+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6642 | |||||||||
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Title | Cryo-EM structure of the WRC-Rac1 complex | |||||||||
Map data | Reconstruction of WRC-Rac1 | |||||||||
Sample |
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Keywords | cryoelectron microscopy / Wave Regulatory Complex / GTPase / actin nucleation | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 7.0 Å | |||||||||
Authors | Chen B / Chou H-T / Xing W / Henry L / Walz T / Rosen MK | |||||||||
Citation | Journal: Elife / Year: 2017 Title: Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites. Authors: Baoyu Chen / Hui-Ting Chou / Chad A Brautigam / Wenmin Xing / Sheng Yang / Lisa Henry / Lynda K Doolittle / Thomas Walz / Michael K Rosen / Abstract: The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a ...The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly. | |||||||||
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_6642.map.gz | 7.6 MB | EMDB map data format | |
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Header (meta data) | emd-6642-v30.xml emd-6642.xml | 11.9 KB 11.9 KB | Display Display | EMDB header |
Images | 400_6642.gif 80_6642.gif | 40.6 KB 3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6642 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6642 | HTTPS FTP |
-Validation report
Summary document | emd_6642_validation.pdf.gz | 78.5 KB | Display | EMDB validaton report |
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Full document | emd_6642_full_validation.pdf.gz | 77.6 KB | Display | |
Data in XML | emd_6642_validation.xml.gz | 493 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6642 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6642 | HTTPS FTP |
-Related structure data
Similar structure data |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_6642.map.gz / Format: CCP4 / Size: 58.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of WRC-Rac1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.24 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Complex of WAVE regulatory complex (WRC) and Rac1
Entire | Name: Complex of WAVE regulatory complex (WRC) and Rac1 |
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Components |
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-Supramolecule #1000: Complex of WAVE regulatory complex (WRC) and Rac1
Supramolecule | Name: Complex of WAVE regulatory complex (WRC) and Rac1 / type: sample / ID: 1000 / Details: The sample was monodisperse. Oligomeric state: One heteropentamer of WRC binds to one Rac1 Number unique components: 2 |
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Molecular weight | Theoretical: 375 KDa |
-Macromolecule #1: WAVE regulatory complex
Macromolecule | Name: WAVE regulatory complex / type: protein_or_peptide / ID: 1 / Name.synonym: WRC Details: WRC is composed of 5 subunits: Sra1, Nap1, WAVE1, Abi2, and HSPC300. Number of copies: 1 / Oligomeric state: heterohexamer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human |
Molecular weight | Theoretical: 375 KDa |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: sf9 |
-Macromolecule #2: Rac1
Macromolecule | Name: Rac1 / type: protein_or_peptide / ID: 2 / Details: Rac1 is fused to C-terminal WAVE1. / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: sf9 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.1 mg/mL |
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Buffer | pH: 7 / Details: 10 mM HEPES, 100 mM NaCl, 1 mM MgCl2, 2 mM TCEP |
Grid | Details: glow-discharged Quantifoil holey carbon grids (400 copper mesh, R1.2/1.3) |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 103 K / Instrument: GATAN CRYOPLUNGE 3 Method: WRC-Rac1 complex (3.5 microliter at 0.1 mg/ml) was applied to glow-discharged Quantifoil holey carbon grids (400 copper mesh, R1.2/1.3). The grids were blotted for 3 seconds and quick-frozen ...Method: WRC-Rac1 complex (3.5 microliter at 0.1 mg/ml) was applied to glow-discharged Quantifoil holey carbon grids (400 copper mesh, R1.2/1.3). The grids were blotted for 3 seconds and quick-frozen in liquid ethane using a Gatan CryoPlunge 3. |
-Electron microscopy
Microscope | FEI TECNAI 20 |
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Temperature | Average: 88 K |
Date | Jun 16, 2015 |
Image recording | Category: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 2173 / Average electron dose: 38.4 e/Å2 Details: There were a total of 2,173 image stacks. For 1,366 stacks, 30 frames were recorded at 200 ms per frame (total exposure time of 6 seconds). For 407 stacks, 34 frames were recorded at 300 ms ...Details: There were a total of 2,173 image stacks. For 1,366 stacks, 30 frames were recorded at 200 ms per frame (total exposure time of 6 seconds). For 407 stacks, 34 frames were recorded at 300 ms per frame (total exposure time of 10.2 seconds). For 400 stacks, 51 frames were recorded at 200 ms per frame (total exposure time of 10.2 seconds). |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 40410 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 29000 |
Sample stage | Specimen holder: This holder operates at liquid nitrogen temperature. Specimen holder model: OTHER |
-Image processing
Details | Initial model was low-pass filtered crystal structure of the WRC complex (PDB ID 3P8C). Automatic particle picking, 2D classification, 3D classification, refinement, and subsequent reconstruction were performed using RELION. |
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CTF correction | Details: whole micrograph |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.0 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 29784 |
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: Chimera |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
-Atomic model buiding 2
Initial model | PDB ID: |
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Software | Name: Chimera |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |