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- EMDB-21952: Subtomogram average of ribosomes from tomograms of cryoFIB-SEM-ge... -

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Entry
Database: EMDB / ID: EMD-21952
TitleSubtomogram average of ribosomes from tomograms of cryoFIB-SEM-generated lamellae of yeast cells under heat shock stress.
Map dataSubtomogram average of ribosomes from tomograms of cryoFIB-SEM-generated lamellae of yeast cells under heat shock stress.
Sample
  • Complex: Yeast ribosome subtomogram average
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsubtomogram averaging / cryo EM / Resolution: 21.0 Å
AuthorsWu GH / Mitchell PG / Galaz-Montoya JG / Hecksel CW / Sontag EM / Gangadharan V / Marshman J / Mankus D / Bisher ME / Lytton-Jean AKR ...Wu GH / Mitchell PG / Galaz-Montoya JG / Hecksel CW / Sontag EM / Gangadharan V / Marshman J / Mankus D / Bisher ME / Lytton-Jean AKR / Frydman J / Czymmek K / Chiu W
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)P01NS092525 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5P41GM103832 United States
National Institutes of Health/National Institute on Aging (NIH/NIA)PO1AG054407 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)F32NS086253 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021600 United States
CitationJournal: Structure / Year: 2020
Title: Multi-scale 3D Cryo-Correlative Microscopy for Vitrified Cells.
Authors: Gong-Her Wu / Patrick G Mitchell / Jesus G Galaz-Montoya / Corey W Hecksel / Emily M Sontag / Vimal Gangadharan / Jeffrey Marshman / David Mankus / Margaret E Bisher / Abigail K R Lytton- ...Authors: Gong-Her Wu / Patrick G Mitchell / Jesus G Galaz-Montoya / Corey W Hecksel / Emily M Sontag / Vimal Gangadharan / Jeffrey Marshman / David Mankus / Margaret E Bisher / Abigail K R Lytton-Jean / Judith Frydman / Kirk Czymmek / Wah Chiu /
Abstract: Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging ...Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labeled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-hydrated specimens in a variety of healthy and diseased conditions with and without treatments.
History
DepositionMay 10, 2020-
Header (metadata) releaseSep 2, 2020-
Map releaseSep 2, 2020-
UpdateSep 2, 2020-
Current statusSep 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21952.png

Downloads & links

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Map

FileDownload / File: emd_21952.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of ribosomes from tomograms of cryoFIB-SEM-generated lamellae of yeast cells under heat shock stress.
Voxel sizeX=Y=Z: 3.458 Å
Density
Contour LevelBy AUTHOR: 1 / Movie #1: 1
Minimum - Maximum-0.7923188 - 7.688226
Average (Standard dev.)0.15580522 (±0.67541677)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 442.624 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.4583.4583.458
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z442.624442.624442.624
α/β/γ90.00090.00090.000
start NX/NY/NZ13112264
NX/NY/NZ123151209
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.7927.6880.156

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Supplemental data

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Half map: Subtomogram average of ribosomes from tomograms of cryoFIB-SEM-generated...

Fileemd_21952_half_map_1.map
AnnotationSubtomogram average of ribosomes from tomograms of cryoFIB-SEM-generated lamellae of yeast cells under heat shock stress. EVEN MAP.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Subtomogram average of ribosomes from tomograms of cryoFIB-SEM-generated...

Fileemd_21952_half_map_2.map
AnnotationSubtomogram average of ribosomes from tomograms of cryoFIB-SEM-generated lamellae of yeast cells under heat shock stress. ODD 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 : Yeast ribosome subtomogram average

EntireName: Yeast ribosome subtomogram average
Components
  • Complex: Yeast ribosome subtomogram average

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Supramolecule #1: Yeast ribosome subtomogram average

SupramoleculeName: Yeast ribosome subtomogram average / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BY4741

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 0.8264 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 5 / Number images used: 3633 / Method: manual / Software - Name: EMAN2
CTF correctionSoftware - Name: IMOD / Details: 3D CTF correction
Final 3D classificationNumber classes: 1 / Avg.num./class: 1459
Final angle assignmentType: NOT APPLICABLE / Software - Name: IMOD
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 / Number subtomograms used: 1459

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