+Open data
-Basic information
Entry | Database: PDB / ID: 6uhc | ||||||||||||
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Title | CryoEM structure of human Arp2/3 complex with bound NPFs | ||||||||||||
Components |
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Keywords | CONTRACTILE PROTEIN / actin / ATPase / actin related protein / arp / cytoskeleton / Arp2-3 complex / actin nucleation / actin branching | ||||||||||||
Function / homology | Function and homology information tubulobulbar complex / meiotic chromosome movement towards spindle pole / cytosolic transport / growth cone leading edge / muscle cell projection membrane / negative regulation of membrane tubulation / meiotic cytokinesis / spindle localization / membrane invagination / positive regulation of clathrin-dependent endocytosis ...tubulobulbar complex / meiotic chromosome movement towards spindle pole / cytosolic transport / growth cone leading edge / muscle cell projection membrane / negative regulation of membrane tubulation / meiotic cytokinesis / spindle localization / membrane invagination / positive regulation of clathrin-dependent endocytosis / actin polymerization-dependent cell motility / plasma membrane tubulation / Arp2/3 protein complex / asymmetric cell division / negative regulation of lymphocyte migration / Arp2/3 complex-mediated actin nucleation / postsynapse organization / actin nucleation / regulation of cell projection assembly / actin cap / vesicle organization / postsynaptic actin cytoskeleton organization / vesicle transport along actin filament / regulation of actin filament polymerization / vesicle budding from membrane / positive regulation of chemotaxis / protein-containing complex localization / dendritic spine morphogenesis / positive regulation of filopodium assembly / regulation of postsynapse organization / establishment or maintenance of cell polarity / positive regulation of actin filament polymerization / cell leading edge / filamentous actin / brush border / positive regulation of double-strand break repair via homologous recombination / cilium assembly / RHO GTPases Activate WASPs and WAVEs / positive regulation of lamellipodium assembly / positive regulation of substrate adhesion-dependent cell spreading / EPHB-mediated forward signaling / cytoskeletal protein binding / actin filament polymerization / cellular response to nerve growth factor stimulus / cell projection / FCGR3A-mediated phagocytosis / response to bacterium / structural constituent of cytoskeleton / Regulation of actin dynamics for phagocytic cup formation / cellular response to type II interferon / response to estrogen / regulation of protein localization / azurophil granule lumen / cell-cell junction / Clathrin-mediated endocytosis / actin cytoskeleton / cell migration / lamellipodium / response to estradiol / site of double-strand break / actin binding / cell cortex / actin cytoskeleton organization / cytoplasmic vesicle / secretory granule lumen / ficolin-1-rich granule lumen / postsynapse / endosome / neuron projection / cell division / Golgi membrane / focal adhesion / glutamatergic synapse / Neutrophil degranulation / protein-containing complex binding / enzyme binding / positive regulation of transcription by RNA polymerase II / extracellular exosome / extracellular region / nucleoplasm / ATP binding / identical protein binding / membrane / nucleus / cytoplasm / cytosol Similarity search - Function | ||||||||||||
Biological species | Homo sapiens (human) Mus musculus (house mouse) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å | ||||||||||||
Authors | Zimmet, A. / van Eeuwen, T. / Dominguez, R. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: Sci Adv / Year: 2020 Title: Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism. Authors: Austin Zimmet / Trevor Van Eeuwen / Malgorzata Boczkowska / Grzegorz Rebowski / Kenji Murakami / Roberto Dominguez / Abstract: Actin-related protein (Arp) 2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including two actin-related subunits (Arp2 and Arp3). Two nucleation- ...Actin-related protein (Arp) 2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including two actin-related subunits (Arp2 and Arp3). Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions, and contribution of each NPF to activation are unresolved. Here, we report the cryo-electron microscopy structure of recombinantly expressed human Arp2/3 complex with two WASP family NPFs bound and address the mechanism of activation. A cross-linking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex. | ||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6uhc.cif.gz | 403.5 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6uhc.ent.gz | 305.7 KB | Display | PDB format |
PDBx/mmJSON format | 6uhc.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6uhc_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 6uhc_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 6uhc_validation.xml.gz | 63.9 KB | Display | |
Data in CIF | 6uhc_validation.cif.gz | 96.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/uh/6uhc ftp://data.pdbj.org/pub/pdb/validation_reports/uh/6uhc | HTTPS FTP |
-Related structure data
Related structure data | 20770MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Actin-related protein ... , 7 types, 7 molecules ABCDEFG
#1: Protein | Mass: 47428.031 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: ACTR3, ARP3 / Plasmid: pBIG2abc / Cell (production host): suspension / Cell line (production host): Sf9 Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths) References: UniProt: P61158 |
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#2: Protein | Mass: 44818.711 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: ACTR2, ARP2 / Plasmid: pBIG2abc / Cell (production host): suspension / Cell line (production host): Sf9 Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths) References: UniProt: P61160 |
#3: Protein | Mass: 41004.781 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: ARPC1B, ARC41 / Plasmid: pBIG2abc / Cell (production host): suspension / Cell line (production host): Sf9 Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths) References: UniProt: O15143 |
#4: Protein | Mass: 34386.043 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: ARPC2, ARC34, PRO2446 / Plasmid: pBIG2abc / Cell (production host): suspension / Cell line (production host): Sf9 Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths) References: UniProt: O15144 |
#5: Protein | Mass: 23196.330 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: C-terminal Flag tag / Source: (gene. exp.) Homo sapiens (human) / Gene: ARPC3, ARC21 / Plasmid: pBIG2abc / Cell (production host): suspension / Cell line (production host): Sf9 Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths) References: UniProt: O15145 |
#6: Protein | Mass: 19697.047 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: ARPC4, ARC20 / Plasmid: pBIG2abc / Cell (production host): suspension / Cell line (production host): Sf9 Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths) References: UniProt: P59998 |
#7: Protein | Mass: 16341.407 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: ARPC5, ARC16 / Plasmid: pBIG2abc / Cell (production host): suspension / Cell line (production host): Sf9 Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths) References: UniProt: O15511 |
-Protein , 1 types, 2 molecules HI
#8: Protein | Mass: 54342.023 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Gene: Wasl / Production host: Escherichia coli (E. coli) / References: UniProt: Q91YD9 |
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-Non-polymers , 2 types, 4 molecules
#9: Chemical | #10: Chemical | |
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-Details
Has ligand of interest | N |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Cryo-EM structure of human Arp2/3 complex with bound NPFs Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT | |||||||||||||||||||||||||||||||||||
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Molecular weight | Value: 243 kDa/nm / Experimental value: NO | |||||||||||||||||||||||||||||||||||
Source (natural) | Organism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths) | |||||||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths) Plasmid: pBIG2abc | |||||||||||||||||||||||||||||||||||
Buffer solution | pH: 7 | |||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse | |||||||||||||||||||||||||||||||||||
Specimen support | Details: Grids glow discharged with easiGLOW glow discharger at 0.3 mBar, 25mA for 1 minute Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2 | |||||||||||||||||||||||||||||||||||
Vitrification | Instrument: LEICA EM CPC / Cryogen name: ETHANE Details: Grids manually blotted for 3 seconds with Whatman 41 filter paper and manually plunged on Leica EM CPC manual plunger. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS Details: Data collected in super resolution mode. Illuminated area of 1.01um. Nominal Dose of 40a/A^2 and a dose rate of 4.87 e-/s/pixel. 2 or 5 exposures per hole by image shift. |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELD / 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 holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 7 sec. / Electron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5004 |
EM imaging optics | Energyfilter slit width: 20 eV |
Image scans | Movie frames/image: 40 / Used frames/image: 1-40 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 627163 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 123198 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 132 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: correlation | ||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 4JD2 Accession code: 4JD2 / Source name: PDB / Type: experimental model |