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- EMDB-2271: Cryo EM map of the Gaalphaq-PLCbeta3 complex. -

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Basic information

Entry
Database: EMDB / ID: EMD-2271
TitleCryo EM map of the Gaalphaq-PLCbeta3 complex.
Map dataReconstruction of the PLCbeta3 distal CTD in contact with the PLCbeta3 catalytic core.
Sample
  • Sample: Cryo EM reconstruction of the Galphaq-PLCbeta3 complex.
  • Protein or peptide: G alpha q
  • Protein or peptide: phospholipase C beta 3
KeywordsGTP-binding protein alpha subunits / phospholipase C beta / coiled-coil domain / PH DOMAIN / EF HAND / C2 DOMAIN / TIM BARREL DOMAIN / phospholipase / GTP hydrolysis / G-protein signaling / membrane targeting / lipase / calcium binding / phospholipids / GTP-BINDING PROTEIN-HYDROLASE complex
Function / homology: / Phosphatidylinositol-4, 5-bisphosphate phosphodiesterase beta / G-protein alpha subunit, group Q / G protein-coupled receptor signaling pathway
Function and homology information
Biological speciesMus musculus (house mouse) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 21.0 Å
AuthorsDutta S / Lyon AM / Tesmer JJG / Skiniotis G
CitationJournal: Nat Struct Mol Biol / Year: 2013
Title: Full-length Gα(q)-phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain.
Authors: Angeline M Lyon / Somnath Dutta / Cassandra A Boguth / Georgios Skiniotis / John J G Tesmer /
Abstract: Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present ...Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM three-dimensional reconstructions of human full-length PLCβ3 in complex with mouse Gαq. The distal CTD forms an extended monomeric helical bundle consisting of three antiparallel segments with structural similarity to membrane-binding bin-amphiphysin-Rvs (BAR) domains. Sequence conservation of the distal CTD suggests putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gαq in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests that the distal CTD has roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis.
History
DepositionJan 3, 2013-
Header (metadata) releaseJan 16, 2013-
Map releaseMar 13, 2013-
UpdateMar 13, 2013-
Current statusMar 13, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.844
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.844
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2271.png

Downloads & links

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Map

FileDownload / File: emd_2271.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the PLCbeta3 distal CTD in contact with the PLCbeta3 catalytic core.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.48 Å/pix.
x 64 pix.
= 286.72 Å		4.48 Å/pix.
x 64 pix.
= 286.72 Å		4.48 Å/pix.
x 64 pix.
= 286.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.48 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.844 / Movie #1: 0.844
Minimum - Maximum-1.97102332 - 6.22964668
Average (Standard dev.)0.0030469 (±0.35487327)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-32-32-32
Dimensions646464
Spacing646464
CellA=B=C: 286.72 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.484.484.48
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z286.720286.720286.720
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS-32-32-32
NC/NR/NS646464
D min/max/mean-1.9716.2300.003

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Supplemental data

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Sample components

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Entire : Cryo EM reconstruction of the Galphaq-PLCbeta3 complex.

EntireName: Cryo EM reconstruction of the Galphaq-PLCbeta3 complex.
Components
  • Sample: Cryo EM reconstruction of the Galphaq-PLCbeta3 complex.
  • Protein or peptide: G alpha q
  • Protein or peptide: phospholipase C beta 3

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Supramolecule #1000: Cryo EM reconstruction of the Galphaq-PLCbeta3 complex.

SupramoleculeName: Cryo EM reconstruction of the Galphaq-PLCbeta3 complex.
type: sample / ID: 1000
Details: The sample was monodisperse and had a molecular weight consistent with the theoretical weight.
Oligomeric state: Dimer / Number unique components: 2
Molecular weightExperimental: 1.8 MDa / Theoretical: 1.8 MDa
Method: Size-exclusion chromatography and multi-angle light scattering

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Macromolecule #1: G alpha q

MacromoleculeName: G alpha q / type: protein_or_peptide / ID: 1 / Name.synonym: Gaq / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: House Mouse / Cell: High 5 / Location in cell: cytosol and plasma membrane
Molecular weightExperimental: 44 KDa / Theoretical: 44 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: pFastBacHTA
SequenceGO: GO: 0007202 / InterPro: G-protein alpha subunit, group Q

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Macromolecule #2: phospholipase C beta 3

MacromoleculeName: phospholipase C beta 3 / type: protein_or_peptide / ID: 2 / Name.synonym: PLCbeta3 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: High 5 / Location in cell: cytosol, plasma membrane
Molecular weightExperimental: 139 KDa / Theoretical: 139 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: pFastBacDual
SequenceGO: G protein-coupled receptor signaling pathway
InterPro: Phosphatidylinositol-4, 5-bisphosphate phosphodiesterase beta

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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

Concentration1 mg/mL
BufferpH: 8
Details: 20 mM HEPES pH 8, 200 mM NaCl, 2 mM DTT, 0.9 mM CaCl2, 50 microM GDP, 30 microM AlCl3, 10 mM NaF, 5 mM MgCl2
GridDetails: glow-discharged Quantifoil R2/200 mesh grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
Details: Vitrification carried out in liquid nitrogen atmosphere.
Method: Blot for 2 seconds at 3 microliter sample.

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 89 K / Max: 95 K / Average: 89 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 135,000 times magnification.
DateOct 20, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 350 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 71138 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Used two holders. / Specimen holder model: OTHER
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN1 / Number images used: 12673
Final angle assignmentDetails: EMAN1

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Atomic model buiding 1

Initial modelPDB ID:

4gnk

RefinementSpace: REAL

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