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- PDB-6w18: Structure of S. pombe Arp2/3 complex in inactive state -

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Basic information

Entry
Database: PDB / ID: 6w18
TitleStructure of S. pombe Arp2/3 complex in inactive state
Components(Actin-related protein ...) x 7
KeywordsSTRUCTURAL PROTEIN / Arp2/3 / actin / cytoskeletal protein / actin regulator
Function / homology
Function and homology information


Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / Clathrin-mediated endocytosis / actin cortical patch organization / Neutrophil degranulation / medial cortex / actin filament branching / cell cortex of cell tip / actin cortical patch assembly / Arp2/3 protein complex ...Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / Clathrin-mediated endocytosis / actin cortical patch organization / Neutrophil degranulation / medial cortex / actin filament branching / cell cortex of cell tip / actin cortical patch assembly / Arp2/3 protein complex / Arp2/3 complex-mediated actin nucleation / actin cortical patch / cell tip / regulation of actin filament polymerization / mating projection tip / cortical actin cytoskeleton organization / cell division site / establishment or maintenance of cell polarity / actin filament polymerization / structural constituent of cytoskeleton / endocytosis / actin filament binding / cell cortex / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Actin-related protein 2/3 complex subunit 5 / ARP2/3 complex 16 kDa subunit (p16-Arc) / 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 ...Actin-related protein 2/3 complex subunit 5 / ARP2/3 complex 16 kDa subunit (p16-Arc) / 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 20 kDa subunit (ARPC4) / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / 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 2/3 complex subunit 2 / Actin-related protein 3 / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 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
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)227,84710
Polymers226,8087
Non-polymers1,0393
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16070 Å2
ΔGint-106 kcal/mol
Surface area92190 Å2

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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)
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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Non-polymers , 2 types, 3 molecules

#8: 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
#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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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: Arp2/3 complex / Type: COMPLEX / Details: Inactive state of S. pombe Arp2/3 complex / Entity ID: #1-#7 / Source: NATURAL
Molecular weightValue: 0.225 MDa / Experimental value: NO
Source (natural)Organism: Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Strain: 972 / ATCC 24843 / Cellular location: cytoplasm
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium chlorideNaCl1
21 mMmagnesium chlorideMgCl21
31 mMadenosine triphosphateC10H16N5O13P31
410 mMtris(hydroxymethyl)aminomethaneC4H11NO31
51 mMdithiothreitolC4H10O2S21
SpecimenConc.: 2.5 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
Details: 4 uL of sample was applied to freshly glow-discharged grid. Excess sample was manually blotted off with a dry Whatman No.1 filter paper for 5-7 seconds. Immediately after the blotting step ...Details: 4 uL of sample was applied to freshly glow-discharged grid. Excess sample was manually blotted off with a dry Whatman No.1 filter paper for 5-7 seconds. Immediately after the blotting step the sample containing grid was rapidly vitrified by plunge freezing into liquid ethane.

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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 combining untilted and tilted (20, 30 and 40 degrees) images. Stage shifting to the targeted exposure position was used for navigation during data acquisition.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 120000 X / Nominal defocus max: -1300 nm / Nominal defocus min: -700 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: 40 sec. / Electron dose: 44.34 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 5309
Details: Each micrograph was collected as dose-fractionated movies consisting of 62 fractions per movie.
Image scansSampling size: 14 µm / Width: 4000 / Height: 4000

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC2.12.4particle selection
2EPU2.2.0.65image acquisition
4cryoSPARC2.12.4CTF correction
7NAMDmodel fitting
8Cootmodel fitting
9UCSF Chimeramodel fittingrigid body fitting
12cryoSPARC2.12.4final Euler assignment
13cryoSPARC2.12.4classification
14cryoSPARC2.12.43D reconstruction
15PHENIXmodel refinement
CTF correctionDetails: Local CTF estimation was performed for all micrographs using the CTF estimation package within cryoSPARC.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3336981 / Details: Automated Gaussian-based blob picker was used.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 112170 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 3DWL

3dwl


Accession code: 3DWL / Source name: PDB / Type: experimental model
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 51.2 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002513425
ELECTRON MICROSCOPYf_angle_d0.513518345
ELECTRON MICROSCOPYf_chiral_restr0.04082116
ELECTRON MICROSCOPYf_plane_restr0.00182410
ELECTRON MICROSCOPYf_dihedral_angle_d19.4844440

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