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- PDB-4u14: Structure of the M3 muscarinic acetylcholine receptor bound to th... -

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Basic information

Entry
Database: PDB / ID: 4u14
TitleStructure of the M3 muscarinic acetylcholine receptor bound to the antagonist tiotropium crystallized with disulfide-stabilized T4 lysozyme (dsT4L)
ComponentsMuscarinic acetylcholine receptor M3,Endolysin,Muscarinic acetylcholine receptor M3
KeywordsMEMBRANE PROTEIN / alpha helix / G protein-coupled receptors (GPCRs) / T4 lysozyme / fusion protein / chimera protein
Function / homology
Function and homology information


negative regulation of heart rate by acetylcholine / G protein-coupled acetylcholine receptor binding / Muscarinic acetylcholine receptors / regulation of vascular associated smooth muscle contraction / saliva secretion / quaternary ammonium group binding / ion channel modulating, G protein-coupled receptor signaling pathway / phospholipase C-activating G protein-coupled acetylcholine receptor signaling pathway / response to acetylcholine / G protein-coupled acetylcholine receptor activity ...negative regulation of heart rate by acetylcholine / G protein-coupled acetylcholine receptor binding / Muscarinic acetylcholine receptors / regulation of vascular associated smooth muscle contraction / saliva secretion / quaternary ammonium group binding / ion channel modulating, G protein-coupled receptor signaling pathway / phospholipase C-activating G protein-coupled acetylcholine receptor signaling pathway / response to acetylcholine / G protein-coupled acetylcholine receptor activity / positive regulation of vascular associated smooth muscle contraction / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / positive regulation of smooth muscle contraction / G protein-coupled serotonin receptor activity / synaptic transmission, cholinergic / G alpha (q) signalling events / acetylcholine binding / acetylcholine receptor signaling pathway / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / ligand-gated ion channel signaling pathway / asymmetric synapse / smooth muscle contraction / axon terminus / positive regulation of vasoconstriction / viral release from host cell by cytolysis / peptidoglycan catabolic process / basal plasma membrane / calcium-mediated signaling / postsynaptic density membrane / G protein-coupled acetylcholine receptor signaling pathway / positive regulation of insulin secretion / cell wall macromolecule catabolic process / presynaptic membrane / lysozyme / lysozyme activity / chemical synaptic transmission / basolateral plasma membrane / host cell cytoplasm / defense response to bacterium / dendrite / glutamatergic synapse / synapse / endoplasmic reticulum membrane / plasma membrane
Similarity search - Function
Muscarinic acetylcholine receptor M3 / Muscarinic acetylcholine receptor family / Endolysin T4 type / T4-type lysozyme / Glycoside hydrolase, family 24 / Lysozyme domain superfamily / Phage lysozyme / Serpentine type 7TM GPCR chemoreceptor Srsx / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like ...Muscarinic acetylcholine receptor M3 / Muscarinic acetylcholine receptor family / Endolysin T4 type / T4-type lysozyme / Glycoside hydrolase, family 24 / Lysozyme domain superfamily / Phage lysozyme / 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) / Lysozyme-like domain superfamily
Similarity search - Domain/homology
Chem-0HK / Endolysin / Muscarinic acetylcholine receptor M3
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Enterobacteria phage T4 (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / molecular replacement / Resolution: 3.57 Å
AuthorsThorsen, T.S. / Matt, R.A. / Weis, W.I. / Kobilka, B.K.
Funding support United States, Denmark, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM08311806 United States
National Science Foundation (NSF, United States)CHE-1223785 United States
Mathers Foundation United States
Benzon Foundation Denmark
CitationJournal: Structure / Year: 2014
Title: Modified T4 Lysozyme Fusion Proteins Facilitate G Protein-Coupled Receptor Crystallogenesis.
Authors: Thorsen, T.S. / Matt, R. / Weis, W.I. / Kobilka, B.K.
History
DepositionJul 15, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 26, 2014Provider: repository / Type: Initial release
Revision 1.1Dec 24, 2014Group: Database references
Revision 1.2Sep 13, 2017Group: Author supporting evidence / Database references ...Author supporting evidence / Database references / Derived calculations / Other / Source and taxonomy
Category: citation / entity_src_gen ...citation / entity_src_gen / pdbx_audit_support / pdbx_database_status / pdbx_struct_oper_list
Item: _citation.journal_id_CSD / _entity_src_gen.pdbx_alt_source_flag ..._citation.journal_id_CSD / _entity_src_gen.pdbx_alt_source_flag / _pdbx_audit_support.funding_organization / _pdbx_database_status.pdb_format_compatible / _pdbx_struct_oper_list.symmetry_operation
Revision 1.3Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Dec 27, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / refine_hist / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Muscarinic acetylcholine receptor M3,Endolysin,Muscarinic acetylcholine receptor M3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,0772
Polymers52,6851
Non-polymers3931
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area21790 Å2
2
A: Muscarinic acetylcholine receptor M3,Endolysin,Muscarinic acetylcholine receptor M3
hetero molecules

A: Muscarinic acetylcholine receptor M3,Endolysin,Muscarinic acetylcholine receptor M3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)106,1554
Polymers105,3702
Non-polymers7852
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation7_557y,x,-z+21
Buried area2400 Å2
ΔGint-18 kcal/mol
Surface area41170 Å2
MethodPISA
Unit cell
Length a, b, c (Å)54.952, 54.952, 348.030
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number92
Space group name H-MP41212
Detailsbiological unit is the same as asym.

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Components

#1: Protein Muscarinic acetylcholine receptor M3,Endolysin,Muscarinic acetylcholine receptor M3 / Lysis protein / Lysozyme / Muramidase


Mass: 52684.773 Da / Num. of mol.: 1
Fragment: UNP P08483 residues 57-259, 482-563, P00720 residues 1-161
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat), (gene. exp.) Enterobacteria phage T4 (virus)
Gene: Chrm3, Chrm-3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P08483, UniProt: P00720, lysozyme
#2: Chemical ChemComp-0HK / (1R,2R,4S,5S,7S)-7-{[hydroxy(dithiophen-2-yl)acetyl]oxy}-9,9-dimethyl-3-oxa-9-azoniatricyclo[3.3.1.0~2,4~]nonane / Tiotropium / Tiotropium bromide


Mass: 392.512 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H22NO4S2 / Comment: antagonist*YM
Sequence detailsThe fusion protein is a chimeric of M3 and T4: T4L lysozyme was inserted into a intracellular loop ...The fusion protein is a chimeric of M3 and T4: T4L lysozyme was inserted into a intracellular loop of the receptor. The fusion protein is made of M 3 ( residues 57-259) - T4L (residues 1001-1161)- M3 (residues 482-563). These two proteins were numbered separately in order to maintain original (UNP P08483 and P00720) numbering.

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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: 2.49 Å3/Da / Density % sol: 50.55 %
Crystal growTemperature: 298 K / Method: lipidic cubic phase / pH: 8.1
Details: 45% PEG 300, 110 mM ammonium sulfate, 113.5 mM lithium citrate, 100 mM Tris

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Data collection

DiffractionMean temperature: 80 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-D / Wavelength: 1.033 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Jun 10, 2013
RadiationMonochromator: Si-111 crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.033 Å / Relative weight: 1
ReflectionResolution: 3.57→31.62 Å / Num. obs: 6315 / % possible obs: 89.4 % / Redundancy: 3.1 % / Biso Wilson estimate: 99.37 Å2 / Rmerge(I) obs: 0.204 / Net I/σ(I): 4 / Num. measured all: 19754
Reflection shell

Diffraction-ID: 1 / Rejects: 0

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique allΧ2% possible all
3.57-3.730.8271.16201.12589.3
3.7-3.843.20.6711.46331.0394.3
3.84-4.023.30.4821.96141.01691.2
4.02-4.233.10.3872.36311.17489.4
4.23-4.493.10.27736051.18691.5
4.5-4.843.20.2293.36331.26690.6
4.84-5.333.20.1834.66221.2589.5
5.33-6.083.20.22546491.38789.5
6.1-7.663.10.1356.36341.07187.1
7.66-31.622.90.06611.86741.12893.9

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Phasing

PhasingMethod: molecular replacement

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Processing

SoftwareName: PHENIX / Version: (phenix.refine: 1.9_1692) / Classification: refinement
RefinementResolution: 3.57→31.62 Å / SU ML: 0.44 / Cross valid method: FREE R-VALUE / σ(F): 1.45 / Phase error: 31.68 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.3253 320 5.07 %Random selection
Rwork0.2723 ---
obs0.2751 6315 89.42 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.57→31.62 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3416 0 26 0 3442
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0043538
X-RAY DIFFRACTIONf_angle_d0.5614835
X-RAY DIFFRACTIONf_dihedral_angle_d12.2121231
X-RAY DIFFRACTIONf_chiral_restr0.018569
X-RAY DIFFRACTIONf_plane_restr0.003587
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.57-4.49440.34641540.29012950X-RAY DIFFRACTION91
4.4944-31.62580.31371660.26213045X-RAY DIFFRACTION88

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