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- EMDB-8174: CryoET of U2OS Cells and Associated Manual Segmentation of Mitoch... -

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Basic information

Entry
Database: EMDB / ID: EMD-8174
TitleCryoET of U2OS Cells and Associated Manual Segmentation of Mitochondria
Map dataU2OS Cells
Sample
  • Cell: Mitochondria from U2OS Cells
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsHecksel CW / Darrow MC / Dai W / Galaz-Montoya JG / Chin JA / Mitchell PG / Chen S / Jakana J / Schmid MF / Chiu W
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103832 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM079429 United States
National Institutes of Health/National Eye Institute (NIH/NEI)PN2EY016525 United States
Ovarian Cancer Research Fund5-258813 United States
Robert A. Welch FoundationQ1242 United States
CitationJournal: Microsc Microanal / Year: 2016
Title: Quantifying Variability of Manual Annotation in Cryo-Electron Tomograms.
Authors: Corey W Hecksel / Michele C Darrow / Wei Dai / Jesús G Galaz-Montoya / Jessica A Chin / Patrick G Mitchell / Shurui Chen / Jemba Jakana / Michael F Schmid / Wah Chiu /
Abstract: Although acknowledged to be variable and subjective, manual annotation of cryo-electron tomography data is commonly used to answer structural questions and to create a "ground truth" for evaluation ...Although acknowledged to be variable and subjective, manual annotation of cryo-electron tomography data is commonly used to answer structural questions and to create a "ground truth" for evaluation of automated segmentation algorithms. Validation of such annotation is lacking, but is critical for understanding the reproducibility of manual annotations. Here, we used voxel-based similarity scores for a variety of specimens, ranging in complexity and segmented by several annotators, to quantify the variation among their annotations. In addition, we have identified procedures for merging annotations to reduce variability, thereby increasing the reliability of manual annotation. Based on our analyses, we find that it is necessary to combine multiple manual annotations to increase the confidence level for answering structural questions. We also make recommendations to guide algorithm development for automated annotation of features of interest.
History
DepositionMay 9, 2016-
Header (metadata) releaseJun 8, 2016-
Map releaseJun 8, 2016-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Simplified surface model
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8174.png

Downloads & links

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Map

FileDownload / File: emd_8174.map.gz / Format: CCP4 / Size: 16.3 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationU2OS Cells
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
18.21 Å/pix.
x 71 pix.
= 1292.91 Å		18.21 Å/pix.
x 401 pix.
= 7302.209 Å		18.21 Å/pix.
x 301 pix.
= 5481.21 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 18.21 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-1414 - 1509
Average (Standard dev.)39.242565 (±271.32428)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions40130171
Spacing30140171
CellA: 5481.21 Å / B: 7302.2095 Å / C: 1292.9099 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z18.2118.20999750623418.21
M x/y/z30140171
origin x/y/z0.0000.0000.000
length x/y/z5481.2107302.2091292.910
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS30140171
D min/max/mean-1414.0001509.00039.243

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Supplemental data

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Mask #1

Fileemd_8174_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_8174_msk_2.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Mask #3

Fileemd_8174_msk_3.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #4

Fileemd_8174_msk_4.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Mitochondria from U2OS Cells

EntireName: Mitochondria from U2OS Cells
Components
  • Cell: Mitochondria from U2OS Cells

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Supramolecule #1: Mitochondria from U2OS Cells

SupramoleculeName: Mitochondria from U2OS Cells / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Tissue: Bone

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4 / Details: PBS
GridModel: Quantifoil SiO2 R1/4 / Material: GOLD / Mesh: 200 / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 21 K / Instrument: LEICA EM GP / Details: Plunged into liquid ethane (LEICA EM GP).
DetailsIntact mammalian cells
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: EMS / Diameter: 15 nm

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Electron microscopy

MicroscopeJEOL 2100
TemperatureMin: 98.0 K / Max: 98.0 K
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average exposure time: 1.0 sec. / Average electron dose: 2.6 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal magnification: 12000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD
Details: Final reconstruction was binned by two before segmentation.
Number images used: 27

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