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- PDB-7jpn: Cryo-EM structure of Arpin-bound Arp2/3 complex -

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Basic information

Entry
Database: PDB / ID: 7jpn
TitleCryo-EM structure of Arpin-bound Arp2/3 complex
Components
  • (Actin-related protein ...) x 7
  • Arpin
KeywordsCONTRACTILE PROTEIN / actin / ATPase / actin related protein / arp / cytoskeleton / Arp2-3 complex / actin nucleation / actin branching / Arpin
Function / homology
Function and homology information


negative regulation of actin nucleation / negative regulation of lamellipodium morphogenesis / EPHB-mediated forward signaling / Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / Arp2/3 protein complex / Arp2/3 complex-mediated actin nucleation / directional locomotion / regulation of actin filament polymerization / Clathrin-mediated endocytosis ...negative regulation of actin nucleation / negative regulation of lamellipodium morphogenesis / EPHB-mediated forward signaling / Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / Arp2/3 protein complex / Arp2/3 complex-mediated actin nucleation / directional locomotion / regulation of actin filament polymerization / Clathrin-mediated endocytosis / Neutrophil degranulation / positive regulation of double-strand break repair via homologous recombination / cilium assembly / positive regulation of lamellipodium assembly / actin filament polymerization / negative regulation of cell migration / cell projection / structural constituent of cytoskeleton / actin filament binding / cell migration / lamellipodium / site of double-strand break / neuron projection / synapse / positive regulation of transcription by RNA polymerase II / ATP binding / nucleus / cytoplasm / cytosol
Similarity search - Function
Arpin / Arp2/3-interacting proteins Arpin / 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 ...Arpin / Arp2/3-interacting proteins Arpin / 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) / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding 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 2 / Actin-related protein 2/3 complex subunit 3 / Actin-related protein 2/3 complex subunit 1B / Arpin
Similarity search - Component
Biological speciesHomo sapiens (human)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.24 Å
Authorsvan Eeuwen, T. / Fregoso, F.E. / Dominguez, R. / Zimmet, A. / Boczkowska, M. / Rebowski, G.
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: Nat Commun / Year: 2022
Title: Molecular mechanism of Arp2/3 complex inhibition by Arpin.
Authors: Fred E Fregoso / Trevor van Eeuwen / Gleb Simanov / Grzegorz Rebowski / Malgorzata Boczkowska / Austin Zimmet / Alexis M Gautreau / Roberto Dominguez /
Abstract: Positive feedback loops involving signaling and actin assembly factors mediate the formation and remodeling of branched actin networks in processes ranging from cell and organelle motility to ...Positive feedback loops involving signaling and actin assembly factors mediate the formation and remodeling of branched actin networks in processes ranging from cell and organelle motility to mechanosensation. The Arp2/3 complex inhibitor Arpin controls the directional persistence of cell migration by interrupting a feedback loop involving Rac-WAVE-Arp2/3 complex, but Arpin's mechanism of inhibition is unknown. Here, we describe the cryo-EM structure of Arpin bound to Arp2/3 complex at 3.24-Å resolution. Unexpectedly, Arpin binds Arp2/3 complex similarly to WASP-family nucleation-promoting factors (NPFs) that activate the complex. However, whereas NPFs bind to two sites on Arp2/3 complex, on Arp2-ArpC1 and Arp3, Arpin only binds to the site on Arp3. Like NPFs, Arpin has a C-helix that binds at the barbed end of Arp3. Mutagenesis studies in vitro and in cells reveal how sequence differences within the C-helix define the molecular basis for inhibition by Arpin vs. activation by NPFs.
History
DepositionAug 9, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 9, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 24, 2022Group: 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
Revision 1.2May 15, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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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  • Deposited structure unit
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Structure viewerMolecule:
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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: Arpin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)225,82412
Polymers224,7618
Non-polymers1,0634
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: isothermal titration calorimetry
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-2 / Actin-like protein 3


Mass: 46908.355 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: P61157
#2: Protein Actin-related protein 2 / Actin-like protein 2


Mass: 42945.453 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: A7MB62
#3: Protein Actin-related protein 2/3 complex subunit 1B / Arp2/3 complex 41 kDa subunit / p41-ARC


Mass: 41030.766 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: Q58CQ2
#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) / 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) / 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) / References: UniProt: Q148J6
#7: Protein Actin-related protein 2/3 complex subunit 5


Mass: 15473.396 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: G3MXC8

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Protein/peptide , 1 types, 1 molecules H

#8: Protein/peptide Arpin / Arp2/3 inhibition protein


Mass: 3730.892 Da / Num. of mol.: 1 / Fragment: UNP residues 193-226
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARPIN, C15orf38 / Production host: Escherichia coli (E. coli) / References: UniProt: Q7Z6K5

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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


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

Component
IDNameTypeEntity IDParent-IDSource
1bovine Arp2/3 complex with ArpinCOMPLEX#1-#80MULTIPLE SOURCES
2bovine Arp2/3COMPLEX#1-#71NATURAL
3ArpinCOMPLEX#81RECOMBINANT
Molecular weightValue: 0.2489 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrainOrgan
22Bos taurus (cattle)9913brain
33Homo sapiens (human)9606BL21 (DE3)
Source (recombinant)Organism: Escherichia coli (E. coli)
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
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportDetails: unspecified
VitrificationInstrument: LEICA EM CPC / Cryogen name: ETHANE
Details: Grids were manually blotted for 3 seconds with Whatman 41 filter paper and manually plunged using a 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 were collected in super-resolution mode with an illuminated area of 1.01 um, nominal dose of 40 e-/A^2, a dose rate of 4.87 e-/s/pixel, and 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: 2.6 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9661
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2Latitudeimage acquisition
4CTFFIND4.1.13CTF correction
5cryoSPARC2.12.4CTF correction
8Cootmodel fitting
10cryoSPARC2.12.4initial Euler assignment
11cryoSPARC2.12.4final Euler assignment
12cryoSPARC2.12.4classification
13cryoSPARC2.12.43D reconstruction
14PHENIXmodel refinement
Image processingDetails: Super resolution mode; micrographs binned during motion correction
CTF correctionDetails: CTF correction was done in cryoSPARC for intial 2D classification and then repeated in CTFFIND4 (Relion) for classification and final reconstruction.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4485066 / Details: Particles autopicked in cryoSPARC
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 182324 / 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 building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
14JD2

4jd2

14JD21PDBexperimental model
21K8K

1k8k

11K8K2PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315373
ELECTRON MICROSCOPYf_angle_d0.53820807
ELECTRON MICROSCOPYf_dihedral_angle_d4.4822048
ELECTRON MICROSCOPYf_chiral_restr0.0452281
ELECTRON MICROSCOPYf_plane_restr0.0042674

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