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- PDB-8p94: Cryo-EM structure of cortactin stabilized Arp2/3-complex nucleate... -

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Basic information

Entry
Database: PDB / ID: 8p94
TitleCryo-EM structure of cortactin stabilized Arp2/3-complex nucleated actin branches
Components
  • (Actin-related protein ...) x 7
  • Actin, cytoplasmic 1
  • F-actin-capping protein subunit alpha-1
  • Isoform 2 of F-actin-capping protein subunit beta
  • Phalloidin
  • Src substrate cortactin
KeywordsCONTRACTILE PROTEIN / Complex
Function / homology
Function and homology information


cytoskeletal calyx / tubulobulbar complex / meiotic chromosome movement towards spindle pole / cytosolic transport / growth cone leading edge / meiotic cytokinesis / muscle cell projection membrane / RHOF GTPase cycle / lamellipodium organization / Advanced glycosylation endproduct receptor signaling ...cytoskeletal calyx / tubulobulbar complex / meiotic chromosome movement towards spindle pole / cytosolic transport / growth cone leading edge / meiotic cytokinesis / muscle cell projection membrane / RHOF GTPase cycle / lamellipodium organization / Advanced glycosylation endproduct receptor signaling / spindle localization / RHOD GTPase cycle / Gap junction degradation / Formation of annular gap junctions / Regulation of actin dynamics for phagocytic cup formation / EPHB-mediated forward signaling / VEGFA-VEGFR2 Pathway / Cell-extracellular matrix interactions / RHO GTPases Activate WASPs and WAVEs / MAP2K and MAPK activation / site of polarized growth / actin polymerization-dependent cell motility / COPI-independent Golgi-to-ER retrograde traffic / Clathrin-mediated endocytosis / Arp2/3 protein complex / asymmetric cell division / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Arp2/3 complex-mediated actin nucleation / actin nucleation / WASH complex / Arp2/3 complex binding / sperm connecting piece / F-actin capping protein complex / COPI-mediated anterograde transport / negative regulation of filopodium assembly / modification of postsynaptic actin cytoskeleton / modification of postsynaptic structure / mitotic spindle midzone / regulation of cell projection assembly / cellular response to cytochalasin B / actin cap / regulation of mitophagy / regulation of transepithelial transport / structural constituent of postsynaptic actin cytoskeleton / morphogenesis of a polarized epithelium / profilin binding / substrate-dependent cell migration, cell extension / postsynaptic actin cytoskeleton / protein localization to adherens junction / dense body / Factors involved in megakaryocyte development and platelet production / Tat protein binding / positive regulation of smooth muscle contraction / positive regulation of chemotaxis / regulation of actin filament polymerization / cell projection organization / cell junction assembly / apical protein localization / MHC class II antigen presentation / barbed-end actin filament capping / actin polymerization or depolymerization / adherens junction assembly / focal adhesion assembly / podosome / RHO GTPases activate IQGAPs / regulation of cell morphogenesis / RHO GTPases Activate Formins / regulation of lamellipodium assembly / regulation of axon extension / proline-rich region binding / tight junction / regulation of norepinephrine uptake / NuA4 histone acetyltransferase complex / lamellipodium assembly / dendritic spine maintenance / regulation of synaptic vesicle endocytosis / apical junction complex / establishment or maintenance of cell polarity / cortical actin cytoskeleton / cortical cytoskeleton / positive regulation of double-strand break repair via homologous recombination / positive regulation of actin filament polymerization / nitric-oxide synthase binding / filamentous actin / brush border / kinesin binding / calyx of Held / cilium assembly / asymmetric synapse / RHO GTPases Activate WASPs and WAVEs / regulation of protein localization to plasma membrane / voltage-gated potassium channel complex / positive regulation of lamellipodium assembly / positive regulation of substrate adhesion-dependent cell spreading / cytoskeleton organization / extrinsic apoptotic signaling pathway / clathrin-coated pit / ruffle / EPHB-mediated forward signaling / hippocampal mossy fiber to CA3 synapse
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) / F-actin-capping protein subunit beta / F-actin capping protein, beta subunit, conserved site / F-actin-capping protein subunit beta, N-terminal domain / F-actin capping protein, beta subunit / F-actin capping protein beta subunit signature. / F-actin capping protein, alpha subunit, conserved site / F-actin capping protein alpha subunit signature 1. / F-actin capping protein alpha subunit signature 2. / F-actin-capping protein subunit alpha / F-actin-capping protein subunit alpha/beta / F-actin-capping protein subunit alpha/beta, domain 2 / F-actin capping protein, alpha subunit, domain 1 / F-actin capping protein alpha subunit / Variant SH3 domain / Actins signature 1. / Actin, conserved site / Actins signature 2. / 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 / 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
Similarity search - Domain/homology
: / ADENOSINE-5'-DIPHOSPHATE / Actin-related protein 2/3 complex subunit 1B / Actin-related protein 2/3 complex subunit 2 / Actin-related protein 2/3 complex subunit 3 / F-actin-capping protein subunit alpha-1 / F-actin-capping protein subunit beta / Actin-related protein 2/3 complex subunit 4 / Actin-related protein 3 / Actin-related protein 2 ...: / ADENOSINE-5'-DIPHOSPHATE / Actin-related protein 2/3 complex subunit 1B / Actin-related protein 2/3 complex subunit 2 / Actin-related protein 2/3 complex subunit 3 / F-actin-capping protein subunit alpha-1 / F-actin-capping protein subunit beta / Actin-related protein 2/3 complex subunit 4 / Actin-related protein 3 / Actin-related protein 2 / Src substrate cortactin / Actin, cytoplasmic 1 / Actin-related protein 2/3 complex subunit 5-like protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
Sus scrofa (pig)
Amanita phalloides (death cap)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLiu, T. / Moores, C.A.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)810207European Union
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Cortactin stabilizes actin branches by bridging activated Arp2/3 to its nucleated actin filament.
Authors: Tianyang Liu / Luyan Cao / Miroslav Mladenov / Antoine Jegou / Michael Way / Carolyn A Moores /
Abstract: Regulation of the assembly and turnover of branched actin filament networks nucleated by the Arp2/3 complex is essential during many cellular processes, including cell migration and membrane ...Regulation of the assembly and turnover of branched actin filament networks nucleated by the Arp2/3 complex is essential during many cellular processes, including cell migration and membrane trafficking. Cortactin is important for actin branch stabilization, but the mechanism by which this occurs is unclear. Given this, we determined the structure of vertebrate cortactin-stabilized Arp2/3 actin branches using cryogenic electron microscopy. We find that cortactin interacts with the new daughter filament nucleated by the Arp2/3 complex at the branch site, rather than the initial mother actin filament. Cortactin preferentially binds activated Arp3. It also stabilizes the F-actin-like interface of activated Arp3 with the first actin subunit of the new filament, and its central repeats extend along successive daughter-filament subunits. The preference of cortactin for activated Arp3 explains its retention at the actin branch and accounts for its synergy with other nucleation-promoting factors in regulating branched actin network dynamics.
History
DepositionJun 5, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 3, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2024Group: 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.pdbx_database_id_DOI / _citation.title / _citation.year / _citation_author.name
Revision 1.2Feb 7, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name

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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 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-like protein
H: Actin, cytoplasmic 1
I: Src substrate cortactin
J: Actin, cytoplasmic 1
K: Actin, cytoplasmic 1
N: Actin, cytoplasmic 1
O: Actin, cytoplasmic 1
P: Actin, cytoplasmic 1
Q: Actin, cytoplasmic 1
R: Actin, cytoplasmic 1
S: Actin, cytoplasmic 1
U: F-actin-capping protein subunit alpha-1
V: Isoform 2 of F-actin-capping protein subunit beta
W: Actin, cytoplasmic 1
a: Phalloidin
b: Phalloidin
c: Phalloidin
d: Phalloidin
h: Phalloidin
i: Phalloidin
j: Phalloidin
k: Phalloidin
l: Phalloidin
m: Phalloidin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)781,19654
Polymers775,77830
Non-polymers5,41824
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
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 / Production host: unidentified baculovirus / 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 / Production host: unidentified baculovirus / 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 / Production host: unidentified baculovirus / 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 / Production host: unidentified baculovirus / References: UniProt: O15144
#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 genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARPC3, ARC21 / Production host: unidentified baculovirus / 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 / Production host: unidentified baculovirus / References: UniProt: P59998
#7: Protein Actin-related protein 2/3 complex subunit 5-like protein / Arp2/3 complex 16 kDa subunit 2 / ARC16-2


Mass: 16964.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARPC5L / Production host: unidentified baculovirus / References: UniProt: Q9BPX5

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Protein , 4 types, 13 molecules HJKNOPQRSWIUV

#8: Protein
Actin, cytoplasmic 1 / / Beta-actin


Mass: 41782.660 Da / Num. of mol.: 10 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: Q6QAQ1
#9: Protein Src substrate cortactin


Mass: 61340.738 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Cttn, Ems1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q60598
#10: Protein F-actin-capping protein subunit alpha-1 / CapZ alpha-1


Mass: 32980.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Capza1, Cappa1 / Production host: Escherichia coli (E. coli) / References: UniProt: P47753
#11: Protein Isoform 2 of F-actin-capping protein subunit beta / CapZ beta


Mass: 30669.768 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Capzb, Cappb1 / Production host: Escherichia coli (E. coli) / References: UniProt: P47757

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Protein/peptide , 1 types, 10 molecules abcdhijklm

#12: Protein/peptide
Phalloidin / /


Type: Peptide-like / Class: Toxin / Mass: 808.899 Da / Num. of mol.: 10 / Source method: isolated from a natural source / Source: (natural) Amanita phalloides (death cap) / References: BIRD: PRD_002366

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Non-polymers , 2 types, 24 molecules

#13: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#14: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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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

Component
IDNameTypeEntity IDParent-IDSource
1Cortactin stabilizes Arp2/3-complex nucleated actin branches with daughter filament cappedCOMPLEX#1-#120MULTIPLE SOURCES
2Human Arp2/3 C1BC5L complexCOMPLEX#1-#71RECOMBINANT
3Porcine actin, cytoplasmic 1COMPLEX#81NATURAL
4Mouse cortactinCOMPLEX#91RECOMBINANT
5Mouse capping proteinCOMPLEX#10-#111RECOMBINANT
6PhalloidinCOMPLEX#121NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Sus scrofa (pig)9823
54Mus musculus (house mouse)10090
65Mus musculus (house mouse)10090
76Amanita phalloides (death cap)67723
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21unidentified baculovirus10469
32unidentified baculovirus10469
43unidentified baculovirus10469
54Escherichia coli (E. coli)562
65Escherichia coli (E. coli)562
76Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Details: 20 mM HEPES pH 7.5, 50mM KCl, 1mM EGTA, 1mM MgCl2, 0.2 mM ATP and 1 mM DTT
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 98 % / Chamber temperature: 295.15 K / Details: Back blotting

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 900 nm
Image recordingElectron dose: 49.4 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategoryFitting-ID
1cryoSPARCparticle selection
3EPUimage acquisition
5cryoSPARCCTF correction
8UCSF ChimeraXmodel fitting1
12ISOLDEmodel refinement1
14ISOLDEmodel refinement2
15Cootmodel refinement2
16cryoSPARCinitial Euler assignment
18cryoSPARCfinal Euler assignment
21cryoSPARC3D reconstruction
CTF correctionDetails: CryoSPARC Patch CTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2001580
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 130915 / Symmetry type: POINT
Atomic model building
IDProtocol
1OTHER
2
Atomic model building
ID 3D fitting-IDSource nameType
11AlphaFoldin silico model
22

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