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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 35
  • 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 / cellular response to sucrose stimulus / adrenomedullin binding / CGRP receptor complex / positive regulation of protein glycosylation / adrenomedullin receptor activity / adrenomedullin receptor complex / adrenomedullin receptor signaling pathway / amylin receptor activity ...nervous system process involved in regulation of systemic arterial blood pressure / calcitonin gene-related peptide binding / cellular response to sucrose stimulus / adrenomedullin binding / CGRP receptor complex / positive regulation of protein glycosylation / adrenomedullin receptor activity / adrenomedullin receptor complex / adrenomedullin receptor signaling pathway / amylin receptor activity / calcitonin receptor activity / calcitonin gene-related peptide receptor signaling pathway / vascular associated smooth muscle cell proliferation / positive regulation of interleukin-1 alpha production / calcitonin gene-related peptide receptor activity / negative regulation of calcium ion transport into cytosol / positive regulation of macrophage differentiation / amylin receptor 1 signaling pathway / amylin receptor signaling pathway / Calcitonin-like ligand receptors / regulation of G protein-coupled receptor signaling pathway / G protein-coupled receptor internalization / vasculature development / endothelial cell proliferation / negative regulation of bone resorption / leukocyte cell-cell adhesion / negative regulation of osteoclast differentiation / PKA activation in glucagon signalling / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / hair follicle placode formation / developmental growth / regulation of cytosolic calcium ion concentration / D1 dopamine receptor binding / endothelial cell migration / intracellular transport / vascular endothelial cell response to laminar fluid shear stress / renal water homeostasis / cellular response to hormone stimulus / Hedgehog 'off' state / coreceptor activity / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / positive regulation of vascular associated smooth muscle cell proliferation / negative regulation of blood pressure / regulation of insulin secretion / cellular response to glucagon stimulus / adenylate cyclase activator activity / trans-Golgi network membrane / protein localization to plasma membrane / positive regulation of interleukin-8 production / intracellular protein transport / negative regulation of inflammatory response to antigenic stimulus / G protein-coupled receptor activity / hormone activity / bone development / receptor internalization / regulation of blood pressure / G-protein beta/gamma-subunit complex binding / platelet aggregation / Olfactory Signaling Pathway / cognition / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / adenylate cyclase-activating G protein-coupled receptor signaling pathway / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / photoreceptor disc membrane / calcium ion transport / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / cellular response to catecholamine stimulus / vasodilation / ADORA2B mediated anti-inflammatory cytokines production / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / sensory perception of smell / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / protein transport / heterotrimeric G-protein complex / G alpha (12/13) signalling events / sensory perception of taste
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 / Transducin (heterotrimeric G protein), gamma chain / GPCR, family 2, calcitonin receptor family / 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 / Transducin (heterotrimeric G protein), gamma chain / GPCR, family 2, calcitonin receptor family / 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. / : / GPCR, family 2, extracellular hormone receptor domain / G-protein coupled receptors family 2 profile 1. / Domain present in hormone receptors / Hormone receptor domain / 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 / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / Few Secondary Structures / Irregular / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / P-loop containing nucleotide triphosphate hydrolases / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 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
Revision 1.4Nov 13, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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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
Water00
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


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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Details

Has protein modificationY

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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 FIELD / 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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