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- EMDB-11869: Structure of the actin filament Arp2/3 complex branch junction in... -

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Basic information

Entry
Database: EMDB / ID: EMD-11869
TitleStructure of the actin filament Arp2/3 complex branch junction in cells
Map data
SampleActin Filament Arp2/3 Complex Branch Junction:
(Actin-related protein ...) x 7 / Actin, alpha skeletal muscle, ACTA1
Biological speciesMus musculus (house mouse) / House mouse (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 9 Å
AuthorsFaessler F / Dimchev G / Hodirnau VV / Wan W / Schur FKM
Funding support Austria, 1 items
OrganizationGrant numberCountry
Austrian Science FundP33367 Austria
CitationJournal: Nat Commun / Year: 2020
Title: Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction.
Authors: Florian Fäßler / Georgi Dimchev / Victor-Valentin Hodirnau / William Wan / Florian K M Schur /
Abstract: The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves ...The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0 Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Notably, our model reveals a previously undescribed set of interactions of the Arp2/3 complex with the mother filament, significantly different to the previous branch junction model. Our structure also indicates a central role for the ArpC3 subunit in stabilizing the active conformation.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionOct 22, 2020-
Header (metadata) releaseDec 2, 2020-
Map releaseDec 2, 2020-
UpdateJan 6, 2021-
Current statusJan 6, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00404
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.00404
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7aqk
  • Surface level: 0.00404
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7aqk
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11869.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.14 Å/pix.
x 240 pix.
= 512.88 Å
2.14 Å/pix.
x 240 pix.
= 512.88 Å
2.14 Å/pix.
x 240 pix.
= 512.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.137 Å
Density
Contour LevelBy AUTHOR: 0.00404 / Movie #1: 0.00404
Minimum - Maximum-0.0034114548 - 0.014956028
Average (Standard dev.)3.46011e-05 (±0.00058454473)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 512.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.1372.1372.137
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z512.880512.880512.880
α/β/γ90.00090.00090.000
start NX/NY/NZ79740
NX/NY/NZ93103213
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0030.0150.000

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Supplemental data

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Segmentation: #1

Fileemd_11869_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 1

Fileemd_11869_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 2

Fileemd_11869_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Actin Filament Arp2/3 Complex Branch Junction

EntireName: Actin Filament Arp2/3 Complex Branch Junction
Details: Structure obtained from the actin network of extracted and fixed mouse fibroblast lamellipodia
Number of components: 9

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Component #1: cellular-component, Actin Filament Arp2/3 Complex Branch Junction

Cellular-componentName: Actin Filament Arp2/3 Complex Branch Junction
Details: Structure obtained from the actin network of extracted and fixed mouse fibroblast lamellipodia
Recombinant expression: No
SourceSpecies: Mus musculus (house mouse)

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Component #2: protein, Actin-related protein 2, Arp2

ProteinName: Actin-related protein 2, Arp2
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 44.818711 kDa
SourceSpecies: House mouse (house mouse)

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Component #3: protein, Actin-related protein 3, Arp3

ProteinName: Actin-related protein 3, Arp3
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 47.428031 kDa
SourceSpecies: House mouse (house mouse)

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Component #4: protein, Actin-related protein 2/3 complex subunit 1b, ArpC1b

ProteinName: Actin-related protein 2/3 complex subunit 1b, ArpC1b
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 41.016738 kDa
SourceSpecies: House mouse (house mouse)

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Component #5: protein, Actin-related protein 2/3 complex subunit 2, ArpC2

ProteinName: Actin-related protein 2/3 complex subunit 2, ArpC2
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 34.402043 kDa
SourceSpecies: House mouse (house mouse)

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Component #6: protein, Actin-related protein 2/3 complex subunit 3, ArpC3

ProteinName: Actin-related protein 2/3 complex subunit 3, ArpC3
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 20.572666 kDa
SourceSpecies: House mouse (house mouse)

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Component #7: protein, Actin-related protein 2/3 complex subunit 4, ArpC4

ProteinName: Actin-related protein 2/3 complex subunit 4, ArpC4
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 19.697047 kDa
SourceSpecies: House mouse (house mouse)

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Component #8: protein, Actin-related protein 2/3 complex subunit 5, ArpC5

ProteinName: Actin-related protein 2/3 complex subunit 5, ArpC5
Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they ...Details: As models derived from Bos taurus Arp2/3 complexes have been used for fitting, also the Bos taurus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 16.295317 kDa
SourceSpecies: House mouse (house mouse)

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Component #9: protein, Actin, alpha skeletal muscle, ACTA1

ProteinName: Actin, alpha skeletal muscle, ACTA1
Details: As models derived from Oryctolagus cuniculus actin have been used for fitting, also the Oryctolagus cuniculus sequence (as well as the corresponding UniProt identifier) is given here, even ...Details: As models derived from Oryctolagus cuniculus actin have been used for fitting, also the Oryctolagus cuniculus sequence (as well as the corresponding UniProt identifier) is given here, even though they were fit into a mouse structure.
Number of Copies: 11 / Recombinant expression: No
MassTheoretical: 42.096953 kDa
SourceSpecies: House mouse (house mouse)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Cell / Method: cryo EM
Sample solutionBuffer solution: Adjust to pH 6.1 using NaOH / pH: 6.1
Support filmAfter glow discharging of the grid and prior to the seeding of cells, the grid was coated using 25ug/ml Fibronectin
VitrificationCryogen name: ETHANE / Temperature: 277 K / Humidity: 80 %
Details: Leica GP2, 3,5sec back-blotting, sensor on, 0,1mm movement after contact, manually pre-blotted within the chamber prior to the application of fiducials.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 2.79 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 42000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: -1.75 - -5.5 nm / Energy filter: GIF Bioquantum
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: OTHER

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Image acquisition

Image acquisitionDetails: Images were collected in movie-mode at 7 frames per tilt

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Image processing

ProcessingMethod: subtomogram averaging / Applied symmetry: C1 (asymmetric) / Number of subtomograms: 14296 / Number of class averages: 1
3D reconstructionSoftware: RELION / Resolution: 9 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Final reconstruction in RELION was performed after Multiple particle refinement in M version 1.0.9.
Euler angles: Multiple particle refinement in M version 1.0.9 was performed after 3D Refinement in RELION.
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
Input PDB model: 1TYQ, 1TYQ, 1TYQ, 1TYQ, 1TYQ, 1TYQ, 1TYQ, 1TYQ, 1TYQ, 1TYQ, 1TYQ, 1TYQ, 4JD2, 4JD2, 1K8K, 6T20, 6T20, 6T20, 6T20, 6T20
Chain ID: A, A, A, B, C, C, D, D, E, E, F, G, B, B, C, A, B, C, D, E
Output model

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