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Basic information

Entry
Database: PDB / ID: 5tud
TitleStructural Insights into the Extracellular Recognition of the Human Serotonin 2B Receptor by an Antibody
Components
  • 5-hydroxytryptamine receptor 2B,Soluble cytochrome b562 chimera
  • Anti-5-HT2B Fab heavy chain
  • Anti-5-HT2B Fab light chain
KeywordsSIGNALING PROTEIN / 7-TM / GPCR / Fab / complex
Function / homology
Function and homology information


intestine smooth muscle contraction / Gq/11-coupled serotonin receptor activity / phospholipase C-activating serotonin receptor signaling pathway / positive regulation of phosphatidylinositol biosynthetic process / G protein-coupled serotonin receptor signaling pathway / G protein-coupled serotonin receptor complex / protein kinase C signaling / regulation of behavior / Serotonin receptors / serotonin receptor signaling pathway ...intestine smooth muscle contraction / Gq/11-coupled serotonin receptor activity / phospholipase C-activating serotonin receptor signaling pathway / positive regulation of phosphatidylinositol biosynthetic process / G protein-coupled serotonin receptor signaling pathway / G protein-coupled serotonin receptor complex / protein kinase C signaling / regulation of behavior / Serotonin receptors / serotonin receptor signaling pathway / embryonic morphogenesis / cellular response to temperature stimulus / serotonin binding / vasoconstriction / G protein-coupled serotonin receptor activity / G protein-coupled receptor internalization / cardiac muscle hypertrophy / : / neural crest cell differentiation / neural crest cell migration / neurotransmitter receptor activity / cGMP-mediated signaling / positive regulation of cell division / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / G-protein alpha-subunit binding / phosphorylation / heart morphogenesis / release of sequestered calcium ion into cytosol / positive regulation of endothelial cell proliferation / ERK1 and ERK2 cascade / phosphatidylinositol 3-kinase/protein kinase B signal transduction / GTPase activator activity / positive regulation of nitric-oxide synthase activity / positive regulation of cytokine production / calcium-mediated signaling / electron transport chain / positive regulation of MAP kinase activity / intracellular calcium ion homeostasis / chemical synaptic transmission / G alpha (q) signalling events / positive regulation of canonical NF-kappaB signal transduction / periplasmic space / electron transfer activity / positive regulation of ERK1 and ERK2 cascade / iron ion binding / response to xenobiotic stimulus / G protein-coupled receptor signaling pathway / dendrite / heme binding / positive regulation of cell population proliferation / synapse / negative regulation of apoptotic process / nucleoplasm / plasma membrane / cytoplasm
Similarity search - Function
5-Hydroxytryptamine 2B receptor / 5-hydroxytryptamine 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. ...5-Hydroxytryptamine 2B receptor / 5-hydroxytryptamine 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) / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Ergotamine / Soluble cytochrome b562 / 5-hydroxytryptamine receptor 2B
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
Mus musculus (house mouse)
MethodX-RAY DIFFRACTION / FREE ELECTRON LASER / MOLECULAR REPLACEMENT / Resolution: 3 Å
AuthorsIshchenko, A. / Wacker, D. / Kapoor, M. / Zhang, A. / Han, G.W. / Basu, S. / Boutet, S. / James, D. / Wang, D. / Weierstall, U. ...Ishchenko, A. / Wacker, D. / Kapoor, M. / Zhang, A. / Han, G.W. / Basu, S. / Boutet, S. / James, D. / Wang, D. / Weierstall, U. / Liu, W. / Katritch, V. / Stevens, R.C. / Cherezov, V.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM108635 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM094618 United States
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2017
Title: Structural insights into the extracellular recognition of the human serotonin 2B receptor by an antibody.
Authors: Ishchenko, A. / Wacker, D. / Kapoor, M. / Zhang, A. / Han, G.W. / Basu, S. / Patel, N. / Messerschmidt, M. / Weierstall, U. / Liu, W. / Katritch, V. / Roth, B.L. / Stevens, R.C. / Cherezov, V.
History
DepositionNov 5, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 26, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 16, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Sep 13, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Feb 14, 2018Group: Data collection / Category: diffrn_source
Item: _diffrn_source.pdbx_synchrotron_beamline / _diffrn_source.pdbx_synchrotron_site
Revision 1.4Nov 28, 2018Group: Data collection
Category: diffrn / pdbx_serial_crystallography_data_reduction ...diffrn / pdbx_serial_crystallography_data_reduction / pdbx_serial_crystallography_measurement / pdbx_serial_crystallography_sample_delivery / pdbx_serial_crystallography_sample_delivery_injection
Item: _diffrn.pdbx_serial_crystal_experiment
Revision 1.5Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.6Oct 4, 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
Revision 1.7Oct 30, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: 5-hydroxytryptamine receptor 2B,Soluble cytochrome b562 chimera
B: Anti-5-HT2B Fab light chain
C: Anti-5-HT2B Fab heavy chain
D: 5-hydroxytryptamine receptor 2B,Soluble cytochrome b562 chimera
E: Anti-5-HT2B Fab light chain
F: Anti-5-HT2B Fab heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)201,4288
Polymers200,2656
Non-polymers1,1632
Water00
1
A: 5-hydroxytryptamine receptor 2B,Soluble cytochrome b562 chimera
B: Anti-5-HT2B Fab light chain
C: Anti-5-HT2B Fab heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)100,7144
Polymers100,1323
Non-polymers5821
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5420 Å2
ΔGint-31 kcal/mol
Surface area38090 Å2
MethodPISA
2
D: 5-hydroxytryptamine receptor 2B,Soluble cytochrome b562 chimera
E: Anti-5-HT2B Fab light chain
F: Anti-5-HT2B Fab heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)100,7144
Polymers100,1323
Non-polymers5821
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5810 Å2
ΔGint-37 kcal/mol
Surface area37810 Å2
MethodPISA
Unit cell
Length a, b, c (Å)71.290, 118.860, 145.380
Angle α, β, γ (deg.)90.00, 90.37, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein 5-hydroxytryptamine receptor 2B,Soluble cytochrome b562 chimera / 5-HT2B / Serotonin receptor 2B / Cytochrome b-562


Mass: 51497.035 Da / Num. of mol.: 2
Fragment: UNP P41595 residues 36-248 and 314-405 linked by UNP P0ABE7 residues 23-128
Mutation: M144W, M1007W, H1102I, R1106L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (E. coli)
Gene: HTR2B, cybC / Plasmid: pFastBac / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): Sf9 / References: UniProt: P41595, UniProt: P0ABE7
#2: Antibody Anti-5-HT2B Fab light chain


Mass: 23151.842 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm)
#3: Antibody Anti-5-HT2B Fab heavy chain


Mass: 25483.455 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm)
#4: Chemical ChemComp-ERM / Ergotamine


Mass: 581.661 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C33H35N5O5 / Comment: neurotransmitter, alkaloid*YM
Has protein modificationY

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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: 3.07 Å3/Da / Density % sol: 64.21 %
Crystal growTemperature: 293 K / Method: lipidic cubic phase / pH: 7.7
Details: Tris/HCL pH 7.7, 60 mM Sodium/Potassium Tartrate, 25% PEG400.

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Data collection

DiffractionMean temperature: 293 K / Serial crystal experiment: Y
Diffraction sourceSource: FREE ELECTRON LASER / Site: SLAC LCLS / Beamline: CXI / Wavelength: 1.56 Å
DetectorType: CS-PAD CXI-1 / Detector: PIXEL / Date: Jan 31, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.56 Å / Relative weight: 1
ReflectionResolution: 3→35.64 Å / Num. obs: 48606 / % possible obs: 100 % / Redundancy: 310.3 % / Biso Wilson estimate: 90.32 Å2 / Net I/σ(I): 4.3
Reflection shellResolution: 3→3.1 Å / Redundancy: 120.26 % / Mean I/σ(I) obs: 0.4 / CC1/2: 0.34 / % possible all: 100
Serial crystallography measurementPhotons per pulse: 390000000000 Tphotons/pulse / Pulse duration: 35 fsec. / Pulse photon energy: 7.95 keV / XFEL pulse repetition rate: 120 Hz
Serial crystallography sample deliveryMethod: injection
Serial crystallography sample delivery injectionInjector diameter: 1.5 µm
Serial crystallography data reductionFrames failed index: 193328 / Frames indexed: 52291 / Frames total: 1877040

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Processing

Software
NameVersionClassification
BUSTER2.10.2refinement
CrystFELdata reduction
PHASERphasing
CrystFELdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4NC3

4nc3


Resolution: 3→35.64 Å / Cor.coef. Fo:Fc: 0.919 / Cor.coef. Fo:Fc free: 0.896 / Rfactor Rfree error: 0 / Cross valid method: THROUGHOUT / σ(F): 0 / SU R Blow DPI: 1.822 / SU Rfree Blow DPI: 0.357
Details: unmodeled densities (potentially Zn ion) near (1) His232, His235 of chain C & Asp1 of chain E, and (2) His233, His231 of Chain C & His1063 of chain A
RfactorNum. reflection% reflectionSelection details
Rfree0.247 2259 4.65 %RANDOM
Rwork0.227 ---
obs0.228 48606 99.9 %-
Displacement parametersBiso mean: 106.1 Å2
Baniso -1Baniso -2Baniso -3
1-7.2629 Å20 Å22.1576 Å2
2---22.6624 Å20 Å2
3---15.3994 Å2
Refine analyzeLuzzati coordinate error obs: 0.62 Å
Refinement stepCycle: 1 / Resolution: 3→35.64 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12298 0 86 0 12384
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.00512712HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.7617399HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d5649SINUSOIDAL2
X-RAY DIFFRACTIONt_incorr_chiral_ct
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes239HARMONIC2
X-RAY DIFFRACTIONt_gen_planes1910HARMONIC5
X-RAY DIFFRACTIONt_it12712HARMONIC20
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_omega_torsion4.08
X-RAY DIFFRACTIONt_other_torsion2.31
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_chiral_improper_torsion1776SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact14866SEMIHARMONIC4
LS refinement shellResolution: 3→3.08 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.248 167 4.7 %
Rwork0.242 3388 -
all0.243 3555 -
obs--99.58 %
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)
10.1220.44761.08430.89881.5574.38920.1139-0.1398-0.11610.0925-0.0277-0.06510.3326-0.1177-0.0862-0.16450.0496-0.01390.20040.0639-0.195316.0121-29.485962.7126
21.12480.3938-0.20761.0764-1.39184.86410.1025-0.06490.03520.16650.11140.2283-0.1734-0.2297-0.2139-0.05120.00880.0208-0.07990.023-0.154920.4383-10.5337139.413
31.00440.363-1.40690.51-0.155.3519-0.03820.0285-0.0138-0.14680.18340.07290.458-0.5429-0.1452-0.035-0.0764-0.074-0.05560.0022-0.166618.0545-26.5149130.8453
40.01440.4516-1.12680.6978-1.34052.53940.1155-0.26840.09960.1445-0.1052-0.0632-0.37020.2745-0.0103-0.0947-0.0481-0.01750.26820.0301-0.176818.7411-64.038959.9866
51.42030.2840.90520.94520.45185.14890.162-0.052-0.01660.1574-0.1126-0.0160.1822-0.1227-0.0494-0.1409-0.04460.01670.0120.0111-0.216516.9535-86.1553135.8512
61.86730.22322.12020.38340.47944.9011-0.1281-0.01730.1042-0.11620.02990.0414-0.3728-0.38620.0982-0.07040.0344-0.0173-0.087-0.0025-0.12219.5503-69.5515128.7206
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1{ A|54 - A|1106 }
2X-RAY DIFFRACTION2{ B|1 - B|211 }
3X-RAY DIFFRACTION3{ C|1 - C|235 }
4X-RAY DIFFRACTION4{ D|55 - D|1106 }
5X-RAY DIFFRACTION5{ E|1 - E|211 }
6X-RAY DIFFRACTION6{ F|1 - F|233 }

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