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- PDB-6e3y: Cryo-EM structure of the active, Gs-protein complexed, human CGRP... -

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Basic information

Entry
Database: PDB / ID: 6e3y
TitleCryo-EM structure of the active, Gs-protein complexed, human CGRP receptor
Components
  • (Calcitonin gene-related peptide ...) x 2
  • (Guanine nucleotide-binding protein ...) x 3
  • Nanobody 35Single-domain antibody
  • Receptor activity-modifying protein 1
KeywordsSIGNALING PROTEIN / class B G protein-coupled receptor / agonist-receptor-G protein ternary complex / calcitonin gene-related peptide receptor / receptor activity modifying protein 1 / active-state G protein-coupled receptor / phase plate / phase contrast
Function / homology
Function and homology information


: / nervous system process involved in regulation of systemic arterial blood pressure / calcitonin gene-related peptide binding / CGRP receptor complex / calcitonin gene-related peptide receptor signaling pathway / positive regulation of protein glycosylation / adrenomedullin binding / cellular response to sucrose stimulus / adrenomedullin receptor activity / adrenomedullin receptor complex ...: / nervous system process involved in regulation of systemic arterial blood pressure / calcitonin gene-related peptide binding / CGRP receptor complex / calcitonin gene-related peptide receptor signaling pathway / positive regulation of protein glycosylation / adrenomedullin binding / cellular response to sucrose stimulus / adrenomedullin receptor activity / adrenomedullin receptor complex / adrenomedullin receptor signaling pathway / amylin receptor activity / calcitonin receptor activity / positive regulation of interleukin-1 alpha production / vascular associated smooth muscle cell proliferation / calcitonin gene-related peptide receptor activity / response to yeast / amylin receptor signaling pathway / Calcitonin-like ligand receptors / positive regulation of macrophage differentiation / negative regulation of calcium ion transport into cytosol / antifungal humoral response / regulation of G protein-coupled receptor signaling pathway / vasculature development / G protein-coupled receptor internalization / endothelial cell proliferation / negative regulation of bone resorption / leukocyte cell-cell adhesion / negative regulation of osteoclast differentiation / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / PKA activation in glucagon signalling / hair follicle placode formation / intracellular transport / D1 dopamine receptor binding / developmental growth / endothelial cell migration / Hedgehog 'off' state / positive regulation of cAMP-mediated signaling / coreceptor activity / adenylate cyclase-activating adrenergic receptor signaling pathway / cellular response to hormone stimulus / positive regulation of vascular associated smooth muscle cell proliferation / regulation of cytosolic calcium ion concentration / activation of adenylate cyclase activity / adenylate cyclase activator activity / negative regulation of blood pressure / trans-Golgi network membrane / positive regulation of interleukin-8 production / G protein-coupled receptor activity / protein localization to plasma membrane / intracellular protein transport / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / bone development / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / adenylate cyclase-activating G protein-coupled receptor signaling pathway / hormone activity / receptor internalization / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / platelet aggregation / cognition / Glucagon-type ligand receptors / Vasopressin regulates renal water homeostasis via Aquaporins / positive regulation of GTPase activity / G alpha (z) signalling events / regulation of blood pressure / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / vasodilation / adenylate cyclase-activating dopamine receptor signaling pathway / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / sensory perception of taste / GPER1 signaling / G-protein beta-subunit binding / heterotrimeric G-protein complex / Inactivation, recovery and regulation of the phototransduction cascade / extracellular vesicle / calcium ion transport / G alpha (12/13) signalling events / signaling receptor complex adaptor activity / antimicrobial humoral immune response mediated by antimicrobial peptide
Similarity search - Function
Calcitonin gene-related peptide / GPCR, family 2, calcitonin gene-related peptide, type 1 receptor / Receptor activity modifying protein / RAMP domain superfamily / Receptor activity modifying family / GPCR, family 2, calcitonin receptor family / Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / Calcitonin-like / Calcitonin peptide-like ...Calcitonin gene-related peptide / GPCR, family 2, calcitonin gene-related peptide, type 1 receptor / Receptor activity modifying protein / RAMP domain superfamily / Receptor activity modifying family / GPCR, family 2, calcitonin receptor family / Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / Calcitonin-like / Calcitonin peptide-like / Calcitonin, conserved site / Calcitonin / CGRP / IAPP family signature. / calcitonin / Calcitonin/adrenomedullin / Calcitonin / CGRP / IAPP family / G-protein coupled receptors family 2 signature 1. / Hormone receptor domain / GPCR, family 2, extracellular hormone receptor domain / G-protein coupled receptors family 2 profile 1. / Domain present in hormone receptors / GPCR family 2, extracellular hormone receptor domain superfamily / G-protein coupled receptors family 2 signature 2. / GPCR, family 2, secretin-like, conserved site / GPCR, family 2, secretin-like / 7 transmembrane receptor (Secretin family) / GPCR, family 2-like / G-protein coupled receptors family 2 profile 2. / G-protein alpha subunit, group S / YVTN repeat-like/Quinoprotein amine dehydrogenase / 7 Propeller / Methylamine Dehydrogenase; Chain H / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / Few Secondary Structures / Irregular / G-protein beta WD-40 repeat / WD40 repeat, conserved site / P-loop containing nucleotide triphosphate hydrolases / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Receptor activity-modifying protein 1 / Calcitonin gene-related peptide 1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Calcitonin gene-related peptide type 1 receptor
Similarity search - Component
Biological speciesLama glama (llama)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLiang, Y.L. / Khoshouei, M. / Deganutti, G. / Glukhova, A. / Koole, C. / Peat, T.S. / Radjainia, M. / Plitzko, J.M. / Baumeister, W. / Miller, L.J. ...Liang, Y.L. / Khoshouei, M. / Deganutti, G. / Glukhova, A. / Koole, C. / Peat, T.S. / Radjainia, M. / Plitzko, J.M. / Baumeister, W. / Miller, L.J. / Hay, D.L. / Christopoulos, A. / Reynolds, C.A. / Wootten, D. / Sexton, P.M.
Funding support Australia, 2items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1120919 Australia
National Health and Medical Research Council (NHMRC, Australia)1055134 Australia
CitationJournal: Nature / Year: 2018
Title: Cryo-EM structure of the active, G-protein complexed, human CGRP receptor.
Authors: Yi-Lynn Liang / Maryam Khoshouei / Giuseppe Deganutti / Alisa Glukhova / Cassandra Koole / Thomas S Peat / Mazdak Radjainia / Jürgen M Plitzko / Wolfgang Baumeister / Laurence J Miller / ...Authors: Yi-Lynn Liang / Maryam Khoshouei / Giuseppe Deganutti / Alisa Glukhova / Cassandra Koole / Thomas S Peat / Mazdak Radjainia / Jürgen M Plitzko / Wolfgang Baumeister / Laurence J Miller / Deborah L Hay / Arthur Christopoulos / Christopher A Reynolds / Denise Wootten / Patrick M Sexton /
Abstract: Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that has a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like ...Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that has a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and a type 1 transmembrane domain protein, receptor activity-modifying protein 1 (RAMP1). Here we report the structure of the human CGRP receptor in complex with CGRP and the G-protein heterotrimer at 3.3 Å global resolution, determined by Volta phase-plate cryo-electron microscopy. The receptor activity-modifying protein transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilizes CLR extracellular loop 2. RAMP1 makes only limited direct contact with CGRP, consistent with its function in allosteric modulation of CLR. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly in the positioning of the extracellular domain of CLR. This work provides insights into the control of G-protein-coupled receptor function.
History
DepositionJul 16, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 19, 2018Provider: repository / Type: Initial release
Revision 1.1Sep 26, 2018Group: Data collection / Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Oct 10, 2018Group: Data collection / Database references / Structure summary
Category: citation / entity
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _entity.formula_weight
Revision 1.3Jan 15, 2020Group: Author supporting evidence / Structure summary / Category: entity / pdbx_audit_support
Item: _entity.formula_weight / _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
P: Calcitonin gene-related peptide 1
N: Nanobody 35
A: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
R: Calcitonin gene-related peptide type 1 receptor
E: Receptor activity-modifying protein 1


Theoretical massNumber of molelcules
Total (without water)184,3567
Polymers184,3567
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area20890 Å2
ΔGint-140 kcal/mol
Surface area52580 Å2

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Components

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Calcitonin gene-related peptide ... , 2 types, 2 molecules PR

#1: Protein/peptide Calcitonin gene-related peptide 1 / / Alpha-type CGRP / Calcitonin gene-related peptide I / CGRP-I


Mass: 3795.354 Da / Num. of mol.: 1 / Fragment: residues 83-119 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P06881
#6: 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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Guanine nucleotide-binding protein ... , 3 types, 3 molecules ABG

#3: Protein Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-stimulating G alpha protein


Mass: 45683.434 Da / Num. of mol.: 1
Mutation: N54S, A226G, A268E, K271N, D274K, K280R, D284T, T285I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63092
#4: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 38534.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P62873
#5: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7861.143 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P59768

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Antibody / Protein , 2 types, 2 molecules NE

#2: Antibody Nanobody 35 / Single-domain antibody


Mass: 15140.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#7: 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

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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: Complex of a full-length, active-state calcitonin gene-related peptide receptor with calcitonin gene-related peptide ligand, heterotrimeric Gs protein and nanobody35
Type: COMPLEX / Entity ID: all / 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
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: NITROGEN / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 47170 X
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
7Coot0.8.9model fitting
13PHENIXmodel refinement
CTF correctionDetails: phase plate CTF correction / Type: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 407000 / Symmetry type: POINT

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