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- EMDB-8799: Yeast 80S ribosome derived from EMPIAR-10045 using emClarity -

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Basic information

Entry
Database: EMDB / ID: EMD-8799
TitleYeast 80S ribosome derived from EMPIAR-10045 using emClarity
Map dataYeast 80S ribosome derived from EMPIAR-10045 using emClarity
Sample
  • Complex: S. cerevisiae 80S ribosome
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsubtomogram averaging / cryo EM / Resolution: 7.8 Å
AuthorsHimes BA / Zhang P
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM085043 United States
CitationJournal: Nat Methods / Year: 2018
Title: emClarity: software for high-resolution cryo-electron tomography and subtomogram averaging.
Authors: Benjamin A Himes / Peijun Zhang /
Abstract: Macromolecular complexes are intrinsically flexible and often challenging to purify for structure determination by single-particle cryo-electron microscopy (cryo-EM). Such complexes can be studied by ...Macromolecular complexes are intrinsically flexible and often challenging to purify for structure determination by single-particle cryo-electron microscopy (cryo-EM). Such complexes can be studied by cryo-electron tomography (cryo-ET) combined with subtomogram alignment and classification, which in exceptional cases achieves subnanometer resolution, yielding insight into structure-function relationships. However, it remains challenging to apply this approach to specimens that exhibit conformational or compositional heterogeneity or are present in low abundance. To address this, we developed emClarity ( https://github.com/bHimes/emClarity/wiki ), a GPU-accelerated image-processing package featuring an iterative tomographic tilt-series refinement algorithm that uses subtomograms as fiducial markers and a 3D-sampling-function-compensated, multi-scale principal component analysis classification method. We demonstrate that our approach offers substantial improvement in the resolution of maps and in the separation of different functional states of macromolecular complexes compared with current state-of-the-art software.
History
DepositionJun 30, 2017-
Header (metadata) releaseOct 10, 2018-
Map releaseOct 10, 2018-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8799.png

Downloads & links

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Map

FileDownload / File: emd_8799.map.gz / Format: CCP4 / Size: 115.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationYeast 80S ribosome derived from EMPIAR-10045 using emClarity
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.17 Å/pix.
x 312 pix.
= 677.04 Å		2.17 Å/pix.
x 312 pix.
= 677.04 Å		2.17 Å/pix.
x 312 pix.
= 677.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.17 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.5 / Movie #1: 2.5
Minimum - Maximum-15.062231 - 15.681607
Average (Standard dev.)0.010970133 (±0.41644982)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions312312312
Spacing312312312
CellA=B=C: 677.04004 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.172.172.17
M x/y/z312312312
origin x/y/z0.0000.0000.000
length x/y/z677.040677.040677.040
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS312312312
D min/max/mean-15.06215.6820.011

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Supplemental data

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Mask #1

Fileemd_8799_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_8799_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : S. cerevisiae 80S ribosome

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

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Supramolecule #1: S. cerevisiae 80S ribosome

SupramoleculeName: S. cerevisiae 80S ribosome / type: complex / ID: 1 / Parent: 0 / Details: Non-translating yeast 80s ribosome
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
GridModel: Quantifoil R2/2 / Material: COPPER/RHODIUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: Gatan Quantum Energy Filter
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 53000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: emClarity (ver. 1.0)
Software - details: anisotropic "single-particle wiener filter" for final weighting/reconstruction
Details: Independent beyond 3.2 nm as determined by the FSC at the time of dividing the half-sets
Number subtomograms used: 3120
ExtractionNumber tomograms: 4 / Number images used: 3120 / Reference model: EMD-3228 / Method: template matching in emClarity
Details: Template matching using EMD-3228 as an initial reference low-pass filtered to 4.0nm.
CTF correctionSoftware - Name: emClarity (ver. 1.0)
Software - details: included astigmatism and per tilt ctf estimation
Details: Phases were corrected on the projections by multiplication of the astigmatic-CTF determined in emClarity in tiles of 320 sq pix with a strip replacement that depended on the tilt angle.
Final angle assignmentType: OTHER / Software - Name: emClarity (ver. 1.0)
Details: 3D refinement with two half-sets fully separated after template matching, at which point the FSC 0.5 was 3.2 nanometers. Reference and subtomograms filtered dynamically according to the FSC ...Details: 3D refinement with two half-sets fully separated after template matching, at which point the FSC 0.5 was 3.2 nanometers. Reference and subtomograms filtered dynamically according to the FSC at each cycle using the FOM approach.
FSC plot (resolution estimation)

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