[English] 日本語
Yorodumi
- PDB-6uhc: CryoEM structure of human Arp2/3 complex with bound NPFs -

+
Open data


ID or keywords:

Loading...

-
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 / spindle localization / meiotic cytokinesis / positive regulation of clathrin-dependent endocytosis / actin polymerization-dependent cell motility / muscle cell projection membrane ...tubulobulbar complex / meiotic chromosome movement towards spindle pole / cytosolic transport / growth cone leading edge / negative regulation of membrane tubulation / spindle localization / meiotic cytokinesis / positive regulation of clathrin-dependent endocytosis / actin polymerization-dependent cell motility / muscle cell projection membrane / asymmetric cell division / Arp2/3 protein complex / negative regulation of lymphocyte migration / Arp2/3 complex-mediated actin nucleation / actin nucleation / vesicle transport along actin filament / actin cap / vesicle organization / regulation of actin filament polymerization / vesicle budding from membrane / dendritic spine morphogenesis / protein-containing complex localization / regulation of postsynapse organization / positive regulation of filopodium assembly / positive regulation of actin filament polymerization / establishment or maintenance of cell polarity / filamentous actin / brush border / cilium assembly / RHO GTPases Activate WASPs and WAVEs / regulation of synaptic vesicle endocytosis / positive regulation of double-strand break repair via homologous recombination / positive regulation of lamellipodium assembly / actin filament polymerization / EPHB-mediated forward signaling / positive regulation of substrate adhesion-dependent cell spreading / cell projection / response to bacterium / FCGR3A-mediated phagocytosis / cellular response to nerve growth factor stimulus / Regulation of actin dynamics for phagocytic cup formation / cellular response to type II interferon / structural constituent of cytoskeleton / response to estrogen / synaptic vesicle membrane / azurophil granule lumen / cell-cell junction / cell migration / response to estradiol / actin cytoskeleton / lamellipodium / Clathrin-mediated endocytosis / regulation of protein localization / site of double-strand break / actin binding / actin cytoskeleton organization / cytoplasmic vesicle / cell cortex / secretory granule lumen / ficolin-1-rich granule lumen / postsynapse / neuron projection / endosome / cell division / focal adhesion / Neutrophil degranulation / protein-containing complex binding / glutamatergic synapse / enzyme binding / positive regulation of transcription by RNA polymerase II / extracellular exosome / extracellular region / nucleoplasm / ATP binding / nucleus / membrane / cytosol / cytoplasm
Similarity search - Function
Actin-related protein 2/3 complex subunit 3 / Actin-related protein 2/3 complex subunit 5 / Yope Regulator; Chain: A, - #20 / Actin nucleation-promoting factor WAS, C-terminal / WASP family, EVH1 domain / Arp2/3 complex 21 kDa subunit ARPC3 / Actin-related protein 2/3 complex subunit 5 / ARP2/3 complex 16 kDa subunit (p16-Arc) / Yope Regulator; Chain: A, / Actin-related protein 2/3 complex subunit 2 ...Actin-related protein 2/3 complex subunit 3 / Actin-related protein 2/3 complex subunit 5 / Yope Regulator; Chain: A, - #20 / Actin nucleation-promoting factor WAS, C-terminal / WASP family, EVH1 domain / Arp2/3 complex 21 kDa subunit ARPC3 / Actin-related protein 2/3 complex subunit 5 / ARP2/3 complex 16 kDa subunit (p16-Arc) / Yope Regulator; Chain: A, / 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 20 kDa subunit (ARPC4) / Wiskott Aldrich syndrome homology region 2 / WH2 motif / WH1/EVH1 domain / WH1 domain / WH1 domain profile. / WASP homology region 1 / WH2 domain profile. / WH2 domain / CRIB domain superfamily / P21-Rho-binding domain / CRIB domain profile. / P21-Rho-binding domain / CRIB domain / ATPase, substrate binding domain, subdomain 4 / Actin; Chain A, domain 4 / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / YVTN repeat-like/Quinoprotein amine dehydrogenase / 7 Propeller / Methylamine Dehydrogenase; Chain H / ATPase, nucleotide binding domain / Serine Threonine Protein Phosphatase 5, Tetratricopeptide repeat / Alpha Horseshoe / PH-like domain superfamily / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Alpha-Beta Complex / 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

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-20770
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
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
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

-
Components

-
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

-
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

-
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


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

-
Details

Has ligand of interestN

-
Experimental details

-
Experiment

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

-
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 chlorideNaCl1
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.

-
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 FIELD / 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

-
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

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more