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- EMDB-6358: Negative stain (RCT) surface of full-length glucagon receptor FAB... -

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Basic information

Entry
Database: EMDB / ID: EMD-6358
TitleNegative stain (RCT) surface of full-length glucagon receptor FAB complex
Map dataRCT of full-length glucagon receptor FAB complex, alternative orientation
Sample
  • Sample: Full-length GCGR in complex with the antigen-binding fragment (Fab) of the monoclonal antibody mAb2330
  • Protein or peptide: glucagon receptor
  • Protein or peptide: antibody mAb2330 Fab
KeywordsGlucagon Receptor / GPCR
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 40.0 Å
AuthorsYang L / Yang D / de Graaf C / Moeller A / West GM / Dharmarajan V / Wang C / Siu FY / Song G / Reedtz-Runge S ...Yang L / Yang D / de Graaf C / Moeller A / West GM / Dharmarajan V / Wang C / Siu FY / Song G / Reedtz-Runge S / Pascal BD / Wu B / Potter CS / Zhou H / Griffin PR / Carragher B / Yang H / Wang MW / Stevens RC / Jiang H
CitationJournal: Nat Commun / Year: 2015
Title: Conformational states of the full-length glucagon receptor.
Authors: Linlin Yang / Dehua Yang / Chris de Graaf / Arne Moeller / Graham M West / Venkatasubramanian Dharmarajan / Chong Wang / Fai Y Siu / Gaojie Song / Steffen Reedtz-Runge / Bruce D Pascal / ...Authors: Linlin Yang / Dehua Yang / Chris de Graaf / Arne Moeller / Graham M West / Venkatasubramanian Dharmarajan / Chong Wang / Fai Y Siu / Gaojie Song / Steffen Reedtz-Runge / Bruce D Pascal / Beili Wu / Clinton S Potter / Hu Zhou / Patrick R Griffin / Bridget Carragher / Huaiyu Yang / Ming-Wei Wang / Raymond C Stevens / Hualiang Jiang /
Abstract: Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid ...Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism.
History
DepositionJun 18, 2015-
Header (metadata) releaseJul 1, 2015-
Map releaseJul 1, 2015-
UpdateAug 12, 2015-
Current statusAug 12, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6358.jpg

Downloads & links

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Map

FileDownload / File: emd_6358.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRCT of full-length glucagon receptor FAB complex, alternative orientation
Voxel sizeX=Y=Z: 2.7275 Å
Density
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-1.67740023 - 3.36788034
Average (Standard dev.)-0.05319324 (±0.33256277)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-40-40-40
Dimensions808080
Spacing808080
CellA=B=C: 218.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.72752.72752.7275
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z218.200218.200218.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS-40-40-40
NC/NR/NS808080
D min/max/mean-1.6773.368-0.053

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Supplemental data

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Sample components

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Entire : Full-length GCGR in complex with the antigen-binding fragment (Fa...

EntireName: Full-length GCGR in complex with the antigen-binding fragment (Fab) of the monoclonal antibody mAb2330
Components
  • Sample: Full-length GCGR in complex with the antigen-binding fragment (Fab) of the monoclonal antibody mAb2330
  • Protein or peptide: glucagon receptor
  • Protein or peptide: antibody mAb2330 Fab

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Supramolecule #1000: Full-length GCGR in complex with the antigen-binding fragment (Fa...

SupramoleculeName: Full-length GCGR in complex with the antigen-binding fragment (Fab) of the monoclonal antibody mAb2330
type: sample / ID: 1000 / Oligomeric state: heterodimer / Number unique components: 2
Molecular weightTheoretical: 95 KDa

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Macromolecule #1: glucagon receptor

MacromoleculeName: glucagon receptor / type: protein_or_peptide / ID: 1 / Name.synonym: GCGR / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human / Location in cell: plasma membrane
Molecular weightExperimental: 95 KDa / Theoretical: 95 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster) / Recombinant cell: CHO-K1

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Macromolecule #2: antibody mAb2330 Fab

MacromoleculeName: antibody mAb2330 Fab / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Mus musculus (house mouse) / synonym: mouse

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.00095 mg/mL
BufferpH: 7.5 / Details: HEPES LMNG
StainingType: NEGATIVE / Details: stained 3 times with 2% w/v uranyl formate
GridDetails: thin carbon over holes
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 62000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 62000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle min: -50
DateFeb 20, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 648 / Average electron dose: 45 e/Å2
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final two d classificationNumber classes: 1
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 40.0 Å / Resolution method: OTHER / Software - Name: Appion, Xmipp, Spider / Number images used: 343
DetailsRCT

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