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- EMDB-6642: Cryo-EM structure of the WRC-Rac1 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-6642
TitleCryo-EM structure of the WRC-Rac1 complex
Map dataReconstruction of WRC-Rac1
Sample
  • Sample: Complex of WAVE regulatory complex (WRC) and Rac1
  • Protein or peptide: WAVE regulatory complex
  • Protein or peptide: Rac1
Keywordscryoelectron microscopy / Wave Regulatory Complex / GTPase / actin nucleation
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.0 Å
AuthorsChen B / Chou H-T / Xing W / Henry L / Walz T / Rosen MK
CitationJournal: 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
DepositionMay 3, 2016-
Header (metadata) releaseJul 13, 2016-
Map releaseMay 3, 2017-
UpdateMay 3, 2017-
Current statusMay 3, 2017Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_6642.map.gz / Format: CCP4 / Size: 58.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of WRC-Rac1
Voxel sizeX=Y=Z: 1.24 Å
Density
Contour LevelBy EMDB: 0.0217 / Movie #1: 0.02
Minimum - Maximum-0.03580169 - 0.08824085
Average (Standard dev.)0.00030724 (±0.00353338)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 310.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.241.241.24
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z310.000310.000310.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.0360.0880.000

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Supplemental data

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Sample components

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Entire : Complex of WAVE regulatory complex (WRC) and Rac1

EntireName: Complex of WAVE regulatory complex (WRC) and Rac1
Components
  • Sample: Complex of WAVE regulatory complex (WRC) and Rac1
  • Protein or peptide: WAVE regulatory complex
  • Protein or peptide: Rac1

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Supramolecule #1000: Complex of WAVE regulatory complex (WRC) and Rac1

SupramoleculeName: 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
Molecular weightTheoretical: 375 KDa

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Macromolecule #1: WAVE regulatory complex

MacromoleculeName: 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
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 375 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: sf9

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Macromolecule #2: Rac1

MacromoleculeName: Rac1 / type: protein_or_peptide / ID: 2 / Details: Rac1 is fused to C-terminal WAVE1. / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: sf9

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7 / Details: 10 mM HEPES, 100 mM NaCl, 1 mM MgCl2, 2 mM TCEP
GridDetails: glow-discharged Quantifoil holey carbon grids (400 copper mesh, R1.2/1.3)
VitrificationCryogen 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.

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 40410 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 29000
Sample stageSpecimen holder: This holder operates at liquid nitrogen temperature.
Specimen holder model: OTHER
TemperatureAverage: 88 K
DateJun 16, 2015
Image recordingCategory: 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).

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Image processing

CTF correctionDetails: whole micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.0 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 29784
DetailsInitial 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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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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