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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


tubulobulbar complex / meiotic chromosome movement towards spindle pole / cytosolic transport / growth cone leading edge / negative regulation of membrane tubulation / meiotic cytokinesis / muscle cell projection membrane / spindle localization / membrane invagination / positive regulation of clathrin-dependent endocytosis ...tubulobulbar complex / meiotic chromosome movement towards spindle pole / cytosolic transport / growth cone leading edge / negative regulation of membrane tubulation / meiotic cytokinesis / muscle cell projection membrane / spindle localization / membrane invagination / positive regulation of clathrin-dependent endocytosis / actin polymerization-dependent cell motility / plasma membrane tubulation / Arp2/3 protein complex / asymmetric cell division / negative regulation of lymphocyte migration / Arp2/3 complex-mediated actin nucleation / postsynapse organization / actin nucleation / regulation of cell projection assembly / actin cap / vesicle organization / postsynaptic actin cytoskeleton organization / vesicle budding from membrane / regulation of actin filament polymerization / positive regulation of chemotaxis / vesicle transport along actin filament / protein-containing complex localization / dendritic spine morphogenesis / regulation of postsynapse organization / positive regulation of filopodium assembly / establishment or maintenance of cell polarity / positive regulation of actin filament polymerization / cell leading edge / filamentous actin / brush border / positive regulation of double-strand break repair via homologous recombination / cilium assembly / RHO GTPases Activate WASPs and WAVEs / positive regulation of lamellipodium assembly / positive regulation of substrate adhesion-dependent cell spreading / EPHB-mediated forward signaling / actin filament polymerization / cytoskeletal protein binding / cellular response to nerve growth factor stimulus / cell projection / FCGR3A-mediated phagocytosis / response to bacterium / structural constituent of cytoskeleton / Regulation of actin dynamics for phagocytic cup formation / cellular response to type II interferon / response to estrogen / regulation of protein localization / azurophil granule lumen / cell migration / actin filament binding / cell-cell junction / actin cytoskeleton / response to estradiol / lamellipodium / Clathrin-mediated endocytosis / site of double-strand break / actin binding / cytoplasmic vesicle / actin cytoskeleton organization / postsynapse / secretory granule lumen / ficolin-1-rich granule lumen / endosome / neuron projection / cell division / Golgi membrane / focal adhesion / glutamatergic synapse / Neutrophil degranulation / enzyme binding / positive regulation of transcription by RNA polymerase II / extracellular exosome / extracellular region / nucleoplasm / ATP binding / membrane / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Actin-related protein 2/3 complex subunit 3 / Actin nucleation-promoting factor WAS, C-terminal / Actin-related protein 2/3 complex subunit 5 / Yope Regulator; Chain: A, - #20 / WASP family, EVH1 domain / Arp2/3 complex 21 kDa subunit ARPC3 / Actin-related protein 2/3 complex subunit 5 / Actin-related protein 2/3 complex subunit 2 / Actin-related protein 2/3 complex subunit 3 / Actin-related protein 2/3 complex subunit 4 ...Actin-related protein 2/3 complex subunit 3 / Actin nucleation-promoting factor WAS, C-terminal / Actin-related protein 2/3 complex subunit 5 / Yope Regulator; Chain: A, - #20 / WASP family, EVH1 domain / Arp2/3 complex 21 kDa subunit ARPC3 / Actin-related protein 2/3 complex subunit 5 / Actin-related protein 2/3 complex subunit 2 / Actin-related protein 2/3 complex subunit 3 / Actin-related protein 2/3 complex subunit 4 / Actin-related protein 2/3 complex subunit 1 / Arp2/3 complex subunit 2/4 / Actin-related protein 2/3 complex subunit 5 superfamily / Actin-related protein 2/3 complex subunit 3 superfamily / Arp2/3 complex, 34 kD subunit p34-Arc / ARP2/3 complex ARPC3 (21 kDa) subunit / ARP2/3 complex 16 kDa subunit (p16-Arc) / ARP2/3 complex 20 kDa subunit (ARPC4) / Wiskott Aldrich syndrome homology region 2 / Yope Regulator; Chain: A, / WH2 motif / WH1/EVH1 domain / WH1 domain / WH1 domain profile. / WASP homology region 1 / WH2 domain / WH2 domain profile. / CRIB domain superfamily / P21-Rho-binding domain / CRIB domain profile. / P21-Rho-binding domain / CRIB domain / YVTN repeat-like/Quinoprotein amine dehydrogenase / Actin/actin-like conserved site / Actins and actin-related proteins signature. / 7 Propeller / Methylamine Dehydrogenase; Chain H / Actin / Actin family / Actin / Serine Threonine Protein Phosphatase 5, Tetratricopeptide repeat / ATPase, nucleotide binding domain / Alpha Horseshoe / PH-like domain superfamily / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / 2-Layer Sandwich / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / 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 / Actin nucleation-promoting factor WASL
Similarity search - Component
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.
History
DepositionSep 27, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 1, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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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


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
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
Source method: isolated from a genetically manipulated source
Details: C-terminal Flag tag / 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


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-#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 / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
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 / Specimen holder 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

4jd2


Accession code: 4JD2 / Source name: PDB / Type: experimental model

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