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- PDB-7t5q: Cryo-EM Structure of a Transition State of Arp2/3 Complex Activation -

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Basic information

Entry
Database: PDB / ID: 7t5q
TitleCryo-EM Structure of a Transition State of Arp2/3 Complex Activation
Components
  • (Actin-related protein ...) x 7
  • (F-actin-capping protein subunit ...) x 2
  • Actin, alpha skeletal muscle
KeywordsCONTRACTILE PROTEIN / actin / ATPase / actin related protein / arp / cytoskeleton / Arp2-3 complex / actin nucleation / actin branching / CapZ
Function / homology
Function and homology information


negative regulation of membrane tubulation / muscle cell projection membrane / spindle localization / membrane invagination / positive regulation of clathrin-dependent endocytosis / EPHB-mediated forward signaling / Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / plasma membrane tubulation / Arp2/3 protein complex ...negative regulation of membrane tubulation / muscle cell projection membrane / spindle localization / membrane invagination / positive regulation of clathrin-dependent endocytosis / EPHB-mediated forward signaling / Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / plasma membrane tubulation / Arp2/3 protein complex / negative regulation of lymphocyte migration / Arp2/3 complex-mediated actin nucleation / WASH complex / postsynapse organization / sperm connecting piece / actin nucleation / F-actin capping protein complex / negative regulation of filopodium assembly / 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 / Clathrin-mediated endocytosis / cell junction assembly / COPI-independent Golgi-to-ER retrograde traffic / barbed-end actin filament capping / vesicle transport along actin filament / actin polymerization or depolymerization / protein-containing complex localization / dendritic spine morphogenesis / RHOF GTPase cycle / RHOD GTPase cycle / regulation of cell morphogenesis / regulation of lamellipodium assembly / Sensory processing of sound by inner hair cells of the cochlea / Neutrophil degranulation / regulation of postsynapse organization / lamellipodium assembly / positive regulation of filopodium assembly / cytoskeletal motor activator activity / tropomyosin binding / positive regulation of double-strand break repair via homologous recombination / myosin heavy chain binding / cortical cytoskeleton / mesenchyme migration / troponin I binding / cell leading edge / actin filament bundle / filamentous actin / actin filament bundle assembly / brush border / skeletal muscle thin filament assembly / Advanced glycosylation endproduct receptor signaling / striated muscle thin filament / cilium assembly / skeletal muscle myofibril / actin monomer binding / positive regulation of lamellipodium assembly / COPI-mediated anterograde transport / skeletal muscle fiber development / stress fiber / titin binding / cytoskeleton organization / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / hippocampal mossy fiber to CA3 synapse / MHC class II antigen presentation / actin filament polymerization / Gene and protein expression by JAK-STAT signaling after Interleukin-12 stimulation / cytoskeletal protein binding / sarcomere / filopodium / cell projection / actin filament / response to bacterium / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Schaffer collateral - CA1 synapse / cell morphogenesis / structural constituent of cytoskeleton / calcium-dependent protein binding / regulation of protein localization / cell migration / actin filament binding / actin cytoskeleton / lamellipodium / site of double-strand break / cell body / actin binding / Factors involved in megakaryocyte development and platelet production / cytoplasmic vesicle / actin cytoskeleton organization / postsynapse / protein-containing complex assembly / postsynaptic density / cytoskeleton / hydrolase activity / neuron projection / cadherin binding
Similarity search - Function
Actin nucleation-promoting factor WAS, C-terminal / WASP family, EVH1 domain / 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 ...Actin nucleation-promoting factor WAS, C-terminal / WASP family, EVH1 domain / 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. / Wiskott Aldrich syndrome homology region 2 / 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 / 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 / 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 / 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
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Actin-related protein 2 / F-actin-capping protein subunit beta / F-actin-capping protein subunit alpha-1 / Actin-related protein 3 / Actin, alpha skeletal muscle / Actin-related protein 2/3 complex subunit 4 / Actin-related protein 2/3 complex subunit 1A / Actin-related protein 2/3 complex subunit 2 ...ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Actin-related protein 2 / F-actin-capping protein subunit beta / F-actin-capping protein subunit alpha-1 / Actin-related protein 3 / Actin, alpha skeletal muscle / 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 5 / Actin-related protein 2/3 complex subunit 3 / Actin nucleation-promoting factor WASL
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
Bos taurus (cattle)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsRebowski, G. / van Eeuwen, T. / Boczkowska, M. / 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: To Be Published
Title: Cryo-EM structure of an Arp2/3 complex mini-branch capped by capping protein (CapZ)
Authors: Rebowski, G. / van Eeuwen, T. / Dominguez, R. / Boczkowska, M.
History
DepositionDec 13, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 14, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2023Group: Structure summary / Category: struct / Item: _struct.title

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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: Actin, alpha skeletal muscle
I: F-actin-capping protein subunit alpha-1/Actin nucleation-promoting factor WASL chimera
J: F-actin-capping protein subunit beta/Actin nucleation-promoting factor WASL chimera
hetero molecules


Theoretical massNumber of molelcules
Total (without water)345,22516
Polymers343,71110
Non-polymers1,5146
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
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: 47428.031 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: 44818.711 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 1A


Mass: 41594.238 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / 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) / 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 / Arp2/3 complex 16 kDa subunit / p16-ARC


Mass: 16251.308 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: Q3SYX9

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Protein , 1 types, 1 molecules H

#8: Protein Actin, alpha skeletal muscle / / Alpha-actin-1


Mass: 41875.633 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135

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F-actin-capping protein subunit ... , 2 types, 2 molecules IJ

#9: Protein F-actin-capping protein subunit alpha-1/Actin nucleation-promoting factor WASL chimera / CapZ alpha-1 / Neural Wiskott-Aldrich syndrome protein / N-WASP


Mass: 39600.566 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Mus musculus (house mouse)
Gene: CAPZA1, Wasl / Production host: Escherichia coli (E. coli) / References: UniProt: P52907, UniProt: Q91YD9
#10: Protein F-actin-capping protein subunit beta/Actin nucleation-promoting factor WASL chimera / CapZ beta / Neural Wiskott-Aldrich syndrome protein / N-WASP


Mass: 37470.754 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Mus musculus (house mouse)
Gene: CAPZB, Wasl / Production host: Escherichia coli (E. coli) / References: UniProt: P47756, UniProt: Q91YD9

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Non-polymers , 3 types, 6 molecules

#11: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#12: 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
#13: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, 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-IDSourceDetails
1bovine Arp2/3 complex and Actin, CapZCOMPLEX#1-#100MULTIPLE SOURCES
2bovine Arp2/3COMPLEX#1-#71NATURAL
3Actin skeletal muscleCOMPLEX#81NATURAL
4F-actin-capping proteinCOMPLEX#9-#101RECOMBINANTsubunit alpha and beta
Molecular weightValue: 0.2489 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDOrgan
22Bos taurus (cattle)9913brain
33Oryctolagus cuniculus (rabbit)9986
44Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
13Escherichia coli (E. coli)562
24Escherichia coli (E. coli)562
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
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
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 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: 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: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC2.12.4particle selection
2Latitudeimage acquisition
4CTFFIND4.1.13CTF correction
5cryoSPARC2.12.4CTF correction
8Cootmodel fitting
10PHENIXmodel refinement
11RELION3.1initial Euler assignment
12RELION3.1final Euler assignment
13RELION3.1classification
14cryoSPARC43D reconstructionnon-uniform 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: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 269760 / Algorithm: FOURIER SPACE / Details: Composite map after multibody refinement / 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
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00522141
ELECTRON MICROSCOPYf_angle_d0.83429965
ELECTRON MICROSCOPYf_dihedral_angle_d6.0432946
ELECTRON MICROSCOPYf_chiral_restr0.0493295
ELECTRON MICROSCOPYf_plane_restr0.0063845

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