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- PDB-7knt: CryoEM structure of the apo-CGRP receptor in a detergent micelle -

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Basic information

Entry
Database: PDB / ID: 7knt
TitleCryoEM structure of the apo-CGRP receptor in a detergent micelle
Components
  • Calcitonin gene-related peptide type 1 receptor
  • Receptor activity-modifying protein 1
KeywordsSIGNALING PROTEIN / GPCR / CGRP receptor / apo receptor / MEMBRANE PROTEIN
Function / homology
Function and homology information


calcitonin gene-related peptide binding / positive regulation of protein glycosylation / amylin receptor activity / amylin receptor complex 1 / calcitonin gene-related peptide receptor signaling pathway / CGRP receptor complex / adrenomedullin binding / adrenomedullin receptor complex / adrenomedullin receptor activity / adrenomedullin receptor signaling pathway ...calcitonin gene-related peptide binding / positive regulation of protein glycosylation / amylin receptor activity / amylin receptor complex 1 / calcitonin gene-related peptide receptor signaling pathway / CGRP receptor complex / adrenomedullin binding / adrenomedullin receptor complex / adrenomedullin receptor activity / adrenomedullin receptor signaling pathway / calcitonin receptor activity / cellular response to sucrose stimulus / calcitonin gene-related peptide receptor activity / amylin receptor signaling pathway / regulation of G protein-coupled receptor signaling pathway / negative regulation of smooth muscle contraction / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / G protein-coupled peptide receptor activity / coreceptor activity / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G protein-coupled receptor activity / adenylate cyclase-activating G protein-coupled receptor signaling pathway / cellular response to hormone stimulus / protein localization to plasma membrane / calcium ion transport / receptor internalization / intracellular protein transport / positive regulation of smooth muscle cell proliferation / negative regulation of inflammatory response / protein transport / heart development / angiogenesis / positive regulation of protein binding / lysosome / cell surface receptor signaling pathway / receptor complex / endosome / G protein-coupled receptor signaling pathway / endoplasmic reticulum / cell surface / integral component of plasma membrane / extracellular space / plasma membrane / cytoplasm
GPCR, family 2, calcitonin receptor family / GPCR, family 2, extracellular hormone receptor domain / RAMP domain superfamily / GPCR family 2, extracellular hormone receptor domain superfamily / GPCR, family 2, secretin-like, conserved site / GPCR, family 2-like / Receptor activity modifying protein / GPCR, family 2, calcitonin gene-related peptide, type 1 receptor / GPCR, family 2, secretin-like
Receptor activity-modifying protein 1 / Calcitonin gene-related peptide type 1 receptor
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsBelousoff, M.J. / Danev, R.
Funding support Australia, 4items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1061044 Australia
National Health and Medical Research Council (NHMRC, Australia)1065410 Australia
National Health and Medical Research Council (NHMRC, Australia)1126857 Australia
National Health and Medical Research Council (NHMRC, Australia)1055134 Australia
CitationJournal: Science / Year: 2021
Title: Structure and dynamics of the CGRP receptor in apo and peptide-bound forms.
Authors: Tracy M Josephs / Matthew J Belousoff / Yi-Lynn Liang / Sarah J Piper / Jianjun Cao / Daniel J Garama / Katie Leach / Karen J Gregory / Arthur Christopoulos / Debbie L Hay / Radostin Danev / ...Authors: Tracy M Josephs / Matthew J Belousoff / Yi-Lynn Liang / Sarah J Piper / Jianjun Cao / Daniel J Garama / Katie Leach / Karen J Gregory / Arthur Christopoulos / Debbie L Hay / Radostin Danev / Denise Wootten / Patrick M Sexton /
Abstract: G protein-coupled receptors (GPCRs) are key regulators of information transmission between cells and organs. Despite this, we have only limited understanding of the behavior of GPCRs in the apo state ...G protein-coupled receptors (GPCRs) are key regulators of information transmission between cells and organs. Despite this, we have only limited understanding of the behavior of GPCRs in the apo state and the conformational changes upon agonist binding that lead to G protein recruitment and activation. We expressed and purified unmodified apo and peptide-bound calcitonin gene-related peptide (CGRP) receptors to determine their cryo-EM structures and complemented these with analysis of protein conformational dynamics using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and 3D variance analysis of the cryo-EM data. Together with our previously published structure of the active, Gs-bound, CGRP receptor complex, our work provides important insight into mechanisms of class B1 GPCR activation.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 6, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 24, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 3, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Assembly

Deposited unit
E: Receptor activity-modifying protein 1
R: Calcitonin gene-related peptide type 1 receptor


Theoretical massNumber of molelcules
Total (without water)73,3412
Polymers73,3412
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4150 Å2
ΔGint-37 kcal/mol
Surface area24390 Å2

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Components

#1: Protein Receptor activity-modifying protein 1 / Calcitonin-receptor-like receptor activity-modifying protein 1 / CRLR activity-modifying protein 1


Mass: 17066.701 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAMP1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O60894
#2: Protein Calcitonin gene-related peptide type 1 receptor / CGRP type 1 receptor / Calcitonin receptor-like receptor


Mass: 56274.520 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CALCRL, CGRPR / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q16602

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: apo CGRP receptor / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 57.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 542650 / Symmetry type: POINT
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0033823
ELECTRON MICROSCOPYf_angle_d0.6055207
ELECTRON MICROSCOPYf_dihedral_angle_d20.477491
ELECTRON MICROSCOPYf_chiral_restr0.038585
ELECTRON MICROSCOPYf_plane_restr0.004637

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