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- PDB-6uhc: CryoEM structure of human Arp2/3 complex with bound NPFs -

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Basic information

Entry
Database: PDB / ID: 6uhc
TitleCryoEM structure of human Arp2/3 complex with bound NPFs
Components
  • (Actin-related protein ...) x 7
  • Neural Wiskott-Aldrich syndrome protein
KeywordsCONTRACTILE PROTEIN / actin / ATPase / actin related protein / arp / cytoskeleton / Arp2-3 complex / actin nucleation / actin branching
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 speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsZimmet, A. / van Eeuwen, T. / Dominguez, R.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM07391 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32-GM008275 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)F31-HL146077 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 Report
SummaryFull reportAbout validation report
History
DepositionSep 27, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 1, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Actin-related protein 3
B: Actin-related protein 2
C: Actin-related protein 2/3 complex subunit 1B
D: Actin-related protein 2/3 complex subunit 2
E: Actin-related protein 2/3 complex subunit 3
F: Actin-related protein 2/3 complex subunit 4
G: Actin-related protein 2/3 complex subunit 5
H: Neural Wiskott-Aldrich syndrome protein
I: Neural Wiskott-Aldrich syndrome protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)336,61913
Polymers335,5569
Non-polymers1,0634
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Actin-related protein ... , 7 types, 7 molecules ABCDEFG

#1: Protein Actin-related protein 3 / Actin-like protein 3


Mass: 47428.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ACTR3, ARP3 / Plasmid: pBIG2abc / Cell (production host): suspension / Cell line (production host): Sf9
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: P61158
#2: Protein Actin-related protein 2 / Actin-like protein 2


Mass: 44818.711 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ACTR2, ARP2 / Plasmid: pBIG2abc / Cell (production host): suspension / Cell line (production host): Sf9
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: P61160
#3: Protein Actin-related protein 2/3 complex subunit 1B / Arp2/3 complex 41 kDa subunit / p41-ARC


Mass: 41004.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARPC1B, ARC41 / Plasmid: pBIG2abc / Cell (production host): suspension / Cell line (production host): Sf9
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: O15143
#4: Protein Actin-related protein 2/3 complex subunit 2 / Arp2/3 complex 34 kDa subunit / p34-ARC


Mass: 34386.043 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARPC2, ARC34, PRO2446 / Plasmid: pBIG2abc / Cell (production host): suspension / Cell line (production host): Sf9
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: O15144
#5: Protein Actin-related protein 2/3 complex subunit 3 / Arp2/3 complex 21 kDa subunit / p21-ARC


Mass: 23196.330 Da / Num. of mol.: 1 / Details: C-terminal Flag tag
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARPC3, ARC21 / Plasmid: pBIG2abc / Cell (production host): suspension / Cell line (production host): Sf9
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: O15145
#6: Protein Actin-related protein 2/3 complex subunit 4 / Arp2/3 complex 20 kDa subunit / p20-ARC


Mass: 19697.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARPC4, ARC20 / Plasmid: pBIG2abc / Cell (production host): suspension / Cell line (production host): Sf9
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: P59998
#7: Protein Actin-related protein 2/3 complex subunit 5 / Arp2/3 complex 16 kDa subunit / p16-ARC


Mass: 16341.407 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARPC5, ARC16 / Plasmid: pBIG2abc / Cell (production host): suspension / Cell line (production host): Sf9
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: O15511

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Protein , 1 types, 2 molecules HI

#8: Protein Neural Wiskott-Aldrich syndrome protein / N-WASP


Mass: 54342.023 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Wasl / Production host: Escherichia coli (E. coli) / References: UniProt: Q91YD9

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Non-polymers , 2 types, 4 molecules

#9: Chemical ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#10: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Cryo-EM structure of human Arp2/3 complex with bound NPFs
Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7, 8 / Source: RECOMBINANT
Molecular weightValue: 243 kDa/nm / Experimental value: NO
Source (natural)Organism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
Source (recombinant)Organism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
Plasmid: pBIG2abc
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2100 mMsodium chlorideNaClSodium chloride1
31 mMDithiothreitolHSCH2CH(OH)CH(OH)CH2SH1
4.2 mMAdenosine triphosphate disodium saltC10H14N5O13P3Na21
5.2 mMmagnesium chlorideMgCl21
6.00025 V/VTERGITOL1
SpecimenConc.: 5 mg/ml / Details: This sample was monodisperse / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids glow discharged with easiGLOW glow discharger at 0.3 mBar, 25mA for 1 minute
Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
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
MicroscopyModel: 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 / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 165000 X / Nominal defocus max: -3500 nm / Nominal defocus min: -1500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 7 sec. / Electron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5004
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
2Latitudeimage acquisition
4CTFFINDCTF correction
5cryoSPARC2.9.0CTF correction
8Cootmodel fitting
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARC2.9.0classification
13cryoSPARC2.9.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 627163
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 123198 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 132 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: correlation
Atomic model buildingPDB-ID: 4JD2

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