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- PDB-5uz7: Volta phase plate cryo-electron microscopy structure of a calcito... -

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Entry
Database: PDB / ID: 5uz7
TitleVolta phase plate cryo-electron microscopy structure of a calcitonin receptor-heterotrimeric Gs protein complex
Components
  • Calcitonin receptor
  • 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
  • NANOBODY 35
KeywordsSIGNALING PROTEIN / Class B G protein-coupled receptor Agonist-Receptor-G protein ternary complex Calcitonin receptor Active-state G protein-coupled receptor
Function/homologyGPCR, family 2, calcitonin receptor / calcitonin binding / calcitonin receptor activity / GPCR, family 2, calcitonin receptor family / Thromboxane signalling through TP receptor / Olfactory Signaling Pathway / amylin receptor complex / Prostacyclin signalling through prostacyclin receptor / Activation of the phototransduction cascade / Presynaptic function of Kainate receptors ...GPCR, family 2, calcitonin receptor / calcitonin binding / calcitonin receptor activity / GPCR, family 2, calcitonin receptor family / Thromboxane signalling through TP receptor / Olfactory Signaling Pathway / amylin receptor complex / Prostacyclin signalling through prostacyclin receptor / Activation of the phototransduction cascade / Presynaptic function of Kainate receptors / G beta:gamma signalling through PLC beta / positive regulation of adenylate cyclase activity / positive regulation of calcium ion import across plasma membrane / G-protein alpha subunit, group S / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Activation of G protein gated Potassium channels / amylin receptor signaling pathway / Calcitonin-like ligand receptors / Glucagon signaling in metabolic regulation / ADP signalling through P2Y purinoceptor 12 / G-protein activation / Adrenaline,noradrenaline inhibits insulin secretion / G-protein gamma subunit domain profile. / G-protein, gamma subunit / cardiac muscle cell apoptotic process / cellular response to catecholamine stimulus / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / intrinsic component of membrane / G-protein gamma-like domain superfamily / G-protein gamma-like domain / PKA activation in glucagon signalling / G-protein coupled acetylcholine receptor signaling pathway / G-protein coupled receptors family 2 signature 1. / G-protein beta-subunit binding / G alpha (z) signalling events / protein heterotrimerization / sensory perception of taste / GGL domain / alkylglycerophosphoethanolamine phosphodiesterase activity / Glucagon-type ligand receptors / adenylate cyclase-activating adrenergic receptor signaling pathway / GPCR, family 2, extracellular hormone receptor domain / GPCR family 2, extracellular hormone receptor domain superfamily / G-protein coupled receptors family 2 profile 1. / intracellular transport / GPCR, family 2, secretin-like, conserved site / G-protein coupled receptors family 2 signature 2. / GPCR, family 2, secretin-like / adenylate cyclase-activating dopamine receptor signaling pathway / 7 transmembrane receptor (Secretin family) / rhodopsin mediated signaling pathway / hair follicle placode formation / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / positive regulation of cAMP-mediated signaling / photoreceptor outer segment membrane / Vasopressin regulates renal water homeostasis via Aquaporins / bone development / Hormone receptor domain / Guanine nucleotide binding protein (G-protein), alpha subunit / GPCR, family 2-like / G protein alpha subunit, helical insertion / G-protein coupled receptors family 2 profile 2. / spectrin binding / Hedgehog 'off' state / sensory perception of smell / activation of adenylate cyclase activity / G-protein beta/gamma-subunit complex binding / developmental growth / photoreceptor disc membrane / type 1 angiotensin receptor binding / heterotrimeric G-protein complex / adenylate cyclase-activating G-protein coupled receptor signaling pathway / G-protein alpha subunit / positive regulation of cAMP metabolic process / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / cellular response to glucagon stimulus / renal water homeostasis / Inactivation, recovery and regulation of the phototransduction cascade / Wnt signaling pathway, calcium modulating pathway / extracellular vesicle / positive regulation of protein kinase A signaling / cellular response to prostaglandin E stimulus / positive regulation of cAMP biosynthetic process / Ca2+ pathway / ADP signalling through P2Y purinoceptor 1 / trans-Golgi network membrane / G alpha (12/13) signalling events / retina development in camera-type eye / phospholipase C-activating G-protein coupled receptor signaling pathway / photoreceptor inner segment / cognition / GTPase binding / regulation of insulin secretion / protein transporter activity / protein localization to plasma membrane / G alpha (s) signalling events / Thrombin signalling through proteinase activated receptors (PARs) / G alpha (i) signalling events / G beta:gamma signalling through PI3Kgamma
Function and homology information
Specimen sourceHomo sapiens / / human
Lama glama / Llama / mammal /
MethodElectron microscopy (4.1 Å resolution / Particle / Single particle) / Transmission electron microscopy
AuthorsLiang, Y.L. / Khoshouei, M. / Radjainia, M. / Zhang, Y. / Glukhova, A. / Tarrasch, J. / Thal, D.M. / Furness, S.G.B. / Christopoulos, G. / Coudrat, T. / Danev, R. / Baumeister, W. / Miller, L.J. / Christopoulos, A. / Kobilka, B.K. / Wootten, D. / Skiniotis, G. / Sexton, P.M.
CitationJournal: Nature / Year: 2017
Title: Phase-plate cryo-EM structure of a class B GPCR-G-protein complex.
Authors: Yi-Lynn Liang / Maryam Khoshouei / Mazdak Radjainia / Yan Zhang / Alisa Glukhova / Jeffrey Tarrasch / David M Thal / Sebastian G B Furness / George Christopoulos / Thomas Coudrat / Radostin Danev / Wolfgang Baumeister / Laurence J Miller / Arthur Christopoulos / Brian K Kobilka / Denise Wootten / Georgios Skiniotis / Patrick M Sexton
Abstract: Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B ...Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gαβγ protein determined by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gα. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G-protein coupling via an interaction with the Gβ subunit. This structure provides a new framework for understanding G-protein-coupled receptor function.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 24, 2017 / Release: May 3, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0May 3, 2017Structure modelrepositoryInitial release
1.1Jun 14, 2017Structure modelDatabase referencescitation_citation.country / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
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
N: NANOBODY 35
R: Calcitonin receptor


Theoretical massNumber of molelcules
Total (without water)164,2455
Polyers164,2455
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 44326.160 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens / / human / Gene: GNAS, GNAS1, GSP / Production host: Trichoplusia ni / References: UniProt:P63092
#2: Protein/peptide Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 38744.371 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens / / human / Gene: GNB1 / Production host: Trichoplusia ni / References: UniProt:P62873
#3: Protein/peptide Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7563.750 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens / / human / Gene: GNG2 / Production host: Trichoplusia ni / References: UniProt:P59768
#4: Protein/peptide NANOBODY 35


Mass: 15140.742 Da / Num. of mol.: 1 / Source: (gene. exp.) Lama glama / Llama / mammal / / Production host: Escherichia coli
#5: Protein/peptide Calcitonin receptor / CT-R


Mass: 58469.594 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens / / human / Gene: CALCR / Production host: Trichoplusia ni / References: UniProt:P30988

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: Complex of a full-length, active-state calcitonin receptor with peptide ligand, heterotrimeric Gs protein and nano body 35.
Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5 / Source: RECOMBINANT
Molecular weightValue: 0.15 deg. / Units: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Homo sapiens
Source (recombinant)Organism: Trichoplusia ni
Buffer solutionpH: 7.5
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 / Grid type: Quantifuoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / C2 aperture diameter: 50 mm
Specimen holderSpecimen 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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 106838 / Symmetry type: POINT

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