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- PDB-8tah: Cryo-EM structure of Cortactin-bound to Arp2/3 complex -

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Basic information

Entry
Database: PDB / ID: 8tah
TitleCryo-EM structure of Cortactin-bound to Arp2/3 complex
Components
  • (Actin-related protein ...) x 7
  • Src substrate cortactin
KeywordsCONTRACTILE PROTEIN / Complex / migration / actin / cytoskeleton
Function / homology
Function and homology information


lamellipodium organization / EPHB-mediated forward signaling / site of polarized growth / Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / Arp2/3 protein complex / Arp2/3 complex-mediated actin nucleation / Arp2/3 complex binding / modification of postsynaptic actin cytoskeleton / modification of postsynaptic structure ...lamellipodium organization / EPHB-mediated forward signaling / site of polarized growth / Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / Arp2/3 protein complex / Arp2/3 complex-mediated actin nucleation / Arp2/3 complex binding / modification of postsynaptic actin cytoskeleton / modification of postsynaptic structure / mitotic spindle midzone / regulation of cell projection assembly / regulation of mitophagy / postsynaptic actin cytoskeleton / profilin binding / positive regulation of smooth muscle contraction / regulation of actin filament polymerization / positive regulation of chemotaxis / Clathrin-mediated endocytosis / substrate-dependent cell migration, cell extension / focal adhesion assembly / podosome / proline-rich region binding / dendritic spine maintenance / Neutrophil degranulation / regulation of axon extension / cortical actin cytoskeleton / cortical cytoskeleton / positive regulation of actin filament polymerization / positive regulation of double-strand break repair via homologous recombination / cilium assembly / positive regulation of lamellipodium assembly / extrinsic apoptotic signaling pathway / clathrin-coated pit / voltage-gated potassium channel complex / ruffle / actin filament polymerization / neuron projection morphogenesis / receptor-mediated endocytosis / cell projection / cell motility / actin filament / negative regulation of extrinsic apoptotic signaling pathway / intracellular protein transport / structural constituent of cytoskeleton / actin filament binding / cell migration / lamellipodium / cell junction / site of double-strand break / cell cortex / actin cytoskeleton organization / postsynapse / dendritic spine / neuron projection / focal adhesion / glutamatergic synapse / synapse / Golgi apparatus / endoplasmic reticulum / positive regulation of transcription by RNA polymerase II / ATP binding / nucleus / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Hs1/Cortactin / Cortactin, SH3 domain / Repeat in HS1/Cortactin / Cortactin repeat profile. / 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 ...Hs1/Cortactin / Cortactin, SH3 domain / Repeat in HS1/Cortactin / Cortactin repeat profile. / 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) / Variant SH3 domain / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain / Src homology 3 domains / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Actin-related protein 2 / Actin-related protein 2/3 complex subunit 5 / Actin-related protein 3 / Actin-related protein 2/3 complex subunit 4 / Actin-related protein 2/3 complex subunit 1A / Actin-related protein 2/3 complex subunit 2 / Actin-related protein 2/3 complex subunit 3 / Src substrate cortactin
Similarity search - Component
Biological speciesMus musculus (house mouse)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsFregoso, F.E. / van Eeuwen, T. / Dominguez, R.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM073791 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F31 GM148048 United States
CitationJournal: Nat Commun / Year: 2023
Title: Mechanism of synergistic activation of Arp2/3 complex by cortactin and WASP-family proteins.
Authors: Fred E Fregoso / Malgorzata Boczkowska / Grzegorz Rebowski / Peter J Carman / Trevor van Eeuwen / Roberto Dominguez /
Abstract: Cortactin coactivates Arp2/3 complex synergistically with WASP-family nucleation-promoting factors (NPFs) and stabilizes branched networks by linking Arp2/3 complex to F-actin. It is poorly ...Cortactin coactivates Arp2/3 complex synergistically with WASP-family nucleation-promoting factors (NPFs) and stabilizes branched networks by linking Arp2/3 complex to F-actin. It is poorly understood how cortactin performs these functions. We describe the 2.89 Å resolution cryo-EM structure of cortactin's N-terminal domain (Cort) bound to Arp2/3 complex. Cortactin binds Arp2/3 complex through an inverted Acidic domain (D20-V29), which targets the same site on Arp3 as the Acidic domain of NPFs but with opposite polarity. Sequences N- and C-terminal to cortactin's Acidic domain do not increase its affinity for Arp2/3 complex but contribute toward coactivation with NPFs. Coactivation further increases with NPF dimerization and for longer cortactin constructs with stronger binding to F-actin. The results suggest that cortactin contributes to Arp2/3 complex coactivation with NPFs in two ways, by helping recruit the complex to F-actin and by stabilizing the short-pitch (active) conformation, which are both byproducts of cortactin's core function in branch stabilization.
History
DepositionJun 27, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 27, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 8, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 31, 2024Group: Data collection / Structure summary / Category: audit_author / em_author_list

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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 1A
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: Src substrate cortactin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)234,56912
Polymers233,5068
Non-polymers1,0634
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: isothermal titration calorimetry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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

#1: Protein Actin-related protein 3 / Actin-2 / Actin-like protein 3


Mass: 47428.031 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: Brain / References: UniProt: P61157
#2: Protein Actin-related protein 2 / Actin-like protein 2


Mass: 44818.711 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: Brain / References: UniProt: A7MB62
#3: Protein Actin-related protein 2/3 complex subunit 1A


Mass: 41594.238 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: brain / References: UniProt: Q1JP79
#4: Protein Actin-related protein 2/3 complex subunit 2 / Arp2/3 complex 34 kDa subunit / p34-ARC


Mass: 34402.043 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: brain / References: UniProt: Q3MHR7
#5: Protein Actin-related protein 2/3 complex subunit 3 / Arp2/3 complex 21 kDa subunit / p21-ARC


Mass: 20572.666 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: brain / References: UniProt: Q3T035
#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 natural source / Source: (natural) Bos taurus (cattle) / Organ: brain / References: UniProt: Q148J6
#7: Protein Actin-related protein 2/3 complex subunit 5


Mass: 16309.343 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: brain / References: UniProt: G3MXC8

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Protein , 1 types, 1 molecules H

#8: Protein Src substrate cortactin


Mass: 8684.353 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Cttn / Production host: Escherichia coli (E. coli) / References: UniProt: Q60598

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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 / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


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

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Details

Has ligand of interestY

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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: Cortactin NTA bound to Arp2/3 complex / Type: COMPLEX / Entity ID: #1-#8 / Source: MULTIPLE SOURCES
Molecular weightValue: .223 MDa / Experimental value: NO
Source (natural)Organism: Bos taurus (cattle)
Buffer solutionpH: 7
Details: 10 mM imidazole pH 7.0, 50 mM KCl, 2 mM MgCl2, 1 mM EGTA
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMPotassium chlorideKCl1
22 mMMagnesium ChlorideMgCl21
31 mMethylene glycol-bis(beta-aminoethyl ether)-N,N,N,N-tetraacetic acidEGTA1
410 mMImidazoleI1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: monodisperse
Specimen supportGrid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 41.6 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.0.0particle selection
4cryoSPARC4.0.0CTF correction
7Cootmodel fitting
9cryoSPARC4.0.0initial Euler assignment
10RELIONfinal Euler assignment
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1461919
3D reconstructionResolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 241506 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 1K8K

1k8k


Accession code: 1K8K / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00515300
ELECTRON MICROSCOPYf_angle_d0.6520721
ELECTRON MICROSCOPYf_dihedral_angle_d12.6935640
ELECTRON MICROSCOPYf_chiral_restr0.0512286
ELECTRON MICROSCOPYf_plane_restr0.0072663

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