|Entry||Database: EMDB / ID: 6642|
|Title||Cryo-EM structure of the WRC-Rac1 complex|
|Map data||Reconstruction of WRC-Rac1|
|Sample||Complex of WAVE regulatory complex (WRC) and Rac1|
|Keywords||cryoelectron microscopy / Wave Regulatory Complex / GTPase / actin nucleation|
|Source||Homo sapiens / / human|
|Method||Cryo EM / single particle reconstruction / 7 Å resolution|
|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.
|Date||Deposition: May 3, 2016 / Header (metadata) release: Jul 13, 2016 / Map release: May 3, 2017 / Last update: May 3, 2017|
Downloads & links
|File||emd_6642.map.gz (map file in CCP4 format, 61037 KB)|
|Projections & slices|
Images are generated by Spider package.
|Voxel size||X=Y=Z: 1.24 Å|
CCP4 map header:
-Entire Complex of WAVE regulatory complex (WRC) and Rac1
|Entire||Name: Complex of WAVE regulatory complex (WRC) and Rac1 / Details: The sample was monodisperse. / Number of components: 2|
Oligomeric State: One heteropentamer of WRC binds to one Rac1
|Mass||Theoretical: 375 kDa|
-Component #1: protein, WAVE regulatory complex
|Protein||Name: WAVE regulatory complex / a.k.a: WRC / Oligomeric Details: heterohexamer|
Details: WRC is composed of 5 subunits: Sra1, Nap1, WAVE1, Abi2, and HSPC300.
Number of Copies: 1 / Recombinant expression: Yes
|Mass||Theoretical: 375 kDa|
|Source||Species: Homo sapiens / / human|
|Source (engineered)||Expression System: Spodoptera frugiperda / Fall armyworm / arthropod|
Cell of expression system: sf9
-Component #2: protein, Rac1
|Specimen||Specimen state: particle / Method: Cryo EM|
|Sample solution||Specimen conc.: 0.1 mg/ml|
Buffer solution: 10 mM HEPES, 100 mM NaCl, 1 mM MgCl2, 2 mM TCEP
|Support film||glow-discharged Quantifoil holey carbon grids (400 copper mesh, R1.2/1.3)|
|Vitrification||Instrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Temperature: 103 K / Humidity: 80 %|
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 imaging
|Imaging||Microscope: FEI TECNAI 20 / Date: Jun 16, 2015|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 38.4 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 29000 X (nominal), 40410 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 2500 nm|
|Specimen Holder||Holder: This holder operates at liquid nitrogen temperature.|
Model: OTHER / Temperature: 88 K
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Image acquisition||Number of digital images: 2173 / Sampling size: 5 microns|
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).
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 29784|
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.
|3D reconstruction||Algorithm: Cross-common lines / Software: Relion / CTF correction: whole micrograph / Resolution: 7 Å / Resolution method: FSC 0.143, gold-standard|
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