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- PDB-8pok: Cryo-EM structure of cell-free synthesized human histamine H2 rec... -

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Basic information

Entry
Database: PDB / ID: 8pok
TitleCryo-EM structure of cell-free synthesized human histamine H2 receptor coupled to heterotrimeric Gs protein in lipid environment
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • Histamine H2 receptor
  • Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
  • Nanobody35
KeywordsMEMBRANE PROTEIN / GPCR / Cell-free / H2R / human histamine 2 receptor / active conformation / complex / detergent-free / lipid environment
Function / homology
Function and homology information


gastric acid secretion / Histamine receptors / histamine receptor activity / G protein-coupled serotonin receptor activity / neurotransmitter receptor activity / 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 ...gastric acid secretion / Histamine receptors / histamine receptor activity / G protein-coupled serotonin receptor activity / neurotransmitter receptor activity / 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 / Hedgehog 'off' state / positive regulation of cAMP-mediated signaling / adenylate cyclase-activating adrenergic receptor signaling pathway / positive regulation of vasoconstriction / activation of adenylate cyclase activity / adenylate cyclase activator activity / trans-Golgi network membrane / 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 / 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 / Glucagon-type ligand receptors / cognition / Vasopressin regulates renal water homeostasis via Aquaporins / positive regulation of GTPase activity / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / 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 / G alpha (12/13) signalling events / signaling receptor complex adaptor activity / sensory perception of smell / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / GTPase binding / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / positive regulation of cold-induced thermogenesis / G alpha (i) signalling events / fibroblast proliferation / chemical synaptic transmission / G alpha (s) signalling events / G alpha (q) signalling events / Ras protein signal transduction / cell population proliferation / Extra-nuclear estrogen signaling / immune response / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / dendrite / synapse / protein-containing complex binding / GTP binding / signal transduction / extracellular exosome / membrane / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Histamine H2 receptor / G-protein alpha subunit, group S / Serpentine type 7TM GPCR chemoreceptor Srsx / 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. ...Histamine H2 receptor / G-protein alpha subunit, group S / Serpentine type 7TM GPCR chemoreceptor Srsx / 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 / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / G-protein beta WD-40 repeat / WD40 repeat, conserved site / 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
Similarity search - Domain/homology
HISTAMINE / Histamine H2 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
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsSchnelle, K. / Koeck, Z. / Persechino, M. / Umbach, S. / Schihada, H. / Januliene, D. / Parey, K. / Pockes, S. / Kolb, P. / Doetsch, V. ...Schnelle, K. / Koeck, Z. / Persechino, M. / Umbach, S. / Schihada, H. / Januliene, D. / Parey, K. / Pockes, S. / Kolb, P. / Doetsch, V. / Moeller, A. / Hilger, D. / Bernhard, F.
Funding support Germany, 6items
OrganizationGrant numberCountry
Other governmentGLUE
Other privateSK-208/16
German Research Foundation (DFG)944 (P27) Germany
German Research Foundation (DFG)1557 (P11) Germany
German Research Foundation (DFG)INST 190-196-1 FUGG Germany
Other governmentSPRUNG Stay inspired 15-76251-2-04/22 (10743/2022) Germany
CitationJournal: Nat Commun / Year: 2024
Title: Cryo-EM structure of cell-free synthesized human histamine 2 receptor/G complex in nanodisc environment.
Authors: Zoe Köck / Kilian Schnelle / Margherita Persechino / Simon Umbach / Hannes Schihada / Dovile Januliene / Kristian Parey / Steffen Pockes / Peter Kolb / Volker Dötsch / Arne Möller / ...Authors: Zoe Köck / Kilian Schnelle / Margherita Persechino / Simon Umbach / Hannes Schihada / Dovile Januliene / Kristian Parey / Steffen Pockes / Peter Kolb / Volker Dötsch / Arne Möller / Daniel Hilger / Frank Bernhard /
Abstract: Here we describe the cryo-electron microscopy structure of the human histamine 2 receptor (HR) in an active conformation with bound histamine and in complex with G heterotrimeric protein at an ...Here we describe the cryo-electron microscopy structure of the human histamine 2 receptor (HR) in an active conformation with bound histamine and in complex with G heterotrimeric protein at an overall resolution of 3.4 Å. The complex was generated by cotranslational insertion of the receptor into preformed nanodisc membranes using cell-free synthesis in E. coli lysates. Structural comparison with the inactive conformation of HR and the inactive and G-coupled active state of HR together with structure-guided functional experiments reveal molecular insights into the specificity of ligand binding and G protein coupling for this receptor family. We demonstrate lipid-modulated folding of cell-free synthesized HR, its agonist-dependent internalization and its interaction with endogenously synthesized HR and HR in HEK293 cells by applying a recently developed nanotransfer technique.
History
DepositionJul 5, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 6, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 13, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Histamine H2 receptor
B: Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
C: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
D: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
E: Nanobody35
hetero molecules


Theoretical massNumber of molelcules
Total (without water)146,8676
Polymers146,7565
Non-polymers1111
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Histamine H2 receptor / / H2R / HH2R / Gastric receptor I


Mass: 41997.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Description: Cell-free synthesis / Gene: HRH2 / Production host: Escherichia coli (E. coli) / References: UniProt: P25021
#2: Protein Isoform Gnas-2 of 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 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

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Guanine nucleotide-binding protein ... , 2 types, 2 molecules CD

#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37728.152 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
#4: Protein 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 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 / Non-polymers , 2 types, 2 molecules E

#5: Antibody Nanobody35


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)
#6: Chemical ChemComp-HSM / HISTAMINE / Histamine


Mass: 111.145 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H9N3 / Feature type: SUBJECT OF INVESTIGATION / Comment: neurotransmitter, hormone*YM

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Details

Has ligand of interestY

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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

Component
IDNameTypeEntity IDParent-IDSource
1H2 receptor coupled to heterotrimeric Gs proteinCOMPLEX#1-#50MULTIPLE SOURCES
2Histamine H2 receptorCOMPLEX#11RECOMBINANT
3Isoform Gnas-2 of Guanine nucleotide-binding protein G(s) subunit alpha isoforms short, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 and Guanine nucleotide-binding protein subunit gamma (Fragment)COMPLEX#2-#41RECOMBINANT
4Nanobody35COMPLEX#51RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDDetails
22Homo sapiens (human)9606Cell-free synthesis
33Homo sapiens (human)9606
44Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainCellPlasmid
22Escherichia coli (E. coli)562A19Cell-free synthesispET29
33Trichoplusia ni (cabbage looper)7111
44Escherichia coli (E. coli)562
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: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
2EPU2.9image acquisition
4cryoSPARC4CTF correction
10cryoSPARC4initial Euler assignment
11cryoSPARC4final Euler assignment
13cryoSPARC43D reconstruction
CTF correctionDetails: Patch CTF correction / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 30500000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 425000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048193
ELECTRON MICROSCOPYf_angle_d0.51911100
ELECTRON MICROSCOPYf_dihedral_angle_d11.0292964
ELECTRON MICROSCOPYf_chiral_restr0.0411267
ELECTRON MICROSCOPYf_plane_restr0.0031418

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