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- PDB-4nc3: Crystal structure of the 5-HT2B receptor solved using serial femt... -

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Basic information

Entry
Database: PDB / ID: 4nc3
TitleCrystal structure of the 5-HT2B receptor solved using serial femtosecond crystallography in lipidic cubic phase.
ComponentsChimera protein of human 5-hydroxytryptamine receptor 2B and E. Coli soluble cytochrome b562
KeywordsSIGNALING PROTEIN / ELECTRON TRANSPORT / Serial femtosecond crystallography / human 5HT2B receptor / ergotamine / novel protein engineering / GPCR Network / Membrane protein / lipidic cubic phase / PSI-Biology / free electron laser / Structural Genomics / GPCR / membrane
Function / homology
Function and homology information


intestine smooth muscle contraction / Gq/11-coupled serotonin receptor activity / positive regulation of phosphatidylinositol biosynthetic process / G protein-coupled serotonin receptor signaling pathway / regulation of behavior / G protein-coupled serotonin receptor complex / Serotonin receptors / serotonin receptor activity / G protein-coupled serotonin receptor activity / phospholipase C-activating serotonin receptor signaling pathway ...intestine smooth muscle contraction / Gq/11-coupled serotonin receptor activity / positive regulation of phosphatidylinositol biosynthetic process / G protein-coupled serotonin receptor signaling pathway / regulation of behavior / G protein-coupled serotonin receptor complex / Serotonin receptors / serotonin receptor activity / G protein-coupled serotonin receptor activity / phospholipase C-activating serotonin receptor signaling pathway / serotonin receptor signaling pathway / embryonic morphogenesis / neurotransmitter receptor activity / serotonin binding / vasoconstriction / cardiac muscle hypertrophy / neural crest cell migration / neural crest cell differentiation / 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 / heart morphogenesis / positive regulation of endothelial cell proliferation / release of sequestered calcium ion into cytosol / ERK1 and ERK2 cascade / GTPase activator activity / positive regulation of cytokine production / calcium-mediated signaling / electron transport chain / 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 / G protein-coupled receptor signaling pathway / iron ion binding / response to xenobiotic stimulus / positive regulation of cell population proliferation / synapse / heme binding / dendrite / negative regulation of apoptotic process / nucleoplasm / plasma membrane / cytoplasm
Similarity search - Function
5-Hydroxytryptamine 2B receptor / 5-hydroxytryptamine receptor family / Cytochrome c/b562 / Rhopdopsin 7-helix transmembrane proteins / Rhodopsin 7-helix transmembrane proteins / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Serpentine type 7TM GPCR chemoreceptor Srsx / Four Helix Bundle (Hemerythrin (Met), subunit A) ...5-Hydroxytryptamine 2B receptor / 5-hydroxytryptamine receptor family / Cytochrome c/b562 / Rhopdopsin 7-helix transmembrane proteins / Rhodopsin 7-helix transmembrane proteins / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Serpentine type 7TM GPCR chemoreceptor Srsx / Four Helix Bundle (Hemerythrin (Met), subunit A) / 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) / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
CHOLESTEROL / DIACYL GLYCEROL / Ergotamine / OLEIC ACID / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / DI(HYDROXYETHYL)ETHER / PALMITIC ACID / Soluble cytochrome b562 / 5-hydroxytryptamine receptor 2B
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
MethodX-RAY DIFFRACTION / FREE ELECTRON LASER / MOLECULAR REPLACEMENT / Resolution: 2.8 Å
AuthorsLiu, W. / Wacker, D. / Gati, C. / Han, G.W. / James, D. / Wang, D. / Nelson, G. / Weierstall, U. / Katritch, V. / Barty, A. ...Liu, W. / Wacker, D. / Gati, C. / Han, G.W. / James, D. / Wang, D. / Nelson, G. / Weierstall, U. / Katritch, V. / Barty, A. / Zatsepin, N.A. / Li, D. / Messerschmidt, M. / Boutet, S. / Williams, G.J. / Koglin, J.E. / Seibert, M.M. / Wang, C. / Shah, S.T.A. / Basu, S. / Fromme, R. / Kupitz, C. / Rendek, K.N. / Grotjohann, I. / Fromme, P. / Kirian, R.A. / Beyerlein, K.R. / White, T.A. / Chapman, H.N. / Caffrey, M. / Spence, J.C.H. / Stevens, R.C. / Cherezov, V. / GPCR Network (GPCR)
CitationJournal: Science / Year: 2013
Title: Serial femtosecond crystallography of G protein-coupled receptors.
Authors: Liu, W. / Wacker, D. / Gati, C. / Han, G.W. / James, D. / Wang, D. / Nelson, G. / Weierstall, U. / Katritch, V. / Barty, A. / Zatsepin, N.A. / Li, D. / Messerschmidt, M. / Boutet, S. / ...Authors: Liu, W. / Wacker, D. / Gati, C. / Han, G.W. / James, D. / Wang, D. / Nelson, G. / Weierstall, U. / Katritch, V. / Barty, A. / Zatsepin, N.A. / Li, D. / Messerschmidt, M. / Boutet, S. / Williams, G.J. / Koglin, J.E. / Seibert, M.M. / Wang, C. / Shah, S.T. / Basu, S. / Fromme, R. / Kupitz, C. / Rendek, K.N. / Grotjohann, I. / Fromme, P. / Kirian, R.A. / Beyerlein, K.R. / White, T.A. / Chapman, H.N. / Caffrey, M. / Spence, J.C. / Stevens, R.C. / Cherezov, V.
History
DepositionOct 23, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 18, 2013Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2014Group: Database references
Revision 1.2Jun 7, 2017Group: Database references / Structure summary
Revision 1.3Feb 14, 2018Group: Data collection / Category: diffrn_source
Item: _diffrn_source.pdbx_synchrotron_beamline / _diffrn_source.pdbx_synchrotron_site
Revision 1.4Aug 16, 2023Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_related_exp_data_set / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.5Sep 20, 2023Group: Refinement description / Category: pdbx_initial_refinement_model
Revision 1.6Oct 30, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Chimera protein of human 5-hydroxytryptamine receptor 2B and E. Coli soluble cytochrome b562
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,26015
Polymers48,2831
Non-polymers4,97714
Water1267
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)61.500, 122.200, 168.500
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Chimera protein of human 5-hydroxytryptamine receptor 2B and E. Coli soluble cytochrome b562


Mass: 48283.273 Da / Num. of mol.: 1 / Fragment: BRIL / Mutation: M7W
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

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Non-polymers , 9 types, 21 molecules

#2: Chemical ChemComp-PLM / PALMITIC ACID


Mass: 256.424 Da / Num. of mol.: 1 / Fragment: BRIL / Mutation: H102I / Source method: obtained synthetically / Formula: C16H32O2
#3: Chemical ChemComp-ERM / Ergotamine


Mass: 581.661 Da / Num. of mol.: 1 / Fragment: BRIL / Mutation: R106L / Source method: obtained synthetically / Formula: C33H35N5O5 / Comment: neurotransmitter, alkaloid*YM
#4: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O
#5: Chemical
ChemComp-OLC / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / 1-Oleoyl-R-glycerol


Mass: 356.540 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C21H40O4
#6: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O3
#7: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#8: Chemical ChemComp-OLA / OLEIC ACID


Mass: 282.461 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C18H34O2
#9: Chemical ChemComp-DGA / DIACYL GLYCEROL


Mass: 625.018 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C39H76O5
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Formula: H2O

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Details

Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION

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Sample preparation

CrystalDensity Matthews: 3.28 Å3/Da / Density % sol: 62.48 %
Crystal growTemperature: 293 K / Method: lipidic cubic phase / pH: 8
Details: 100mM Tris/HCl pH8.0, 20-80mM MgCl2 and 30% (v/v) PEG400 , Lipidic Cubic Phase (LCP), temperature 293K

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Data collection

DiffractionMean temperature: 294 K
Diffraction sourceSource: FREE ELECTRON LASER / Site: SLAC LCLS / Beamline: CXI / Wavelength: 1.3 Å
DetectorType: Cornell-SLAC Pixel Array Detector (CSPAD) / Detector: PIXEL / Date: Mar 1, 2013 / Details: K-B Mirrors
RadiationMonochromator: K-B Mirrors / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.3 Å / Relative weight: 1
ReflectionResolution: 2.8→35 Å / Num. obs: 16052 / % possible obs: 100 % / Redundancy: 1150 % / Rmerge(I) obs: 0.095 / Net I/σ(I): 5.9
Reflection shellResolution: 2.8→2.9 Å / % possible all: 100

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Processing

Software
NameVersionClassification
PHASERphasing
PHENIX(phenix.refine: 1.8_1069)refinement
CrystFELdata reduction
CrystFELdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 4IB4

4ib4


Resolution: 2.8→34.681 Å / SU ML: 0.44 / σ(F): 1.33 / Phase error: 31.03 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2701 814 5.08 %
Rwork0.2266 --
obs0.2287 16025 99.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.8→34.681 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2837 0 277 7 3121
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0023179
X-RAY DIFFRACTIONf_angle_d0.6014299
X-RAY DIFFRACTIONf_dihedral_angle_d15.4141157
X-RAY DIFFRACTIONf_chiral_restr0.032506
X-RAY DIFFRACTIONf_plane_restr0.002499
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.8001-2.97540.38411370.34092489X-RAY DIFFRACTION100
2.9754-3.2050.33881360.30722470X-RAY DIFFRACTION100
3.205-3.52720.35481290.26292512X-RAY DIFFRACTION100
3.5272-4.0370.26851430.21532534X-RAY DIFFRACTION100
4.037-5.08360.23251330.17842539X-RAY DIFFRACTION100
5.0836-34.68390.26771360.24912667X-RAY DIFFRACTION100
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)
12.24-0.02610.71172.07540.12941.28170.04740.0881-0.1327-0.0432-0.036-0.10610.25150.0323-0.00160.73260.0302-0.00220.66060.07120.6925-21.768-18.163-2.1578
20.18610.1367-0.13240.2899-0.00750.28450.3702-0.0559-0.053-0.0634-0.2081-0.39850.1855-0.7574-01.02640.0854-0.12121.98750.121.1809-48.373-6.3246-45.2049
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resseq 48:400) or chain 'A' and (resseq 1201:1201)
2X-RAY DIFFRACTION2chain 'A' and (resseq 1001:1106)

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