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- PDB-8e9b: Cryo-EM structure of S. pombe Arp2/3 complex in the branch junction -

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Basic information

Entry
Database: PDB / ID: 8e9b
TitleCryo-EM structure of S. pombe Arp2/3 complex in the branch junction
Components
  • (Actin-related protein ...) x 7
  • Actin, alpha skeletal muscle
KeywordsSTRUCTURAL PROTEIN / Arp2-3 complex / branch juction / polymerization
Function / homology
Function and homology information


Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / actin cortical patch organization / Clathrin-mediated endocytosis / Neutrophil degranulation / medial cortex / cell cortex of cell tip / actin cortical patch assembly / Arp2/3 protein complex / Arp2/3 complex-mediated actin nucleation ...Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / actin cortical patch organization / Clathrin-mediated endocytosis / Neutrophil degranulation / medial cortex / cell cortex of cell tip / actin cortical patch assembly / Arp2/3 protein complex / Arp2/3 complex-mediated actin nucleation / actin cortical patch / cell tip / Striated Muscle Contraction / regulation of actin filament polymerization / mating projection tip / cortical actin cytoskeleton organization / establishment or maintenance of cell polarity / cell division site / skeletal muscle thin filament assembly / striated muscle thin filament / mitotic cytokinesis / skeletal muscle fiber development / stress fiber / actin filament polymerization / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / endocytosis / actin filament binding / hydrolase activity / ATP 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 / ADENOSINE-5'-TRIPHOSPHATE / Actin-related protein 2/3 complex subunit 2 / 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)
Gallus gallus (chicken)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsChou, S.Z. / Pollard, T.P.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM026132 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM026338 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Mechanism of actin filament branch formation by Arp2/3 complex revealed by a high-resolution cryo-EM structureof the branch junction.
Authors: Steven Z Chou / Moon Chatterjee / Thomas D Pollard /
Abstract: We reconstructed the structure of actin filament branch junctions formed by fission yeast Arp2/3 complex at 3.5 Å resolution from images collected by electron cryo-microscopy. During specimen ...We reconstructed the structure of actin filament branch junctions formed by fission yeast Arp2/3 complex at 3.5 Å resolution from images collected by electron cryo-microscopy. During specimen preparation, all of the actin subunits and Arp3 hydrolyzed their bound adenosine triphosphate (ATP) and dissociated the γ-phosphate, but Arp2 retained the γ-phosphate. Binding tightly to the side of the mother filament and nucleating the daughter filament growing as a branch requires Arp2/3 complex to undergo a dramatic conformational change where two blocks of structure rotate relative to each other about 25° to align Arp2 and Arp3 as the first two subunits in the branch. During branch formation, Arp2/3 complex acquires more than 8,000 Å of new buried surface, accounting for the stability of the branch. Inactive Arp2/3 complex binds only transiently to the side of an actin filament, because its conformation allows only a subset of the interactions found in the branch junction.
History
DepositionAug 26, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 1, 2023Provider: repository / Type: Initial release

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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 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
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
H: Actin, alpha skeletal muscle
I: Actin, alpha skeletal muscle
hetero molecules


Theoretical massNumber of molelcules
Total (without water)566,40835
Polymers561,81315
Non-polymers4,59520
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron 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-like protein 3


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


Mass: 44286.758 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast) / Strain: 972 / ATCC 24843 / References: UniProt: Q9UUJ1
#3: Protein Actin-related protein 2/3 complex subunit 1 / 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 (fission yeast) / Strain: 972 / ATCC 24843 / References: UniProt: P78774
#4: Protein Actin-related protein 2/3 complex subunit 2 / 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 (fission yeast) / Strain: 972 / ATCC 24843 / References: UniProt: O14241
#5: Protein Actin-related protein 2/3 complex subunit 3 / 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 (fission yeast) / Strain: 972 / ATCC 24843 / References: UniProt: Q9Y7J4
#6: Protein Actin-related protein 2/3 complex subunit 4 / 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 (fission yeast) / Strain: 972 / ATCC 24843 / References: UniProt: Q92352
#7: Protein Actin-related protein 2/3 complex subunit 5 / 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 (fission yeast) / Strain: 972 / ATCC 24843 / References: UniProt: Q10316

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Protein , 1 types, 8 molecules MNOPQRHI

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


Mass: 41875.633 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken) / References: UniProt: P68139

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Non-polymers , 3 types, 20 molecules

#9: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#11: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM

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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
1S. pombe Arp2/3 complex in actin branch junctionCOMPLEX#1-#80MULTIPLE SOURCES
2S. pombe Arp2/3 complex in actin branch junctionCOMPLEX#1-#71NATURAL
3chicken skeletal muscle actin in branch junctionCOMPLEX#81NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Schizosaccharomyces pombe (fission yeast)284812
23Gallus gallus (chicken)9031
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMpotassium chlorideKCl1
21 mMMagnesium chlorideMgCl21
31 mMEGTAC14H24N2O101
410 mMHEPESC8H18N2O4S1
51 mMDTTC4H10O2S21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal magnification: 36657 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 900 nm / Calibrated defocus max: 2900 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: 3.28 sec. / Electron dose: 50.07 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 4200

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Processing

SoftwareName: PHENIX / Version: dev_3965: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM4image acquisition
4Gctf1.06CTF correction
7Coot0.9.8.1model fitting
9RELION4initial Euler assignment
10RELION4final Euler assignment
11RELION4classification
12RELION43D reconstruction
13PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 131393
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 79467 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model building
IDPDB-ID 3D fitting-ID
11K8K

1k8k

1
26DJO

6djo

1
36W17

6w17

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00340028
ELECTRON MICROSCOPYf_angle_d0.64254326
ELECTRON MICROSCOPYf_dihedral_angle_d5.1245524
ELECTRON MICROSCOPYf_chiral_restr0.0456016
ELECTRON MICROSCOPYf_plane_restr0.0046988

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