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- EMDB-12959: Cryo-tomogram of the K5/K14 keratin network in a keratinocyte gho... -

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Basic information

Entry
Database: EMDB / ID: EMD-12959
TitleCryo-tomogram of the K5/K14 keratin network in a keratinocyte ghost cell
Map dataCryo-tomogram of the K5/K14 keratin network in a keratinocyte ghost cell.
Sample
  • Cell: Keratin network in a detergent permeabilized K5/K14_1 cell
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsWeber MS / Eibauer M / Medalia O
Funding support Switzerland, 2 items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A, 179418 Switzerland
University of ZurichFK-18-041 Switzerland
CitationJournal: Elife / Year: 2021
Title: Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy.
Authors: Miriam S Weber / Matthias Eibauer / Suganya Sivagurunathan / Thomas M Magin / Robert D Goldman / Ohad Medalia /
Abstract: Keratin intermediate filaments are an essential and major component of the cytoskeleton in epithelial cells. They form a stable yet dynamic filamentous network extending from the nucleus to the cell ...Keratin intermediate filaments are an essential and major component of the cytoskeleton in epithelial cells. They form a stable yet dynamic filamentous network extending from the nucleus to the cell periphery, which provides resistance to mechanical stresses. Mutations in keratin genes are related to a variety of epithelial tissue diseases. Despite their importance, the molecular structure of keratin filaments remains largely unknown. In this study, we analyzed the structure of keratin 5/keratin 14 filaments within ghost mouse keratinocytes by cryo-electron microscopy and cryo-electron tomography. By averaging a large number of keratin segments, we have gained insights into the helical architecture of the filaments. Two-dimensional classification revealed profound variations in the diameter of keratin filaments and their subunit organization. Computational reconstitution of filaments of substantial length uncovered a high degree of internal heterogeneity along single filaments, which can contain regions of helical symmetry, regions with less symmetry and regions with significant diameter fluctuations. Cross-section views of filaments revealed that keratins form hollow cylinders consisting of multiple protofilaments, with an electron dense core located in the center of the filament. These findings shed light on the complex and remarkable heterogenic architecture of keratin filaments, suggesting that they are highly flexible, dynamic cytoskeletal structures.
History
DepositionMay 17, 2021-
Header (metadata) releaseAug 18, 2021-
Map releaseAug 18, 2021-
UpdateAug 25, 2021-
Current statusAug 25, 2021Processing site: PDBe / 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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Supplemental images

emd_12959.png

Downloads & links

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Map

FileDownload / File: emd_12959.map.gz / Format: CCP4 / Size: 318.6 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationCryo-tomogram of the K5/K14 keratin network in a keratinocyte ghost cell.
Voxel sizeX=Y=Z: 7.00289 Å
Density
Minimum - Maximum-128.0 - 126.0
Average (Standard dev.)-89.95236 (±6.2312665)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions928960375
Spacing960928375
CellA: 6722.7695 Å / B: 6498.6772 Å / C: 2626.0818 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z7.00288541666677.00288469827597.0028853333333
M x/y/z960928375
origin x/y/z0.0000.0000.000
length x/y/z6722.7706498.6772626.082
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ250250250
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS960928375
D min/max/mean-128.000126.000-89.952

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Supplemental data

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Sample components

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Entire : Keratin network in a detergent permeabilized K5/K14_1 cell

EntireName: Keratin network in a detergent permeabilized K5/K14_1 cell
Components
  • Cell: Keratin network in a detergent permeabilized K5/K14_1 cell

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Supramolecule #1: Keratin network in a detergent permeabilized K5/K14_1 cell

SupramoleculeName: Keratin network in a detergent permeabilized K5/K14_1 cell
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse) / Organ: Skin / Tissue: Epidermis

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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
GridDetails: Cells were grown on grids overnight at 32 degree and 5% CO2 and permeabilized and plunge frozen on the next day.
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Aurion, Netherlands / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 28571
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 0.8 sec. / Average electron dose: 2.17 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: IMOD (ver. 4.10.51)
Final reconstructionSoftware - Name: IMOD (ver. 4.10.51) / Number images used: 41

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