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- EMDB-15137: Tomogram of a lamellipodium of a WT B16-F1 mouse melanoma cell -

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Basic information

Entry
Database: EMDB / ID: EMD-15137
TitleTomogram of a lamellipodium of a WT B16-F1 mouse melanoma cell
Map dataTomogram of a lamellipodium of a WT B16-F1 mouse melanoma cell
Sample
  • Cell: Tomogram of a lamellipodium of a WT B16-F1 mouse melanoma cell
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsFaessler F / Javoor MG / Datler J / Doering H / Hofer FW / Dimchev G / Hodirnau VV / Rottner K / Schur FKM
Funding support Austria, 1 items
OrganizationGrant numberCountry
Austrian Science FundP33367 Austria
CitationJournal: Sci Adv / Year: 2023
Title: ArpC5 isoforms regulate Arp2/3 complex-dependent protrusion through differential Ena/VASP positioning.
Authors: Florian Fäßler / Manjunath G Javoor / Julia Datler / Hermann Döring / Florian W Hofer / Georgi Dimchev / Victor-Valentin Hodirnau / Jan Faix / Klemens Rottner / Florian K M Schur /
Abstract: Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. ...Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. We have combined reverse genetics and cellular structural biology to describe how ArpC5 and ArpC5L differentially affect cell migration. Both define the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, affect protein dynamics and actin network ultrastructure. ArpC5 isoforms also affect the positioning of members of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongators, which mediate ArpC5 isoform-specific effects on the actin assembly level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling pathway enhancing cell migration.
History
DepositionJun 10, 2022-
Header (metadata) releaseFeb 1, 2023-
Map releaseFeb 1, 2023-
UpdateFeb 1, 2023-
Current statusFeb 1, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15137.png

Downloads & links

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Map

FileDownload / File: emd_15137.map.gz / Format: CCP4 / Size: 281.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomogram of a lamellipodium of a WT B16-F1 mouse melanoma cell
Voxel sizeX=Y=Z: 13.544 Å
Density
Minimum - Maximum-0.49811822 - 0.47268265
Average (Standard dev.)1.7543996e-05 (±0.052847598)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions720512200
Spacing512720200
CellA: 6934.528 Å / B: 9751.68 Å / C: 2708.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Tomogram of a lamellipodium of a WT B16-F1 mouse melanoma cell

EntireName: Tomogram of a lamellipodium of a WT B16-F1 mouse melanoma cell
Components
  • Cell: Tomogram of a lamellipodium of a WT B16-F1 mouse melanoma cell

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Supramolecule #1: Tomogram of a lamellipodium of a WT B16-F1 mouse melanoma cell

SupramoleculeName: Tomogram of a lamellipodium of a WT B16-F1 mouse melanoma cell
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse) / Strain: B16-F1 / Tissue: Melanoma

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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: 6.2
Component:
ConcentrationName
10.0 mMMES
150.0 mMSodium
5.0 mMEGTA
5.0 mMGlucose
5.0 mMMagnedium chloride
200.0 mMGlutaraldehyde

Details: Adjust to pH 6.2 using NaOH Immediately prior to use, add Phalloidin to a final concentration of 1ug/ml
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR
Details: After glow discharging of the grid and prior to the seeding of cells, the grid was coated using 25ug/ml Laminin for 60min
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277 K / Instrument: LEICA EM GP
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Aurion / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 53000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 67 / Average electron dose: 2.79 e/Å2
Details: Images were collected in movie-mode with 8 frames per tilt
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: RELION (ver. 3.0.8) / Number images used: 65

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