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- EMDB-15140: In-cell structure of the actin filament Arp2/3 complex branch jun... -

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Basic information

Entry
Database: EMDB / ID: EMD-15140
TitleIn-cell structure of the actin filament Arp2/3 complex branch junction in lamellipodia of ArpC5L knockout B16-F1 mouse melanoma cells
Map dataStructure of the Arp2/3 actin branch junction as found in ArpC5L knockout B16-F1 mouse melanoma cell lamellipodia
Sample
  • Complex: In-cell structure of the actin filament Arp2/3 complex branch junction in lamellipodia of ArpC5L knockout B16-F1 mouse melanoma cells
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 7.8 Å
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_15140.png

Downloads & links

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Map

FileDownload / File: emd_15140.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the Arp2/3 actin branch junction as found in ArpC5L knockout B16-F1 mouse melanoma cell lamellipodia
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
1.69 Å/pix.
x 240 pix.
= 406.32 Å		1.69 Å/pix.
x 240 pix.
= 406.32 Å		1.69 Å/pix.
x 240 pix.
= 406.32 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.693 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00219
Minimum - Maximum-0.006911459 - 0.015045546
Average (Standard dev.)3.5172292e-05 (±0.00041575587)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 406.31998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15140_msk_1.map
Projections & Slices
AxesZYX

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Half map: Halfmap 1 after subtomogram averaging and multi-particle refinemnt

Fileemd_15140_half_map_1.map
AnnotationHalfmap 1 after subtomogram averaging and multi-particle refinemnt
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap 2 after subtomogram averaging and multi-particle refinemnt

Fileemd_15140_half_map_2.map
AnnotationHalfmap 2 after subtomogram averaging and multi-particle refinemnt
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 : In-cell structure of the actin filament Arp2/3 complex branch jun...

EntireName: In-cell structure of the actin filament Arp2/3 complex branch junction in lamellipodia of ArpC5L knockout B16-F1 mouse melanoma cells
Components
  • Complex: In-cell structure of the actin filament Arp2/3 complex branch junction in lamellipodia of ArpC5L knockout B16-F1 mouse melanoma cells

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Supramolecule #1: In-cell structure of the actin filament Arp2/3 complex branch jun...

SupramoleculeName: In-cell structure of the actin filament Arp2/3 complex branch junction in lamellipodia of ArpC5L knockout B16-F1 mouse melanoma cells
type: complex / ID: 1 / Chimera: Yes / Parent: 0
Source (natural)Organism: Mus musculus (house mouse) / Strain: B16-F1 / Tissue: Melanoma / Location in cell: Lamellipodium

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
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: The grids were coated with 25ug/ml laminin for 1 hour prior to seeding the cells
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277 K / Instrument: LEICA EM GP

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Electron microscopy

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

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Image processing

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number subtomograms used: 9831
ExtractionNumber tomograms: 61 / Number images used: 24400 / Reference model: Generated from manually selected particles / Method: Template matching / Software: (Name: Dynamo (ver. 1.1.333), Warp (ver. 1.0.9))
Final 3D classificationNumber classes: 1 / Avg.num./class: 9831 / Software - Name: RELION (ver. 3.0.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
FSC plot (resolution estimation)

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