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- EMDB-20770: CryoEM structure of human Arp2/3 complex with bound NPFs -

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Entry
Database: EMDB / ID: EMD-20770
TitleCryoEM structure of human Arp2/3 complex with bound NPFs
Map data
SampleCryo-EM structure of human Arp2/3 complex with bound NPFs:
(Actin-related protein ...) x 7 / Neural Wiskott-Aldrich syndrome protein / (ligand) x 2
Function / homology
Function and homology information


cytosolic transport / microtubule organizing center localization / growth cone leading edge / tubulobulbar complex / podosome core / meiotic chromosome movement towards spindle pole / muscle cell projection membrane / meiotic cytokinesis / lamellipodium organization / negative regulation of membrane tubulation ...cytosolic transport / microtubule organizing center localization / growth cone leading edge / tubulobulbar complex / podosome core / meiotic chromosome movement towards spindle pole / muscle cell projection membrane / meiotic cytokinesis / lamellipodium organization / negative regulation of membrane tubulation / spindle localization / membrane invagination / Arp2/3 protein complex / cellular response to trichostatin A / Arp2/3 complex-mediated actin nucleation / positive regulation of clathrin-dependent endocytosis / actin polymerization-dependent cell motility / positive regulation of Arp2/3 complex-mediated actin nucleation / actin filament network formation / asymmetric cell division / actin cortical patch assembly / plasma membrane tubulation / actin cortical patch localization / negative regulation of lymphocyte migration / orbitofrontal cortex development / invadopodium / vesicle transport along actin filament / postsynaptic actin cytoskeleton organization / vesicle organization / actin cap / vesicle budding from membrane / positive regulation of actin nucleation / actin filament-based movement / positive regulation of dendritic spine morphogenesis / maintenance of cell polarity / cellular protein-containing complex localization / positive regulation of double-strand break repair via homologous recombination / actin nucleation / dendritic spine morphogenesis / positive regulation of filopodium assembly / positive regulation of lamellipodium assembly / smooth muscle cell migration / brush border / establishment or maintenance of cell polarity / filamentous actin / cilium assembly / cell leading edge / associative learning / positive regulation of substrate adhesion-dependent cell spreading / cellular response to nerve growth factor stimulus / actin filament polymerization / cytoskeletal protein binding / response to immobilization stress / cell projection / actin filament / cellular response to interferon-gamma / response to bacterium / regulation of protein localization / structural constituent of cytoskeleton / ephrin receptor signaling pathway / actin cytoskeleton organization / site of double-strand break / postsynapse / azurophil granule lumen / endocytosis / actin cytoskeleton / Fc-gamma receptor signaling pathway involved in phagocytosis / actin filament binding / growth cone / cell-cell junction / lamellipodium / cell migration / actin binding / membrane organization / response to estradiol / cytoplasmic vesicle / regulation of cell shape / response to ethanol / secretory granule lumen / ficolin-1-rich granule lumen / response to estrogen / postsynaptic density / endosome / cell division / Golgi membrane / neuron projection / glutamatergic synapse / focal adhesion / protein-containing complex binding / neutrophil degranulation / enzyme binding / positive regulation of transcription by RNA polymerase II / go:0005623: / extracellular exosome / membrane / extracellular region / ATP binding / identical protein binding / nucleus / cytosol
Actin-related protein 2/3 complex subunit 4 / WH2 domain / Actin-related protein 2/3 complex subunit 1B / PH-like domain superfamily / Wiscott-Aldrich syndrome protein, C-terminal / Actin-related protein 2 / CRIB domain / WH1/EVH1 domain / Actin-related protein 3 / Actin/actin-like conserved site ...Actin-related protein 2/3 complex subunit 4 / WH2 domain / Actin-related protein 2/3 complex subunit 1B / PH-like domain superfamily / Wiscott-Aldrich syndrome protein, C-terminal / Actin-related protein 2 / CRIB domain / WH1/EVH1 domain / Actin-related protein 3 / Actin/actin-like conserved site / WD40-repeat-containing domain / Actin-related protein 2/3 complex subunit 1 / WD40 repeat / WASP family, EVH1 domain / WD40/YVTN repeat-like-containing domain superfamily / Actin family / Arp2/3 complex subunit 2/4 / Actin-related protein 2/3 complex subunit 5 / WD40-repeat-containing domain superfamily / Actin-related protein 2/3 complex subunit 5 superfamily / Actin-related protein 2/3 complex subunit 3 superfamily / CRIB domain superfamily / ATPase, nucleotide binding domain / Actin-related protein 2/3 complex subunit 2 / Actin-related protein 2/3 complex subunit 3 / Neural Wiskott-Aldrich syndrome protein
Actin-related protein 2/3 complex subunit 1B / Actin-related protein 2/3 complex subunit 2 / Actin-related protein 2/3 complex subunit 3 / Actin-related protein 2/3 complex subunit 5 / Actin-related protein 2/3 complex subunit 4 / Actin-related protein 3 / Actin-related protein 2 / Neural Wiskott-Aldrich syndrome protein
Biological speciesSpodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths) / Homo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsZimmet A / van Eeuwen T / Dominguez R
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM07391 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)F31-HL146077 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32-GM008275 United States
CitationJournal: Sci Adv / Year: 2020
Title: Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism.
Authors: Austin Zimmet / Trevor Van Eeuwen / Malgorzata Boczkowska / Grzegorz Rebowski / Kenji Murakami / Roberto Dominguez /
Abstract: Actin-related protein (Arp) 2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including two actin-related subunits (Arp2 and Arp3). Two nucleation- ...Actin-related protein (Arp) 2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including two actin-related subunits (Arp2 and Arp3). Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions, and contribution of each NPF to activation are unresolved. Here, we report the cryo-electron microscopy structure of recombinantly expressed human Arp2/3 complex with two WASP family NPFs bound and address the mechanism of activation. A cross-linking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex.
Validation ReportPDB-ID: 6uhc

SummaryFull reportAbout validation report
History
DepositionSep 27, 2019-
Header (metadata) releaseNov 20, 2019-
Map releaseJul 1, 2020-
UpdateJul 1, 2020-
Current statusJul 1, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.475
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.475
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6uhc
  • Surface level: 0.475
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20770.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 320 pix.
= 267.52 Å
0.84 Å/pix.
x 320 pix.
= 267.52 Å
0.84 Å/pix.
x 320 pix.
= 267.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.836 Å
Density
Contour LevelBy AUTHOR: 0.475 / Movie #1: 0.475
Minimum - Maximum-1.97515 - 3.0070715
Average (Standard dev.)0.00638433 (±0.08849225)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 267.52002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8360.8360.836
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z267.520267.520267.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-1.9753.0070.006

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Supplemental data

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Additional map: Cryo-EM structure of human Arp2/3 complex bound to...

Fileemd_20770_additional.map
AnnotationCryo-EM structure of human Arp2/3 complex bound to two nucleation promoting factors
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Cryo-EM structure of human Arp2/3 complex with bound NPFs

EntireName: Cryo-EM structure of human Arp2/3 complex with bound NPFs
Number of components: 11

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Component #1: protein, Cryo-EM structure of human Arp2/3 complex with bound NPFs

ProteinName: Cryo-EM structure of human Arp2/3 complex with bound NPFs
Recombinant expression: No
MassTheoretical: 243 MDa
SourceSpecies: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
Source (engineered)Expression System: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
Vector: pBIG2abc

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Component #2: protein, Actin-related protein 3

ProteinName: Actin-related protein 3 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 47.428031 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)

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Component #3: protein, Actin-related protein 2

ProteinName: Actin-related protein 2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 44.818711 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)

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

ProteinName: Actin-related protein 2/3 complex subunit 1B / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 41.004781 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)

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

ProteinName: Actin-related protein 2/3 complex subunit 2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 34.386043 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)

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

ProteinName: Actin-related protein 2/3 complex subunit 3 / Details: C-terminal Flag tag / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 23.19633 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)

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

ProteinName: Actin-related protein 2/3 complex subunit 4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 19.697047 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)

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

ProteinName: Actin-related protein 2/3 complex subunit 5 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 16.341407 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)

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Component #9: protein, Neural Wiskott-Aldrich syndrome protein

ProteinName: Neural Wiskott-Aldrich syndrome protein / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 54.342023 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #10: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #11: ligand, ADENOSINE-5'-TRIPHOSPHATE

LigandName: ADENOSINE-5'-TRIPHOSPHATE / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.507181 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 5 mg/mL / pH: 7
Support filmGrids glow discharged with easiGLOW glow discharger at 0.3 mBar, 25mA for 1 minute
VitrificationInstrument: LEICA EM CPC / Cryogen name: ETHANE
Details: Grids manually blotted for 3 seconds with Whatman 41 filter paper and manually plunged on Leica EM CPC manual plunger..

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Details: Data collected in super resolution mode. Illuminated area of 1.01um. Nominal Dose of 40a/A^2 and a dose rate of 4.87 e-/s/pixel. 2 or 5 exposures per hole by image shift.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: SPOT SCAN
LensMagnification: 165000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: -1500.0 - -3500.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 5004

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 123198
3D reconstructionAlgorithm: FOURIER SPACE / Software: cryoSPARC / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target criteria: correlation / Refinement space: REAL
Input PDB model: 4JD2
Overall bvalue: 132
Output model

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