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

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Basic information

Entry
Database: EMDB / ID: 6642
TitleCryo-EM structure of the WRC-Rac1 complex
Keywordscryoelectron microscopy / Wave Regulatory Complex / GTPase / actin nucleation
SampleComplex of WAVE regulatory complex (WRC) and Rac1
SourceHomo sapiens / human
Map dataReconstruction of WRC-Rac1
Methodsingle particle reconstruction, at 7 Å resolution
AuthorsChen B / Chou H-T / Xing W / Henry L / Walz T / Rosen MK
CitationElife, 2017, 6

Elife, 2017, 6 Yorodumi Papers
Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites.
Baoyu Chen / Hui-Ting Chou / Chad A Brautigam / Wenmin Xing / Sheng Yang / Lisa Henry / Lynda K Doolittle / Thomas Walz / Michael K Rosen

DateDeposition: May 3, 2016 / Header (metadata) release: Jul 13, 2016 / Map release: May 3, 2017 / Last update: May 3, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF CHIMERA
  • Download
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Supplemental images

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Map

Fileemd_6642.map.gz (map file in CCP4 format, 61037 KB)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
250 pix
1.24 Å/pix.
= 310. Å
250 pix
1.24 Å/pix.
= 310. Å
250 pix
1.24 Å/pix.
= 310. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 1.24 Å
Density
Contour Level:0.0217 (by emdb), 0.02 (movie #1):
Minimum - Maximum-0.03580169 - 0.08824085
Average (Standard dev.)0.00030724 (0.00353338)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions250250250
Origin000
Limit249249249
Spacing250250250
CellA=B=C: 310 Å
α=β=γ: 90 deg.

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
start NX/NY/NZ
NX/NY/NZ
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 / Details: The sample was monodisperse. / Number of components: 2
Oligomeric State: One heteropentamer of WRC binds to one Rac1
MassTheoretical: 375 kDa

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Component #1: protein, WAVE regulatory complex

ProteinName: 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
MassTheoretical: 375 kDa
SourceSpecies: Homo sapiens / human
Source (engineered)Expression System: Spodoptera frugiperda / arthropod / Cell of expression system: sf9

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Component #2: protein, Rac1

ProteinName: Rac1 / Details: Rac1 is fused to C-terminal WAVE1. / Recombinant expression: Yes / Number of Copies: 1
SourceSpecies: Homo sapiens / human
Source (engineered)Expression System: Spodoptera frugiperda / arthropod / Cell of expression system: sf9

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Experimental details

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Sample preparation

Specimen stateparticle
Sample solutionSpecimen conc.: 0.1 mg/ml
Buffer solution: 10 mM HEPES, 100 mM NaCl, 1 mM MgCl2, 2 mM TCEP
pH: 7
Support filmglow-discharged Quantifoil holey carbon grids (400 copper mesh, R1.2/1.3)
VitrificationInstrument: 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.

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Electron microscopy imaging

ImagingMicroscope: FEI TECNAI 20 / Date: Jun 16, 2015
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 38.4 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 29000 X (nominal), 40410 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 2500 nm
Specimen HolderHolder: This holder operates at liquid nitrogen temperature.
Model: OTHER / Temperature: 88 K
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber 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).

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Image processing

ProcessingMethod: 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 reconstructionAlgorithm: Cross-common lines / Software: Relion / CTF correction: whole micrograph / Resolution: 7 Å / Resolution method: FSC 0.143, gold-standard

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Atomic model buiding

Modeling #1Software: Chimera / Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 3P8C
Modeling #2Software: Chimera / Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 3SBD

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