+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-20770 | ||||||||||||
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Title | CryoEM structure of human Arp2/3 complex with bound NPFs | ||||||||||||
Map data | Cryo-EM structure of human Arp2/3 complex bound to two nucleation promoting factors (B-factor sharpened). | ||||||||||||
Sample |
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Keywords | actin / ATPase / actin related protein / arp / cytoskeleton / Arp2-3 complex / actin nucleation / actin branching / CONTRACTILE PROTEIN | ||||||||||||
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 / plasma membrane tubulation / actin polymerization-dependent cell motility / Arp2/3 protein complex / asymmetric cell division / Arp2/3 complex-mediated actin nucleation / postsynapse organization / actin nucleation / negative regulation of lymphocyte migration / regulation of cell projection assembly / vesicle organization / actin cap / postsynaptic actin cytoskeleton organization / vesicle transport along actin filament / regulation of actin filament polymerization / vesicle budding from membrane / positive regulation of chemotaxis / dendritic spine morphogenesis / protein-containing complex localization / 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 / cilium assembly / positive regulation of double-strand break repair via homologous recombination / RHO GTPases Activate WASPs and WAVEs / positive regulation of lamellipodium assembly / positive regulation of substrate adhesion-dependent cell spreading / cytoskeletal protein binding / EPHB-mediated forward signaling / actin filament polymerization / cellular response to nerve growth factor stimulus / cell projection / response to bacterium / FCGR3A-mediated phagocytosis / structural constituent of cytoskeleton / cellular response to type II interferon / Regulation of actin dynamics for phagocytic cup formation / response to estrogen / azurophil granule lumen / cell-cell junction / actin cytoskeleton / cell migration / lamellipodium / response to estradiol / Clathrin-mediated endocytosis / regulation of protein localization / 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 / Golgi membrane / cell division / 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 / cytosol / cytoplasm Similarity search - Function | ||||||||||||
Biological species | Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths) / Homo sapiens (human) / Mus musculus (house mouse) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.9 Å | ||||||||||||
Authors | Zimmet A / van Eeuwen T | ||||||||||||
Funding support | United States, 3 items
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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 | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_20770.map.gz | 117.9 MB | EMDB map data format | |
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Header (meta data) | emd-20770-v30.xml emd-20770.xml | 28.7 KB 28.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_20770_fsc.xml | 11.5 KB | Display | FSC data file |
Images | emd_20770.png | 41.8 KB | ||
Filedesc metadata | emd-20770.cif.gz | 8.6 KB | ||
Others | emd_20770_additional.map.gz | 62.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-20770 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-20770 | HTTPS FTP |
-Validation report
Summary document | emd_20770_validation.pdf.gz | 525.5 KB | Display | EMDB validaton report |
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Full document | emd_20770_full_validation.pdf.gz | 525.1 KB | Display | |
Data in XML | emd_20770_validation.xml.gz | 12.4 KB | Display | |
Data in CIF | emd_20770_validation.cif.gz | 16.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-20770 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-20770 | HTTPS FTP |
-Related structure data
Related structure data | 6uhcMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_20770.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Cryo-EM structure of human Arp2/3 complex bound to two nucleation promoting factors (B-factor sharpened). | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.836 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Additional map: Cryo-EM structure of human Arp2/3 complex bound to...
File | emd_20770_additional.map | ||||||||||||
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Annotation | Cryo-EM structure of human Arp2/3 complex bound to two nucleation promoting factors | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : Cryo-EM structure of human Arp2/3 complex with bound NPFs
+Supramolecule #1: Cryo-EM structure of human Arp2/3 complex with bound NPFs
+Macromolecule #1: Actin-related protein 3
+Macromolecule #2: Actin-related protein 2
+Macromolecule #3: Actin-related protein 2/3 complex subunit 1B
+Macromolecule #4: Actin-related protein 2/3 complex subunit 2
+Macromolecule #5: Actin-related protein 2/3 complex subunit 3
+Macromolecule #6: Actin-related protein 2/3 complex subunit 4
+Macromolecule #7: Actin-related protein 2/3 complex subunit 5
+Macromolecule #8: Neural Wiskott-Aldrich syndrome protein
+Macromolecule #9: MAGNESIUM ION
+Macromolecule #10: ADENOSINE-5'-TRIPHOSPHATE
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 5 mg/mL | |||||||||||||||||||||
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Buffer | pH: 7 Component:
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Grid | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR Details: Grids glow discharged with easiGLOW glow discharger at 0.3 mBar, 25mA for 1 minute | |||||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Instrument: LEICA EM CPC Details: Grids manually blotted for 3 seconds with Whatman 41 filter paper and manually plunged on Leica EM CPC manual plunger.. | |||||||||||||||||||||
Details | This sample was monodisperse |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Slit width: 20 eV |
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. |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 2 / Number real images: 5004 / Average exposure time: 7.0 sec. / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 100.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -3.5 µm / Nominal defocus min: -1.5 µm / Nominal magnification: 165000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |