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- PDB-6w17: Structure of Dip1-activated Arp2/3 complex with nucleated actin f... -

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Basic information

Entry
Database: PDB / ID: 6w17
TitleStructure of Dip1-activated Arp2/3 complex with nucleated actin filament
Components
  • (Actin-related protein ...) x 7
  • Actin, alpha skeletal muscle
  • Phalloidin
  • Protein dip1
KeywordsSTRUCTURAL PROTEIN / Arp2/3 / actin / Dip1 / cytoskeletal protein / actin regulator
Function / homology
Function and homology information


protein localization to actin cortical patch / Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / actin cortical patch organization / cell cortex of cell tip / medial cortex / Neutrophil degranulation / establishment or maintenance of cell polarity regulating cell shape / actin cortical patch localization / positive regulation of Arp2/3 complex-mediated actin nucleation ...protein localization to actin cortical patch / Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / actin cortical patch organization / cell cortex of cell tip / medial cortex / Neutrophil degranulation / establishment or maintenance of cell polarity regulating cell shape / actin cortical patch localization / positive regulation of Arp2/3 complex-mediated actin nucleation / actin cortical patch assembly / Arp2/3 protein complex / Arp2/3 complex-mediated actin nucleation / Arp2/3 complex binding / cell tip / actin cortical patch / regulation of actin filament polymerization / cell septum / mating projection tip / cytoskeletal motor activator activity / cortical actin cytoskeleton organization / cell division site / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament / mitotic cytokinesis / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / endocytosis / calcium-dependent protein binding / actin filament binding / lamellipodium / cell body / toxin activity / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
SPIN90/Ldb17, leucine-rich domain / SPIN90/Ldb17 / SPIN90/Ldb17, leucine-rich domain / Amanitin/phalloidin toxin / 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 ...SPIN90/Ldb17, leucine-rich domain / SPIN90/Ldb17 / SPIN90/Ldb17, leucine-rich domain / Amanitin/phalloidin toxin / 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) / 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 / 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 2/3 complex subunit 2 / Protein dip1 / Phalloidin proprotein / Actin-related protein 3 / Actin, alpha skeletal muscle / Actin-related protein 2/3 complex subunit 1 / Actin-related protein 2/3 complex subunit 5 / Actin-related protein 2/3 complex subunit 4 ...ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Actin-related protein 2/3 complex subunit 2 / Protein dip1 / Phalloidin proprotein / Actin-related protein 3 / Actin, alpha skeletal muscle / Actin-related protein 2/3 complex subunit 1 / Actin-related protein 2/3 complex subunit 5 / Actin-related protein 2/3 complex subunit 4 / Actin-related protein 2 / Actin-related protein 2/3 complex subunit 3
Similarity search - Component
Biological speciesSchizosaccharomyces pombe (fission yeast)
Oryctolagus cuniculus (rabbit)
Amanita phalloides (death cap)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsShaaban, M. / Nolen, B.J. / Chowdhury, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM127440, R01GM092917, S10OD012272 United States
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Cryo-EM reveals the transition of Arp2/3 complex from inactive to nucleation-competent state.
Authors: Mohammed Shaaban / Saikat Chowdhury / Brad J Nolen /
Abstract: Arp2/3 complex, a crucial actin filament nucleator, undergoes structural rearrangements during activation by nucleation-promoting factors (NPFs). However, the conformational pathway leading to the ...Arp2/3 complex, a crucial actin filament nucleator, undergoes structural rearrangements during activation by nucleation-promoting factors (NPFs). However, the conformational pathway leading to the nucleation-competent state is unclear due to lack of high-resolution structures of the activated state. Here we report a ~3.9 Å resolution cryo-EM structure of activated Schizosaccharomyces pombe Arp2/3 complex bound to the S. pombe NPF Dip1 and attached to the end of the nucleated actin filament. The structure reveals global and local conformational changes that allow the two actin-related proteins in Arp2/3 complex to mimic a filamentous actin dimer and template nucleation. Activation occurs through a clamp-twisting mechanism, in which Dip1 forces two core subunits in Arp2/3 complex to pivot around one another, shifting half of the complex into a new activated position. By showing how Dip1 stimulates activation, the structure reveals how NPFs can activate Arp2/3 complex in diverse cellular processes.
History
DepositionMar 3, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 12, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 9, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Nov 18, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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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 1
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: Protein dip1
I: Actin, alpha skeletal muscle
J: Actin, alpha skeletal muscle
K: Actin, alpha skeletal muscle
L: Actin, alpha skeletal muscle
M: Phalloidin
N: Phalloidin
O: Phalloidin
P: Phalloidin
Q: Phalloidin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)445,87729
Polymers443,00817
Non-polymers2,86912
Water0
1


  • Idetical with deposited unit
  • defined by author
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 / Arp3 / Actin-like protein 3


Mass: 47427.137 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Plasmid details: Fission yeast / Strain: 972 / ATCC 24843 / References: UniProt: P32390
#2: Protein Actin-related protein 2 / Arp2 / Actin-like protein 2


Mass: 44286.758 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Plasmid details: Fission yeast / Strain: 972 / ATCC 24843 / References: UniProt: Q9UUJ1
#3: Protein Actin-related protein 2/3 complex subunit 1 / ARPC1 / Arp2/3 complex 41 kDa subunit / p41-ARC


Mass: 41643.465 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Plasmid details: Fission yeast / Strain: 972 / ATCC 24843 / References: UniProt: P78774
#4: Protein Actin-related protein 2/3 complex subunit 2 / ARPC2 / Arp2/3 complex 34 kDa subunit / p34-ARC


Mass: 37025.230 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Plasmid details: Fission yeast / Strain: 972 / ATCC 24843 / References: UniProt: O14241
#5: Protein Actin-related protein 2/3 complex subunit 3 / ARPC3 / Arp2/3 complex 21 kDa subunit / p21-ARC


Mass: 19865.746 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Plasmid details: Fission yeast / Strain: 972 / ATCC 24843 / References: UniProt: Q9Y7J4
#6: Protein Actin-related protein 2/3 complex subunit 4 / ARPC4 / Arp2/3 complex 20 kDa / p20-ARC


Mass: 19637.695 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Plasmid details: Fission yeast / Strain: 972 / ATCC 24843 / References: UniProt: Q92352
#7: Protein Actin-related protein 2/3 complex subunit 5 / ARPC5 / Arp2/3 complex 16 kDa subunit / p16-ARC


Mass: 16922.059 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Plasmid details: Fission yeast / Strain: 972 / ATCC 24843 / References: UniProt: Q10316

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Protein , 2 types, 5 molecules HIJKL

#8: Protein Protein dip1


Mass: 43715.137 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Fission yeast
Source: (gene. exp.) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Strain: 972 / ATCC 24843 / Gene: dip1, SPBC24C6.10c / Plasmid: pGV67-Dip1 / Production host: Escherichia coli (E. coli) / References: UniProt: O74771
#9: Protein
Actin, alpha skeletal muscle / / Alpha-actin-1


Mass: 42109.973 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / Plasmid details: Rabbit / References: UniProt: P68135

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Protein/peptide , 1 types, 5 molecules MNOPQ

#10: Protein/peptide
Phalloidin /


Mass: 808.899 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Amanita phalloides (death cap) / References: UniProt: P0CU63*PLUS

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Non-polymers , 3 types, 12 molecules

#11: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / 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.: 4 / 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

ComponentName: Complex consisting of actin-filament nucleator, Arp2/3 complex associated with nucleation promoting factor Dip1 and first four actin subunits from the pointed end of the nucleated actin-filament
Type: COMPLEX / Entity ID: #1-#10 / Source: NATURAL
Molecular weightValue: 0.446 MDa / Experimental value: NO
Source (natural)Organism: Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Strain: 972 / ATCC 24843
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMpotassium chlorideKCl1
21 mMegtazic acidC14H24N2O101
31 mMmagnesium chlorideMgCl21
40.2 mMadenosine triphosphateC10H16N5O13P31
520 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidC8H18N2O4S1
61 mMdithiothreitolC4H10O2S21
710 microMphalloidinC35H48N8O11S1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 20mA current / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 98 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Details: Data were collected by stage shifting to targeted exposure positions.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 92000 X / Nominal defocus max: -1750 nm / Nominal defocus min: -1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 36.35 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5109
Details: Each micrograph was collected as dose-fractionated movies consisting of 45 fractions per movie.
Image scansSampling size: 14 µm / Width: 4000 / Height: 4000

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3.0.6particle selection
2EPU2.1image acquisition
4Gctf1.06CTF correction
7NAMDmodel fitting
8Cootmodel fitting
9UCSF Chimeramodel fittingrigid body fitting
12RELION3.0.6final Euler assignment
13RELION3.0.6classification
14RELION3.0.63D reconstruction
15PHENIXmodel refinement
CTF correctionDetails: Particles were CTF corrected during projection matching and back projection.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3500000
Details: Particles were picked using Laplacian Gaussian auto-picking.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110433 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
13DWL

3dwl

1
23J8I

3j8i

1
36D8C

6d8c

N1
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 102.46 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002227981
ELECTRON MICROSCOPYf_angle_d0.551638042
ELECTRON MICROSCOPYf_chiral_restr0.04094284
ELECTRON MICROSCOPYf_plane_restr0.00254894
ELECTRON MICROSCOPYf_dihedral_angle_d19.599410142

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