applied to samples purified to near homogeneity as current reconstruction algorithms are not designed to handle heterogeneous mixtures of structures from many distinct macromolecular complexes. We extend on long established methods and demonstrate that relating two-dimensional projection images by their common lines in a graphical framework is sufficient for partitioning distinct protein and multiprotein complexes within the same data set. The feasibility of this approach is first demonstrated on a large set of synthetic reprojections from 35 unique macromolecular structures spanning a mass range of hundreds to thousands of kilodaltons. We then apply our algorithm on cryo-EM data collected from a mixture of five protein complexes and use existing methods to solve multiple three-dimensional structures ab initio. Incorporating methods to sort single particle cryo-EM data from extremely heterogeneous mixtures will alleviate the need for stringent purification and pave the way toward investigation of samples containing many unique structures.
History
DepositionApr 15, 2019-
Header (metadata) releaseMay 8, 2019-
Map releaseNov 27, 2019-
UpdateJan 22, 2020-
Current statusJan 22, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20111.png

Downloads & links

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Map

FileDownload / File: emd_20111.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of 80S from mixture
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 384 pix.
= 422.4 Å		1.1 Å/pix.
x 384 pix.
= 422.4 Å		1.1 Å/pix.
x 384 pix.
= 422.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.30785504 - 0.8342499
Average (Standard dev.)0.01366421 (±0.064351596)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 422.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z422.400422.400422.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.3080.8340.014

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Supplemental data

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Sample components

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Entire : 80S ribosome

EntireName: 80S ribosome
Components
  • Complex: 80S ribosome

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Supramolecule #1: 80S ribosome

SupramoleculeName: 80S ribosome / type: complex / ID: 1 / Parent: 0 / Details: From a complex mixture of defined assemblies
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 3.2 MDa

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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

Concentration1 mg/mL
BufferpH: 7.4
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
Detailsa mixture of 40S, 60S, 80S, and apoferritin

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-20 / Number grids imaged: 1 / Number real images: 2400 / Average exposure time: 6.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -3.0 µm / Nominal defocus min: -2.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsDrift-correction using MotionCorr2
Particle selectionNumber selected: 522000
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 1) / Number images used: 30000
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 1)
Final 3D classificationNumber classes: 4 / Details: SLICEM

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