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- PDB-6os1: Structure of synthetic nanobody-stabilized angiotensin II type 1 ... -

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Basic information

Entry
Database: PDB / ID: 6os1
TitleStructure of synthetic nanobody-stabilized angiotensin II type 1 receptor bound to TRV023
Components
  • Nanobody Nb.AT110i1_le
  • TRV023 peptide
  • Type-1 angiotensin II receptor,Soluble cytochrome b562 BRIL fusion protein
KeywordsMEMBRANE PROTEIN / GPCR
Function / homology
Function and homology information


angiotensin type I receptor activity / positive regulation of phospholipase A2 activity / angiotensin type II receptor activity / phospholipase C-activating angiotensin-activated signaling pathway / regulation of renal sodium excretion / maintenance of blood vessel diameter homeostasis by renin-angiotensin / bradykinin receptor binding / renin-angiotensin regulation of aldosterone production / positive regulation of blood vessel endothelial cell proliferation involved in sprouting angiogenesis / positive regulation of macrophage derived foam cell differentiation ...angiotensin type I receptor activity / positive regulation of phospholipase A2 activity / angiotensin type II receptor activity / phospholipase C-activating angiotensin-activated signaling pathway / regulation of renal sodium excretion / maintenance of blood vessel diameter homeostasis by renin-angiotensin / bradykinin receptor binding / renin-angiotensin regulation of aldosterone production / positive regulation of blood vessel endothelial cell proliferation involved in sprouting angiogenesis / positive regulation of macrophage derived foam cell differentiation / C-C chemokine receptor activity / C-C chemokine binding / positive regulation of CoA-transferase activity / low-density lipoprotein particle remodeling / regulation of systemic arterial blood pressure by renin-angiotensin / Rho protein signal transduction / regulation of vasoconstriction / positive regulation of protein metabolic process / blood vessel diameter maintenance / Peptide ligand-binding receptors / neurogenesis / cell chemotaxis / kidney development / angiotensin-activated signaling pathway / regulation of cell growth / calcium-mediated signaling / electron transport chain / brain development / positive regulation of inflammatory response / positive regulation of reactive oxygen species metabolic process / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / phospholipase C-activating G protein-coupled receptor signaling pathway / regulation of cell population proliferation / positive regulation of cytosolic calcium ion concentration / regulation of inflammatory response / G alpha (q) signalling events / periplasmic space / electron transfer activity / symbiont entry into host cell / inflammatory response / iron ion binding / immune response / G protein-coupled receptor signaling pathway / protein heterodimerization activity / external side of plasma membrane / heme binding / membrane / plasma membrane
Similarity search - Function
Angiotensin II receptor type 1 / Angiotensin II receptor family / 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
CHOLESTEROL / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / 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 / Resolution: 2.794 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 1.2Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _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_le
B: TRV023 peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,78110
Polymers63,2553
Non-polymers2,5267
Water30617
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5000 Å2
ΔGint-28 kcal/mol
Surface area23840 Å2
MethodPISA
Unit cell
Length a, b, c (Å)60.670, 101.990, 227.590
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number23
Space group name H-MI222

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Components

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Protein / Antibody / Protein/peptide , 3 types, 3 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_le


Mass: 13796.137 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) / Strain (production host): BL21(DE3)
#3: Protein/peptide TRV023 peptide


Mass: 944.111 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 24 molecules

#4: Chemical
ChemComp-OLC / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / 1-Oleoyl-R-glycerol


Mass: 356.540 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C21H40O4
#5: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 17 / 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: 2.78 Å3/Da / Density % sol: 55.73 %
Crystal growTemperature: 293 K / Method: lipidic cubic phase / pH: 8
Details: Protein complex was reconstituted with a 10:1 (w/w) mixture of monoolein and cholesterol. Crystals were grown in 100 mM Tris pH 8, 50-70 mM MgCl2, 30% PEG 300, 2-4% 1,3-butanediol

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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: Nov 21, 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: 2.79→47.403 Å / Num. obs: 17795 / % possible obs: 98.8 % / Redundancy: 10.545 % / Biso Wilson estimate: 85.736 Å2 / CC1/2: 0.997 / Rmerge(I) obs: 0.206 / Rrim(I) all: 0.217 / Χ2: 0.986 / Net I/σ(I): 8.1 / Num. measured all: 187653 / Scaling rejects: 432
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
2.79-2.878.6141.8820.8510234131211880.5361.98490.5
2.87-2.9410.3771.6881.1812722125612260.6411.76897.6
2.94-3.0311.2371.5381.4114203126412640.6681.611100
3.03-3.1211.3481.3471.7413765121312130.7081.411100
3.12-3.2310.8921.1552.112569115411540.6951.212100
3.23-3.3410.3050.9232.7311676113411330.7790.97399.9
3.34-3.4710.950.7713.412166111211110.8620.80999.9
3.47-3.6110.2740.5744.6110736104710450.9210.60599.8
3.61-3.7710.8860.426.4111147102510240.9520.44199.9
3.77-3.9510.8330.3238.17104979709690.9840.34199.9
3.95-4.1610.9840.25910.72101059239200.9740.27399.7
4.16-4.4210.8790.22512.2396178858840.9660.23899.9
4.42-4.7210.150.15215.8584048298280.990.16199.9
4.72-5.110.3840.12517.1380797807780.9960.13199.7
5.1-5.5910.5620.14215.7873727226980.9930.1596.7
5.59-6.2511.0730.1416.2672756596570.9920.14799.7
6.25-7.2110.8740.1218.9361985735700.9920.12699.5
7.21-8.839.810.07923.549155075010.9950.08398.8
8.83-12.499.7460.05828.1738794053980.9980.06198.3
12.49-47.4038.9490.05828.2220942462340.9980.06295.1

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Processing

Software
NameVersionClassification
PHENIX(1.14_3260)refinement
XDSdata reduction
XSCALEdata scaling
PDB_EXTRACT3.25data extraction
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6OS2

6os2


Resolution: 2.794→47.403 Å / SU ML: 0.48 / Cross valid method: THROUGHOUT / σ(F): 1.39 / Phase error: 36.69 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2822 888 5 %
Rwork0.2389 16876 -
obs0.2412 17764 98.63 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 216.96 Å2 / Biso mean: 92.5509 Å2 / Biso min: 40.58 Å2
Refinement stepCycle: final / Resolution: 2.794→47.403 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3879 0 94 17 3990
Biso mean--108.81 67.49 -
Num. residues----516
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 6

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.7936-2.96860.4041390.35392662280195
2.9686-3.19770.37351470.324928002947100
3.1977-3.51950.39941470.298427982945100
3.5195-4.02850.31471490.265528222971100
4.0285-5.07450.22231500.197228583008100
5.0745-47.40940.25791560.21432936309298
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.5778-0.1309-0.43091.2809-0.75552.10880.00760.17370.1566-0.0682-0.1514-0.1924-0.15470.08060.15290.4776-0.009-0.01380.5117-0.0220.4377-14.9126-13.4647-57.0137
24.3521-0.96641.2029.8076-4.62347.1062-0.5003-0.1448-0.7505-0.93660.4577-0.2351.2556-0.47120.01231.09850.00790.12331.14050.13460.9498-18.0178-47.8622-16.9204
31.9525-0.1873-0.27611.2424-0.56383.7121-0.1617-0.3018-0.17820.132-0.0553-0.13470.5260.17110.30640.6222-0.006-0.00530.53690.01120.5517-17.6911-29.6058-43.3914
44.53135.23635.69036.72575.79048.1230.4726-0.83240.01480.6658-0.64920.36751.3302-0.02530.42160.94230.00960.15581.19910.0060.626-11.1454-14.5006-10.374
53.9509-0.649-1.58765.841-5.30367.20880.0176-0.0901-0.3875-0.6406-0.15770.139-0.1298-0.23570.01380.88680.0456-0.08530.7113-0.08850.6067-2.5126-16.0348-17.9898
64.10293.16872.66287.8770.84856.07640.3710.1587-0.36720.5379-0.1126-0.2323-0.01540.1693-0.16140.7446-0.0136-0.0220.919-0.00360.4839-1.7737-16.3493-11.3948
75.7003-3.95074.07376.185-4.21686.97570.10951.41670.14350.1042-0.44150.0668-0.4567-0.98780.23360.40020.0425-0.11360.95870.02170.5547-20.2127-19.649-29.3444
84.70145.75523.87177.08894.85373.4648-0.63250.05980.6214-0.0276-0.12640.4778-0.9627-0.05191.07030.9456-0.0573-0.20520.63840.02160.6763-3.7202-7.3726-12.4364
96.83091.28391.08687.6897-6.041.9998-0.718-1.4146-0.72971.4271.15841.0731-1.1315-0.9407-0.48990.78280.1457-0.04720.84110.1540.3804-14.8409-16.707-72.5748
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 11 through 225 )A11 - 225
2X-RAY DIFFRACTION2chain 'A' and (resid 226 through 266 )A226 - 266
3X-RAY DIFFRACTION3chain 'A' and (resid 267 through 1318 )A267 - 1318
4X-RAY DIFFRACTION4chain 'D' and (resid 1 through 32 )D1 - 32
5X-RAY DIFFRACTION5chain 'D' and (resid 33 through 59 )D33 - 59
6X-RAY DIFFRACTION6chain 'D' and (resid 60 through 98 )D60 - 98
7X-RAY DIFFRACTION7chain 'D' and (resid 99 through 113 )D99 - 113
8X-RAY DIFFRACTION8chain 'D' and (resid 114 through 127 )D114 - 127
9X-RAY DIFFRACTION9chain 'B' and (resid 2 through 8 )B2 - 8

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