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- EMDB-8173: CryoET of MEF Cells and Associated Manual Segmentation of Microtu... -

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Basic information

Entry
Database: EMDB / ID: EMD-8173
TitleCryoET of MEF Cells and Associated Manual Segmentation of Microtubule and Actin
Map dataMouse Embryonic Fibroblast Cells
Sample
  • Cell: Microtubule and Actin from Mouse Embryonic Fibroblast Cells
Biological speciesMus musculus (house mouse)
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 10, 2016-
Header (metadata) releaseJun 8, 2016-
Map releaseJun 8, 2016-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

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  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8173.png

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Map

FileDownload / File: emd_8173.map.gz / Format: CCP4 / Size: 28.4 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationMouse Embryonic Fibroblast Cells
Voxel sizeX=Y=Z: 18.21 Å
Density
Minimum - Maximum-28525 - 21216
Average (Standard dev.)201.44069 (±2240.8333)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions42041286
Spacing41242086
CellA: 7502.5195 Å / B: 7648.1997 Å / C: 1566.0599 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z18.2118.2118.21
M x/y/z41242086
origin x/y/z0.0000.0000.000
length x/y/z7502.5207648.2001566.060
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/NS41242086
D min/max/mean-28525.00021216.000201.441

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Supplemental data

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Sample components

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Entire : Microtubule and Actin from Mouse Embryonic Fibroblast Cells

EntireName: Microtubule and Actin from Mouse Embryonic Fibroblast Cells
Components
  • Cell: Microtubule and Actin from Mouse Embryonic Fibroblast Cells

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Supramolecule #1: Microtubule and Actin from Mouse Embryonic Fibroblast Cells

SupramoleculeName: Microtubule and Actin from Mouse Embryonic Fibroblast Cells
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse) / Tissue: embryonic fibroblast

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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
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
5.0 mMKClpotassium chloride
1.0 mMCaCl2calcium chloride
1.0 mMMgCl2magnesium chloride
20.0 mMC8H18N2O4SHEPES
25.0 uMRho kinase inhibitor (Y27632)

Details: PBS
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
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
Cryo protectantRinger's Solution
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: EMS / Diameter: 15 nm

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Electron microscopy

MicroscopeJEOL 2100
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 12000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
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

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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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