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Yorodumi- PDB-7use: Cryo-EM structure of WAVE regulatory complex with Rac1 bound on b... -
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-Basic information
Entry | Database: PDB / ID: 7use | ||||||
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Title | Cryo-EM structure of WAVE regulatory complex with Rac1 bound on both A and D site | ||||||
Components |
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Keywords | CELL INVASION / actin regulator / GTPase binding protein / cytoskeletal regulator | ||||||
Function / homology | Function and homology information peripheral region of growth cone / negative regulation of synaptic vesicle recycling / intracellular protein-containing complex / SCAR complex / positive regulation of neurotrophin TRK receptor signaling pathway / lamellipodium morphogenesis / positive regulation of Arp2/3 complex-mediated actin nucleation / Arp2/3 complex binding / modification of synaptic structure / regulation of actin polymerization or depolymerization ...peripheral region of growth cone / negative regulation of synaptic vesicle recycling / intracellular protein-containing complex / SCAR complex / positive regulation of neurotrophin TRK receptor signaling pathway / lamellipodium morphogenesis / positive regulation of Arp2/3 complex-mediated actin nucleation / Arp2/3 complex binding / modification of synaptic structure / regulation of actin polymerization or depolymerization / regulation of respiratory burst / central region of growth cone / modification of postsynaptic actin cytoskeleton / negative regulation of interleukin-23 production / dendrite extension / regulation of neutrophil migration / localization within membrane / Activated NTRK2 signals through CDK5 / regulation of translation at postsynapse, modulating synaptic transmission / NADPH oxidase complex / negative regulation of receptor-mediated endocytosis / regulation of hydrogen peroxide metabolic process / ruffle assembly / NTRK2 activates RAC1 / engulfment of apoptotic cell / Inactivation of CDC42 and RAC1 / filopodium tip / WNT5:FZD7-mediated leishmania damping / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / : / regulation of modification of postsynaptic actin cytoskeleton / cortical cytoskeleton organization / respiratory burst / regulation of actin filament polymerization / hepatocyte growth factor receptor signaling pathway / RNA 7-methylguanosine cap binding / ruffle organization / cell projection assembly / thioesterase binding / negative regulation of fibroblast migration / regulation of stress fiber assembly / RHO GTPases activate CIT / Nef and signal transduction / axon extension / sphingosine-1-phosphate receptor signaling pathway / PCP/CE pathway / motor neuron axon guidance / RHO GTPases activate KTN1 / regulation of nitric oxide biosynthetic process / regulation of lamellipodium assembly / positive regulation of neutrophil chemotaxis / Azathioprine ADME / Activation of RAC1 / positive regulation of ruffle assembly / positive regulation of dendrite development / DCC mediated attractive signaling / positive regulation of cell-substrate adhesion / MET activates RAP1 and RAC1 / Wnt signaling pathway, planar cell polarity pathway / Sema4D mediated inhibition of cell attachment and migration / CD28 dependent Vav1 pathway / Ephrin signaling / protein kinase A binding / lamellipodium assembly / regulation of myelination / cortical actin cytoskeleton organization / positive regulation of Rho protein signal transduction / establishment or maintenance of cell polarity / regulation of cell size / DSCAM interactions / Activation of RAC1 downstream of NMDARs / small GTPase-mediated signal transduction / Rho GDP-dissociation inhibitor binding / positive regulation of actin filament polymerization / NRAGE signals death through JNK / Rac protein signal transduction / protein kinase A regulatory subunit binding / filamentous actin / RHO GTPases activate PAKs / dendritic growth cone / positive regulation of focal adhesion assembly / lamellipodium membrane / semaphorin-plexin signaling pathway / excitatory synapse / ficolin-1-rich granule membrane / Sema3A PAK dependent Axon repulsion / RHOG GTPase cycle / EPH-ephrin mediated repulsion of cells / RHO GTPases Activate NADPH Oxidases / RHO GTPases Activate WASPs and WAVEs / anatomical structure morphogenesis / RAC3 GTPase cycle / RAC2 GTPase cycle / RHO GTPases activate IQGAPs / positive regulation of axon extension / localization / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / positive regulation of lamellipodium assembly / axonal growth cone / response to electrical stimulus Similarity search - Function | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å | ||||||
Authors | Ding, B. / Yang, S. / Chen, B. / Chowdhury, S. | ||||||
Funding support | United States, 1items
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Citation | Journal: Nat Commun / Year: 2022 Title: Structures reveal a key mechanism of WAVE regulatory complex activation by Rac1 GTPase. Authors: Bojian Ding / Sheng Yang / Matthias Schaks / Yijun Liu / Abbigale J Brown / Klemens Rottner / Saikat Chowdhury / Baoyu Chen / Abstract: The Rho-family GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization in many essential processes. Rac1 binds to WRC at two distinct sites-the ...The Rho-family GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization in many essential processes. Rac1 binds to WRC at two distinct sites-the A and D sites. Precisely how Rac1 binds and how the binding triggers WRC activation remain unknown. Here we report WRC structures by itself, and when bound to single or double Rac1 molecules, at ~3 Å resolutions by cryogenic-electron microscopy. The structures reveal that Rac1 binds to the two sites by distinct mechanisms, and binding to the A site, but not the D site, drives WRC activation. Activation involves a series of unique conformational changes leading to the release of sequestered WCA (WH2-central-acidic) polypeptide, which stimulates the Arp2/3 complex to polymerize actin. Together with biochemical and cellular analyses, the structures provide a novel mechanistic understanding of how the Rac1-WRC-Arp2/3-actin signaling axis is regulated in diverse biological processes and diseases. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7use.cif.gz | 534.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7use.ent.gz | 429.3 KB | Display | PDB format |
PDBx/mmJSON format | 7use.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/us/7use ftp://data.pdbj.org/pub/pdb/validation_reports/us/7use | HTTPS FTP |
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-Related structure data
Related structure data | 26734MC 7uscC 7usdC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 5 types, 5 molecules ABCDE
#1: Protein | Mass: 145363.750 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the ...Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts. Source: (gene. exp.) Homo sapiens (human) / Gene: CYFIP1, KIAA0068 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q7L576 |
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#2: Protein | Mass: 128940.727 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the ...Details: This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts. Source: (gene. exp.) Homo sapiens (human) / Gene: NCKAP1, HEM2, KIAA0587, NAP1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9Y2A7 |
#3: Protein | Mass: 37009.406 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: Residues 231-248 are inserted as a flexible linker sequence. This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification ...Details: Residues 231-248 are inserted as a flexible linker sequence. This construct contains two additional uncleaved residues "GA" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts. Source: (gene. exp.) Homo sapiens (human) / Gene: WASF1, KIAA0269, SCAR1, WAVE1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q92558 |
#4: Protein | Mass: 8756.915 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: This construct contains uncleaved residues "GHMGAA" in the N terminus from the construct design and purification procedure. Densities for the residues are not observed in the map and were ...Details: This construct contains uncleaved residues "GHMGAA" in the N terminus from the construct design and purification procedure. Densities for the residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts. Source: (gene. exp.) Homo sapiens (human) / Gene: BRK1, C3orf10, HSPC300, MDS027 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8WUW1 |
#5: Protein | Mass: 18041.482 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: The sequence only contains residues 1-158. Also, there are two additional uncleaved residues "GH" in the N terminus from the construct design and purification procedure. Densities for these ...Details: The sequence only contains residues 1-158. Also, there are two additional uncleaved residues "GH" in the N terminus from the construct design and purification procedure. Densities for these residues are not observed in the map and were not included in the sample sequence to avoid numbering shifts. Source: (gene. exp.) Homo sapiens (human) / Gene: ABI2 / Production host: Escherichia coli (E. coli) / References: UniProt: E9PEZ7 |
-Ras-related C3 botulinum toxin substrate ... , 2 types, 2 molecules FG
#6: Protein | Mass: 21025.459 Da / Num. of mol.: 1 / Mutation: P29S Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RAC1, TC25, MIG5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63000, small monomeric GTPase |
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#7: Protein | Mass: 21010.486 Da / Num. of mol.: 1 / Mutation: P29S, Q61L Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RAC1, TC25, MIG5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63000, small monomeric GTPase |
-Non-polymers , 3 types, 4 molecules
#8: Chemical | ChemComp-GNP / | ||
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#9: Chemical | #10: Chemical | ChemComp-GTP / | |
-Details
Has ligand of interest | Y |
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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: WAVE regulatory complex with Rac1 bound to A and D sites Type: COMPLEX / Entity ID: #1-#7 / Source: MULTIPLE SOURCES | ||||||||||||
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Molecular weight | Value: 0.38 MDa / Experimental value: NO | ||||||||||||
Source (natural) | Organism: Homo sapiens (human) | ||||||||||||
Source (recombinant) |
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Buffer solution | pH: 7 | ||||||||||||
Specimen | Conc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||
Specimen support | Details: 15 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3 | ||||||||||||
Vitrification | Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K |
-Electron microscopy imaging
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA Details: Data were collected by shifting the stage to target exposure positions. |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 120000 X / Nominal defocus max: 1200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 40 sec. / Electron dose: 41.34 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1285 Details: Each micrograph was acquired as dose-fractionated movies consisting of 62 frames per movie. |
Image scans | Sampling size: 14 µm / Width: 4096 / Height: 4096 |
-Processing
EM software |
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CTF correction | Details: Particles were CTF-corrected during projection matching and back projection Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 666417 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 139296 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||
Atomic model building |
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