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- PDB-6niy: A high-resolution cryo-electron microscopy structure of a calcito... -

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Entry
Database: PDB / ID: 6niy
TitleA high-resolution cryo-electron microscopy structure of a calcitonin receptor-heterotrimeric Gs protein complex
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Calcitonin receptor
  • Calcitonin
  • Nanobody35
KeywordsMEMBRANE PROTEIN / GPCR / transmembrane / receptor / calcitonin
Function / homologyActivation of the phototransduction cascade / Adrenaline,noradrenaline inhibits insulin secretion / G beta:gamma signalling through PI3Kgamma / GPCR, family 2, secretin-like, conserved site / Prostacyclin signalling through prostacyclin receptor / WD40-repeat-containing domain / Calcitonin, conserved site / WD40 repeat, conserved site / G-protein beta WD-40 repeat / Guanine nucleotide-binding protein, beta subunit ...Activation of the phototransduction cascade / Adrenaline,noradrenaline inhibits insulin secretion / G beta:gamma signalling through PI3Kgamma / GPCR, family 2, secretin-like, conserved site / Prostacyclin signalling through prostacyclin receptor / WD40-repeat-containing domain / Calcitonin, conserved site / WD40 repeat, conserved site / G-protein beta WD-40 repeat / Guanine nucleotide-binding protein, beta subunit / Ca2+ pathway / Calcitonin/adrenomedullin / Calcitonin-like / Calcitonin / P-loop containing nucleoside triphosphate hydrolase / G-protein gamma-like domain superfamily / GPCR, family 2-like / WD40/YVTN repeat-like-containing domain superfamily / G beta:gamma signalling through PLC beta / Inactivation, recovery and regulation of the phototransduction cascade / GPCR, family 2, secretin-like / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein, beta subunit / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / WD40 repeat / Olfactory Signaling Pathway / GPCR, family 2, calcitonin receptor / G-protein gamma-like domain / Calcitonin peptide-like / ADP signalling through P2Y purinoceptor 12 / G-protein, gamma subunit / GPCR, family 2, extracellular hormone receptor domain / GPCR, family 2, calcitonin receptor family / G protein alpha subunit, helical insertion / G alpha (q) signalling events / G alpha (12/13) signalling events / WD40-repeat-containing domain superfamily / Presynaptic function of Kainate receptors / Trp-Asp (WD) repeats profile. / Thromboxane signalling through TP receptor / Vasopressin regulates renal water homeostasis via Aquaporins / G-protein coupled receptors family 2 profile 1. / Thrombin signalling through proteinase activated receptors (PARs) / G-protein coupled receptors family 2 profile 2. / Trp-Asp (WD) repeats circular profile. / Calcitonin-like ligand receptors / Hedgehog 'off' state / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Activation of G protein gated Potassium channels / Glucagon signaling in metabolic regulation / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / PKA activation in glucagon signalling / G-protein activation / Glucagon-type ligand receptors / G alpha (z) signalling events / G-protein alpha subunit, group S / GPCR family 2, extracellular hormone receptor domain superfamily / Trp-Asp (WD) repeats signature. / G-protein coupled receptors family 2 signature 2. / G-protein coupled receptors family 2 signature 1. / Calcitonin / CGRP / IAPP family signature. / Hormone receptor domain / GGL domain / G-protein alpha subunit / G-protein gamma subunit domain profile. / WD domain, G-beta repeat / G alpha (s) signalling events / G alpha (i) signalling events / ADP signalling through P2Y purinoceptor 1 / Calcitonin / CGRP / IAPP family / 7 transmembrane receptor (Secretin family) / amylin receptor complex 3 / calcitonin binding / cross-receptor inhibition within G protein-coupled receptor heterodimer / amylin receptor complex 2 / amylin receptor activity / amylin receptor complex 1 / calcitonin receptor activity / calcitonin gene-related peptide receptor activity / positive regulation of calcium ion import across plasma membrane / amylin receptor signaling pathway / intracellular transport / positive regulation of adenylate cyclase activity / hair follicle placode formation / intrinsic component of membrane / cellular response to catecholamine stimulus / cardiac muscle cell apoptotic process / G protein-coupled acetylcholine receptor signaling pathway / adenylate cyclase-activating adrenergic receptor signaling pathway / sensory perception of taste / protein heterotrimerization / alkylglycerophosphoethanolamine phosphodiesterase activity / G-protein beta-subunit binding / adenylate cyclase-activating dopamine receptor signaling pathway / rhodopsin mediated signaling pathway / photoreceptor outer segment membrane / sensory perception of smell / bone development / developmental growth / G-protein beta/gamma-subunit complex binding
Function and homology information
Specimen sourceHomo sapiens (human)
Lama glama (llama)
Oncorhynchus sp. (fish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.34 Å resolution
Authorsdal Maso, E. / Glukhova, A. / Zhu, Y. / Garcia-Nafria, J. / Tate, C.G. / Atanasio, S. / Reynolds, C.A. / Ramirez-Aportela, E. / Carazo, J.-M. / Hick, C.A. / Furness, S.G.B. / Hay, D.L. / Liang, Y.-L. / Miller, L.J. / Christopoulos, A. / Wang, M.-W. / Wootten, D. / Sexton, P.M.
CitationJournal: to be published
Title: The molecular control of calcitonin receptor (CTR) signaling
Authors: dal Maso, E. / Glukhova, A. / Zhu, Y. / Garcia-Nafria, J. / Tate, C.G. / Atanasio, S. / Reynolds, C.A. / Ramirez-Aportela, E. / Carazo, J.-M. / Hick, C.A. / Furness, S.G.B. / Hay, D.L. / Liang, Y.-L. / Miller, L.J. / Christopoulos, A. / Wang, M.-W. / Wootten, D. / Sexton, P.M.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jan 2, 2019 / Release: Jan 23, 2019

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
N: Nanobody35
R: Calcitonin receptor
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
P: Calcitonin


Theoretical massNumber of molelcules
Total (without water)164,9716
Polyers164,9716
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)14720
ΔGint (kcal/M)-85
Surface area (Å2)42880

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Components

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules ABG

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


Mass: 45725.520 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: GNAS, GNAS1, GSP / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63092
#4: Protein/peptide Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37416.930 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P62873
#5: Protein/peptide 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: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P59768

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Protein/peptide , 3 types, 3 molecules NRP

#2: Protein/peptide Nanobody35


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


Mass: 55387.316 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: CALCR / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P30988
#6: Protein/peptide Calcitonin /


Mass: 3438.886 Da / Num. of mol.: 1 / Source: (synth.) Oncorhynchus sp. (fish) / References: UniProt: Q92163

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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 nanobody 35
Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6 / 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: Quantifoil 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 / Calibrated magnification: 47170 / Cs: 2.7 mm / C2 aperture diameter: 50 microns
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)
EM imaging opticsPhase plate: The images were taken at 500 defocus

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Processing

Software

Classification: refinement / Contact author: Paul D. Adams / Contact author email: pdadams[at]lbl.gov / Language: Python/C++ / Location: https://www.phenix-online.org/ / Type: program / Version: 1.14_3260

Name
phenix.real_space_refine
PHENIX
EM software
IDNameVersionCategory
1RELIONparticle selection
4GctfCTF correction
7Coot0.8.9.1model fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
12RELION3D reconstruction
13PHENIX1.14-3260model refinement
CTF correctionDetails: Phase Plate CTF correction / Type: NONE
Particle selectionNumber of particles selected: 1445614
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 417949 / Symmetry type: POINT
Atomic model buildingRef space: REAL
Atomic model buildingPDB-ID: 5UZ7
RefineStereochemistry target values: CDL v1.2
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00837893
ELECTRON MICROSCOPYf_angle_d0.980810679
ELECTRON MICROSCOPYf_chiral_restr0.06421198
ELECTRON MICROSCOPYf_plane_restr0.00651346
ELECTRON MICROSCOPYf_dihedral_angle_d3.86974665

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