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- PDB-6os0: Structure of synthetic nanobody-stabilized angiotensin II type 1 ... -

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Basic information

Entry
Database: PDB / ID: 6os0
TitleStructure of synthetic nanobody-stabilized angiotensin II type 1 receptor bound to angiotensin II
Components
  • Angiotensinogen
  • Nanobody Nb.AT110i1
  • Type-1 angiotensin II receptor,Soluble cytochrome b562 BRIL fusion protein
KeywordsMEMBRANE PROTEIN / GPCR / nanobody
Function / homology
Function and homology information


angiotensin type I receptor activity / positive regulation of phospholipase A2 activity / angiotensin type II receptor activity / regulation of blood volume by renin-angiotensin / phospholipase C-activating angiotensin-activated signaling pathway / response to muscle activity involved in regulation of muscle adaptation / type 2 angiotensin receptor binding / : / regulation of renal sodium excretion / maintenance of blood vessel diameter homeostasis by renin-angiotensin ...angiotensin type I receptor activity / positive regulation of phospholipase A2 activity / angiotensin type II receptor activity / regulation of blood volume by renin-angiotensin / phospholipase C-activating angiotensin-activated signaling pathway / response to muscle activity involved in regulation of muscle adaptation / type 2 angiotensin receptor binding / : / regulation of renal sodium excretion / maintenance of blood vessel diameter homeostasis by renin-angiotensin / negative regulation of neurotrophin TRK receptor signaling pathway / regulation of extracellular matrix assembly / bradykinin receptor binding / positive regulation of activation of Janus kinase activity / regulation of renal output by angiotensin / G protein-coupled receptor signaling pathway coupled to cGMP nucleotide second messenger / renal system process / renin-angiotensin regulation of aldosterone production / positive regulation of blood vessel endothelial cell proliferation involved in sprouting angiogenesis / positive regulation of branching involved in ureteric bud morphogenesis / positive regulation of extracellular matrix assembly / positive regulation of macrophage derived foam cell differentiation / C-C chemokine receptor activity / C-C chemokine binding / vasoconstriction / positive regulation of CoA-transferase activity / type 1 angiotensin receptor binding / low-density lipoprotein particle remodeling / positive regulation of extrinsic apoptotic signaling pathway / response to angiotensin / positive regulation of epidermal growth factor receptor signaling pathway / positive regulation of cardiac muscle hypertrophy / Rho protein signal transduction / regulation of systemic arterial blood pressure by renin-angiotensin / positive regulation of gap junction assembly / positive regulation of protein tyrosine kinase activity / regulation of vasoconstriction / regulation of cardiac conduction / blood vessel remodeling / Metabolism of Angiotensinogen to Angiotensins / positive regulation of epithelial to mesenchymal transition / nitric oxide-cGMP-mediated signaling / positive regulation of protein metabolic process / positive regulation of endothelial cell migration / blood vessel diameter maintenance / Peptide ligand-binding receptors / neurogenesis / cell chemotaxis / negative regulation of MAP kinase activity / kidney development / positive regulation of cytokine production / angiotensin-activated signaling pathway / regulation of cell growth / calcium-mediated signaling / electron transport chain / growth factor activity / serine-type endopeptidase inhibitor activity / hormone activity / brain development / PPARA activates gene expression / regulation of blood pressure / positive regulation of miRNA transcription / positive regulation of inflammatory response / positive regulation of reactive oxygen species metabolic process / positive regulation of fibroblast proliferation / Cargo recognition for clathrin-mediated endocytosis / cell-cell signaling / Clathrin-mediated endocytosis / phospholipase C-activating G protein-coupled receptor signaling pathway / positive regulation of NF-kappaB transcription factor activity / regulation of cell population proliferation / G alpha (i) signalling events / positive regulation of cytosolic calcium ion concentration / regulation of inflammatory response / G alpha (q) signalling events / regulation of apoptotic process / collagen-containing extracellular matrix / periplasmic space / electron transfer activity / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / blood microparticle / inflammatory response / symbiont entry into host cell / iron ion binding / immune response / G protein-coupled receptor signaling pathway / protein heterodimerization activity / external side of plasma membrane / heme binding / positive regulation of DNA-templated transcription / extracellular space / extracellular exosome / extracellular region / membrane / plasma membrane / cytosol
Similarity search - Function
Angiotensin II receptor type 1 / Angiotensin II receptor family / Angiotensinogen, serpin domain / Angiotensinogen / Serpin, conserved site / Serpins signature. / Serpin superfamily, domain 2 / Serpin family / Serpin domain / Serpin superfamily ...Angiotensin II receptor type 1 / Angiotensin II receptor family / Angiotensinogen, serpin domain / Angiotensinogen / Serpin, conserved site / Serpins signature. / Serpin superfamily, domain 2 / Serpin family / Serpin domain / Serpin superfamily / Serpin superfamily, domain 1 / Serpin (serine protease inhibitor) / SERine Proteinase INhibitors / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Serpentine type 7TM GPCR chemoreceptor Srsx / 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)
Similarity search - Domain/homology
Angiotensinogen / Soluble cytochrome b562 / Type-1 angiotensin II receptor
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.9 Å
AuthorsWingler, L.M. / Staus, D.P. / Skiba, M.A. / McMahon, C. / Kleinhenz, A.L.W. / Lefkowitz, R.J. / Kruse, A.C.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL16037 United States
National Institutes of Health/Office of the Director5DP5OD021345 United States
Other private United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Science / Year: 2020
Title: Angiotensin and biased analogs induce structurally distinct active conformations within a GPCR.
Authors: Wingler, L.M. / Skiba, M.A. / McMahon, C. / Staus, D.P. / Kleinhenz, A.L.W. / Suomivuori, C.M. / Latorraca, N.R. / Dror, R.O. / Lefkowitz, R.J. / Kruse, A.C.
History
DepositionMay 1, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 19, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 4, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _entity.pdbx_description ..._chem_comp.name / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.3Oct 11, 2023Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Type-1 angiotensin II receptor,Soluble cytochrome b562 BRIL fusion protein
D: Nanobody Nb.AT110i1
B: Angiotensinogen
hetero molecules


Theoretical massNumber of molelcules
Total (without water)63,5676
Polymers63,2753
Non-polymers2923
Water32418
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5550 Å2
ΔGint-51 kcal/mol
Surface area25120 Å2
MethodPISA
Unit cell
Length a, b, c (Å)135.530, 37.830, 111.920
Angle α, β, γ (deg.)90.000, 94.410, 90.000
Int Tables number5
Space group name H-MC121

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Components

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Protein / Antibody / Protein/peptide / Sugars , 4 types, 4 molecules ADB

#1: Protein Type-1 angiotensin II receptor,Soluble cytochrome b562 BRIL fusion protein / AT1AR / AT1BR / Angiotensin II type-1 receptor / AT1 / Cytochrome b-562


Mass: 48514.918 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (E. coli)
Gene: AGTR1, AGTR1A, AGTR1B, AT2R1, AT2R1B, cybC / Plasmid: pcDNA-Zeo-tetO / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: P30556, UniProt: P0ABE7
#2: Antibody Nanobody Nb.AT110i1


Mass: 13711.979 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): BL21(DE3)
#3: Protein/peptide Angiotensinogen / Serpin A8


Mass: 1048.195 Da / Num. of mol.: 1 / Fragment: residues 34-41 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P01019
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 2 types, 20 molecules

#5: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 18 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.26 Å3/Da / Density % sol: 45.59 %
Crystal growTemperature: 293 K / Method: lipidic cubic phase / pH: 7.6
Details: Protein complex was reconstituted with a 10:1 (w/w) mixture of monoolein and cholesterol. Crystals were grown in 100 mM Tris pH 7.6, 10 mM MgCl2, 25-26% PEG 300

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-B / Wavelength: 1.033 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jun 29, 2018
RadiationMonochromator: Double Crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.033 Å / Relative weight: 1
ReflectionResolution: 2.9→37.196 Å / Num. obs: 11842 / % possible obs: 91.2 % / Redundancy: 2.349 % / Biso Wilson estimate: 65.057 Å2 / CC1/2: 0.926 / Rmerge(I) obs: 0.276 / Rrim(I) all: 0.341 / Χ2: 0.961 / Net I/σ(I): 2.25 / Num. measured all: 27811 / Scaling rejects: 7
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsCC1/2Rrim(I) all% possible all
2.9-2.982.3730.8060.6120369228580.3541.00293.1
2.98-3.062.4090.70.7521279398830.4210.86994
3.06-3.152.3430.6340.8919319118240.5270.78990.5
3.15-3.242.3120.5530.9918438477970.5760.68894.1
3.24-3.352.2580.5211.117708487840.5190.65192.5
3.35-3.472.3120.4851.4817438427540.6290.59689.5
3.47-3.62.3380.4421.7916347616990.7070.54991.9
3.6-3.742.420.409217307877150.7430.50790.9
3.74-3.912.4180.3972.3216327276750.6540.48892.8
3.91-4.12.3690.3722.6515427006510.7160.46393
4.1-4.322.370.3493.0114486736110.6930.43190.8
4.32-4.592.4170.2843.4713686345660.8360.34989.3
4.59-4.92.2440.2313.7212035985360.9090.28489.6
4.9-5.292.2990.2313.7111545635020.8760.28689.2
5.29-5.82.4390.2483.6511565114740.860.30292.8
5.8-6.482.4470.2233.810354834230.8780.27787.6
6.48-7.492.260.2024.088704263850.8780.25390.4
7.49-9.172.190.1484.557163583270.950.18391.3
9.17-12.972.3390.1285.185522832360.9660.15683.4
12.97-37.1962.2610.0975.133211731420.990.12582.1

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
PHENIX(1.14_3260)refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6DO1

6do1


Resolution: 2.9→37.196 Å / SU ML: 0.55 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 35.6 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.318 1172 9.94 %
Rwork0.2593 10622 -
obs0.2652 11794 90.88 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 178.82 Å2 / Biso mean: 79.6207 Å2 / Biso min: 36.06 Å2
Refinement stepCycle: final / Resolution: 2.9→37.196 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4105 0 16 18 4139
Biso mean--120.21 61.8 -
Num. residues----529
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 8

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.9-3.0320.40421340.34741324145892
3.032-3.19170.38161580.33091297145591
3.1917-3.39160.40461490.31471336148592
3.3916-3.65330.34241420.29821312145491
3.6533-4.02050.34081470.26211338148592
4.0205-4.60140.29411480.2281310145890
4.6014-5.79370.33441420.23671338148090
5.7937-37.19950.25011520.23131367151988

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