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- PDB-7tpt: Single-particle Cryo-EM structure of Arp2/3 complex at branched-a... -

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Basic information

Entry
Database: PDB / ID: 7tpt
TitleSingle-particle Cryo-EM structure of Arp2/3 complex at branched-actin junction.
Components
  • (Actin-related protein ...) x 7
  • Actin, alpha skeletal muscle
  • Phalloidin
KeywordsSTRUCTURAL PROTEIN / Arp2/3 / actin / cytoskeletal protein / actin regulator
Function / homology
Function and homology information


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 / regulation of actin filament polymerization / Clathrin-mediated endocytosis / Neutrophil degranulation / cytoskeletal motor activator activity / tropomyosin binding ...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 / regulation of actin filament polymerization / Clathrin-mediated endocytosis / Neutrophil degranulation / cytoskeletal motor activator activity / tropomyosin binding / mesenchyme migration / troponin I binding / myosin heavy chain binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle assembly / skeletal muscle myofibril / positive regulation of double-strand break repair via homologous recombination / actin monomer binding / cilium assembly / skeletal muscle fiber development / positive regulation of lamellipodium assembly / stress fiber / titin binding / actin filament polymerization / filopodium / cell projection / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / calcium-dependent protein binding / actin filament binding / cell migration / lamellipodium / site of double-strand break / actin binding / cell cortex / cell body / hydrolase activity / neuron projection / protein domain specific binding / synapse / calcium ion binding / positive regulation of gene expression / magnesium ion binding / positive regulation of transcription by RNA polymerase II / ATP binding / identical protein binding / nucleus / cytoplasm / cytosol
Similarity search - Function
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 ...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 / 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'-DIPHOSPHATE / Actin-related protein 2 / Actin-related protein 3 / Actin, alpha skeletal muscle / Actin-related protein 2/3 complex subunit 4 / 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-related protein 2/3 complex subunit 1B
Similarity search - Component
Biological speciesBos taurus (cattle)
Oryctolagus cuniculus (rabbit)
Amanita phalloides (death cap)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsDing, B. / Narvaez-Ortiz, H.Y. / 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: Proc Natl Acad Sci U S A / Year: 2022
Title: Structure of Arp2/3 complex at a branched actin filament junction resolved by single-particle cryo-electron microscopy.
Authors: Bojian Ding / Heidy Y Narvaez-Ortiz / Yuvraj Singh / Glen M Hocky / Saikat Chowdhury / Brad J Nolen /
Abstract: Arp2/3 complex nucleates branched actin filaments that provide pushing forces to drive cellular processes such as lamellipodial protrusion and endocytosis. Arp2/3 complex is intrinsically inactive, ...Arp2/3 complex nucleates branched actin filaments that provide pushing forces to drive cellular processes such as lamellipodial protrusion and endocytosis. Arp2/3 complex is intrinsically inactive, and multiple classes of nucleation promoting factors (NPFs) stimulate its nucleation activity. When activated by WASP family NPFs, the complex must bind to the side of a preexisting (mother) filament of actin to complete the nucleation process, ensuring that WASP-mediated activation creates branched rather than linear actin filaments. How actin filaments contribute to activation is currently not understood, largely due to the lack of high-resolution structures of activated Arp2/3 complex bound to the side of a filament. Here, we present the 3.9-Å cryo-electron microscopy structure of the Arp2/3 complex at a branch junction. The structure reveals contacts between Arp2/3 complex and the side of the mother actin filament that likely stimulate subunit flattening, a conformational change that allows the actin-related protein subunits in the complex (Arp2 and Arp3) to mimic filamentous actin subunits. In contrast, limited contact between the bottom half of the complex and the mother filament suggests that clamp twisting, a second major conformational change observed in the active state, is not stimulated by actin filaments, potentially explaining why actin filaments are required but insufficient to trigger nucleation during WASP-mediated activation. Along with biochemical and live-cell imaging data and molecular dynamics simulations, the structure reveals features critical for the interaction of Arp2/3 complex with actin filaments and regulated assembly of branched actin filament networks in cells.
History
DepositionJan 26, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 25, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 8, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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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
H: Actin, alpha skeletal muscle
I: Actin, alpha skeletal muscle
J: Actin, alpha skeletal muscle
K: Actin, alpha skeletal muscle
L: Actin, alpha skeletal muscle
M: Actin, alpha skeletal muscle
N: Actin, alpha skeletal muscle
O: Actin, alpha skeletal muscle
P: Actin, alpha skeletal muscle
Q: Actin, alpha skeletal muscle
R: Actin, alpha skeletal muscle
S: Actin, alpha skeletal muscle
T: Actin, alpha skeletal muscle
U: Actin, alpha skeletal muscle
a: Phalloidin
b: Phalloidin
c: Phalloidin
d: Phalloidin
e: Phalloidin
f: Phalloidin
g: Phalloidin
h: Phalloidin
i: Phalloidin
j: Phalloidin
k: Phalloidin
l: Phalloidin
m: Phalloidin
n: Phalloidin
o: Phalloidin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)833,09868
Polymers825,87436
Non-polymers7,22432
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
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
Details: Missing sequences correspond to unmodeled regions due to poor density map
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
Details: Missing sequences correspond to unmodelled regions due to poor density map
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
Details: Missing sequences correspond to unmodeled regions due to poor density map
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
Details: Missing sequences correspond to unmodeled regions due to poor density map
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
Details: Missing sequences correspond to unmodeled regions due to poor density map
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
Details: Missing sequences correspond to unmodeled regions due to poor density map
Source: (natural) Bos taurus (cattle) / References: UniProt: Q3SYX9

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Protein / Protein/peptide , 2 types, 29 molecules HIJKLMNOPQRSTUabcdefghijklmno

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


Mass: 42109.973 Da / Num. of mol.: 14 / Source method: isolated from a natural source
Details: Missing sequences correspond to unmodeled regions due to poor density map. Actin subunits corresponding to chains J,K,L,M,T and U have been trimmed to c-beta due to lack of density for ...Details: Missing sequences correspond to unmodeled regions due to poor density map. Actin subunits corresponding to chains J,K,L,M,T and U have been trimmed to c-beta due to lack of density for modeling sidechains. These subunits were rigid body fitted into the map.
Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135
#9: Protein/peptide
Phalloidin


Type: Peptide-like / Class: Toxin / Mass: 808.899 Da / Num. of mol.: 15 / Source method: isolated from a natural source
Details: Phalloidin from Amanita phalloides is a rigid bicyclic heptapeptide. This is a small molecule and does not have conventional planar peptide bonds present in proteins.
Source: (natural) Amanita phalloides (death cap) / References: BIRD: PRD_002366

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Non-polymers , 2 types, 32 molecules

#10: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Mg
#11: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 16 / 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 Arp2/3 complex-mediated branched actin junction
Type: COMPLEX / Entity ID: #1-#9 / Source: NATURAL
Molecular weightValue: 0.834 MDa / Experimental value: NO
Source (natural)Organism: Bos taurus (cattle)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 30 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / 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 shifting of stage to targeted exposure position. Stage was tilted to different angles: including 40 degree, 36 degree, 30 degree, 25 degree, 15 degree, 0 degree, -20 ...Details: Data were collected by shifting of stage to targeted exposure position. Stage was tilted to different angles: including 40 degree, 36 degree, 30 degree, 25 degree, 15 degree, 0 degree, -20 degree, and -33 degree during data collection.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 92000 X / Nominal defocus max: 1200 nm / Nominal defocus min: 800 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: 70 sec. / Electron dose: 41.97 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 7436
Details: Each micrograph was acquired as dose-fractionated movies consisting of 82 frames per movie.
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCversion 2particle selection
2EPU2.1image acquisition
4cryoSPARCversion 2CTF correctionPatch CTF
7NAMDmodel fitting
8Cootmodel fitting
9UCSF Chimeramodel fitting
12cryoSPARCVersion 2final Euler assignment
14cryoSPARCversion 23D 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: 2290562
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 127093 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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