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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 35Single-domain antibody
KeywordsSIGNALING PROTEIN / Class B G protein-coupled receptor Agonist-Receptor-G protein ternary complex Calcitonin receptor Active-state G protein-coupled receptor
Function / homology7 transmembrane receptor (Secretin family) / Trp-Asp (WD) repeats signature. / GPCR, family 2, secretin-like / G-protein alpha subunit, group S / G-protein alpha subunit / GGL domain / Hormone receptor domain / G-protein coupled receptors family 2 signature 1. / G-protein coupled receptors family 2 signature 2. / G-protein gamma subunit domain profile. ...7 transmembrane receptor (Secretin family) / Trp-Asp (WD) repeats signature. / GPCR, family 2, secretin-like / G-protein alpha subunit, group S / G-protein alpha subunit / GGL domain / Hormone receptor domain / G-protein coupled receptors family 2 signature 1. / G-protein coupled receptors family 2 signature 2. / G-protein gamma subunit domain profile. / G-protein, beta subunit / Trp-Asp (WD) repeats profile. / G-protein coupled receptors family 2 profile 1. / G-protein coupled receptors family 2 profile 2. / Trp-Asp (WD) repeats circular profile. / Activation of G protein gated Potassium channels / WD domain, G-beta repeat / PKA activation in glucagon signalling / Guanine nucleotide binding protein (G-protein), alpha subunit / WD40 repeat / Activation of the phototransduction cascade / GPCR, family 2, secretin-like, conserved site / GPCR family 2, extracellular hormone receptor domain superfamily / WD40-repeat-containing domain superfamily / G-protein gamma-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase / G-protein beta WD-40 repeat / WD40 repeat, conserved site / WD40-repeat-containing domain / GPCR, family 2-like / GPCR, family 2, calcitonin receptor / Guanine nucleotide-binding protein, beta subunit / WD40/YVTN repeat-like-containing domain superfamily / G-protein gamma-like domain / G protein alpha subunit, helical insertion / GPCR, family 2, calcitonin receptor family / GPCR, family 2, extracellular hormone receptor domain / G-protein, gamma subunit / G-protein activation / Glucagon signaling in metabolic regulation / Hedgehog 'off' state / Olfactory Signaling Pathway / Glucagon-type ligand receptors / Vasopressin regulates renal water homeostasis via Aquaporins / Adrenaline,noradrenaline inhibits insulin secretion / Prostacyclin signalling through prostacyclin receptor / Thrombin signalling through proteinase activated receptors (PARs) / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / G beta:gamma signalling through PI3Kgamma / G alpha (12/13) signalling events / Thromboxane signalling through TP receptor / Presynaptic function of Kainate receptors / ADP signalling through P2Y purinoceptor 12 / Ca2+ pathway / G beta:gamma signalling through PLC beta / G alpha (s) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / Inactivation, recovery and regulation of the phototransduction cascade / ADP signalling through P2Y purinoceptor 1 / G alpha (i) signalling events / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G alpha (q) signalling events / G alpha (z) signalling events / Calcitonin-like ligand receptors / calcitonin binding / calcitonin receptor activity / amylin receptor complex / positive regulation of adenylate cyclase activity / positive regulation of calcium ion import across plasma membrane / amylin receptor signaling pathway / intracellular transport / intrinsic component of membrane / cardiac muscle cell apoptotic process / type 1 angiotensin receptor binding / hair follicle placode formation / cellular response to catecholamine stimulus / G-protein coupled acetylcholine receptor signaling pathway / adenylate cyclase-activating adrenergic receptor signaling pathway / protein heterotrimerization / G-protein beta-subunit binding / sensory perception of taste / alkylglycerophosphoethanolamine phosphodiesterase activity / adenylate cyclase-activating dopamine receptor signaling pathway / rhodopsin mediated signaling pathway / positive regulation of cAMP-mediated signaling / photoreceptor outer segment membrane / bone development / spectrin binding / sensory perception of smell / activation of adenylate cyclase activity / developmental growth / G-protein beta/gamma-subunit complex binding / photoreceptor disc membrane / go:0004872: / heterotrimeric G-protein complex / adenylate cyclase-activating G-protein coupled receptor signaling pathway / go:0030816: / cellular response to glucagon stimulus / go:0030819: / renal water homeostasis
Function and homology information
Specimen sourceHomo sapiens (human)
Lama Glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4.1 Å resolution
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
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 (cabbage looper) / 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 (cabbage looper) / 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 (cabbage looper) / References: UniProt: P59768
#4: Protein/peptide NANOBODY 35 / Single-domain antibody


Mass: 15140.742 Da / Num. of mol.: 1 / Source: (gene. exp.) Lama Glama (llama) / Production host: Escherichia coli (E. 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 (cabbage looper) / 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 reconstruction

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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 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
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 FIELDBright-field microscopy / 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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