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- EMDB-4045: 13-protofilament microtubule structure determined in situ from U2... -

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Basic information

Entry
Database: EMDB / ID: EMD-4045
Title13-protofilament microtubule structure determined in situ from U2OS cell
Map data
Sample
  • Cell: Microtubule
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 25.0 Å
AuthorsGrange M / Vasishtan D / Gruenewald K
CitationJournal: J Struct Biol / Year: 2017
Title: Cellular electron cryo tomography and in situ sub-volume averaging reveal the context of microtubule-based processes.
Authors: Michael Grange / Daven Vasishtan / Kay Grünewald /
Abstract: Electron cryo-tomography (cryoET) is currently the only technique that allows the direct observation of proteins in their native cellular environment. Sub-volume averaging of electron tomograms ...Electron cryo-tomography (cryoET) is currently the only technique that allows the direct observation of proteins in their native cellular environment. Sub-volume averaging of electron tomograms offers a route to increase the signal-to-noise of repetitive biological structures, such improving the information content and interpretability of tomograms. We discuss the potential for sub-volume averaging in highlighting and investigating specific processes in situ, focusing on microtubule structure and viral infection. We show that (i) in situ sub-volume averaging from single tomograms can guide and complement segmentation of biological features, (ii) the in situ determination of the structure of individual viruses is possible as they infect a cell, and (iii) novel, transient processes can be imaged with high levels of detail.
History
DepositionJun 30, 2016-
Header (metadata) releaseJul 13, 2016-
Map releaseJul 13, 2016-
UpdateAug 30, 2017-
Current statusAug 30, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4045.png

Downloads & links

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Map

FileDownload / File: emd_4045.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.6 Å/pix.
x 72 pix.
= 331.2 Å		4.6 Å/pix.
x 72 pix.
= 331.2 Å		4.6 Å/pix.
x 72 pix.
= 331.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.6 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2. / Movie #1: 2
Minimum - Maximum-6.9646897 - 6.982263
Average (Standard dev.)-0.46713483 (±2.4142387)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions727272
Spacing727272
CellA=B=C: 331.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.64.64.6
M x/y/z727272
origin x/y/z0.0000.0000.000
length x/y/z331.200331.200331.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS727272
D min/max/mean-6.9656.982-0.467

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Supplemental data

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Sample components

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Entire : Microtubule

EntireName: Microtubule
Components
  • Cell: Microtubule

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Supramolecule #1: Microtubule

SupramoleculeName: Microtubule / type: cell / ID: 1 / Parent: 0 / Details: microtubule from U2OS
Source (natural)Organism: Homo sapiens (human) / Strain: U2OS / Tissue: Cell

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statefilament

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Sample preparation

BufferpH: 7 / Details: Cell
GridModel: C-flat 2/2 holey carbon gold supported grids (EMS) / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 60 % / Chamber temperature: 298 K / Instrument: HOMEMADE PLUNGER
Details: Adherent cells grown on electron microscopy grid, manually blotted for 3s and plunge frozen.

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 77.0 K / Max: 110.0 K
Specialist opticsEnergy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3836 pixel / Average electron dose: 2.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 5.0 µm / Calibrated defocus min: 5.0 µm / Calibrated magnification: 21739 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 95000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 9.2 Å
Applied symmetry - Helical parameters - Δ&Phi: 27.6923 °
Applied symmetry - Helical parameters - Axial symmetry: C13 (13 fold cyclic)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: PEET (ver. 1.10.0 Patch 3) / Number subtomograms used: 1924
ExtractionNumber tomograms: 1 / Number images used: 1924 / Software - Name: IMOD (ver. 4.8.41) / Software - details: 3dmod
CTF correctionSoftware - Name: IMOD (ver. 4.8.41) / Software - details: ctfplotter
Final angle assignmentType: NOT APPLICABLE / Software - Name: PEET (ver. 1.10.0 Patch 3)
FSC plot (resolution estimation)

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