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- PDB-4a4m: Crystal structure of the light-activated constitutively active N2... -

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Basic information

Entry
Database: PDB / ID: 4a4m
TitleCrystal structure of the light-activated constitutively active N2C, M257Y,D282C rhodopsin mutant in complex with a peptide resembling the C-terminus of the Galpha-protein subunit (GaCT)
Components
  • GUANINE NUCLEOTIDE-BINDING PROTEIN G(T) SUBUNIT ALPHA-3
  • RHODOPSIN
KeywordsSIGNALING PROTEIN / G-PROTEIN / G-PROTEIN-COUPLED RECEPTORS / SIGNAL TANSDUCTION / VISUAL SYSTEM / METARHODOPSIN-II
Function / homology
Function and homology information


Adenylate cyclase inhibitory pathway / Opsins / VxPx cargo-targeting to cilium / sensory perception of umami taste / rod photoreceptor outer segment / rod bipolar cell differentiation / sperm head plasma membrane / negative regulation of cyclic-nucleotide phosphodiesterase activity / podosome assembly / absorption of visible light ...Adenylate cyclase inhibitory pathway / Opsins / VxPx cargo-targeting to cilium / sensory perception of umami taste / rod photoreceptor outer segment / rod bipolar cell differentiation / sperm head plasma membrane / negative regulation of cyclic-nucleotide phosphodiesterase activity / podosome assembly / absorption of visible light / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / opsin binding / The canonical retinoid cycle in rods (twilight vision) / sensory perception of sweet taste / detection of chemical stimulus involved in sensory perception of bitter taste / : / G protein-coupled photoreceptor activity / photoreceptor inner segment membrane / rhodopsin mediated signaling pathway / 11-cis retinal binding / cellular response to light stimulus / G protein-coupled receptor complex / Inactivation, recovery and regulation of the phototransduction cascade / phototransduction, visible light / thermotaxis / Activation of the phototransduction cascade / detection of temperature stimulus involved in thermoception / outer membrane / arrestin family protein binding / photoreceptor cell maintenance / acyl binding / ADP signalling through P2Y purinoceptor 12 / Extra-nuclear estrogen signaling / photoreceptor outer segment membrane / G alpha (i) signalling events / response to light stimulus / phototransduction / photoreceptor outer segment / G-protein alpha-subunit binding / sperm midpiece / visual perception / photoreceptor inner segment / guanyl-nucleotide exchange factor activity / G protein-coupled receptor binding / G-protein beta/gamma-subunit complex binding / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / microtubule cytoskeleton organization / photoreceptor disc membrane / GDP binding / heterotrimeric G-protein complex / cell-cell junction / gene expression / G protein-coupled receptor signaling pathway / Golgi membrane / GTPase activity / GTP binding / protein kinase binding / zinc ion binding / membrane / identical protein binding / metal ion binding / plasma membrane / cytoplasm
Similarity search - Function
Rhodopsin, N-terminal / Amino terminal of the G-protein receptor rhodopsin / Rhodopsin / Opsin / Visual pigments (opsins) retinal binding site / Visual pigments (opsins) retinal binding site. / Rhopdopsin 7-helix transmembrane proteins / Rhodopsin 7-helix transmembrane proteins / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx ...Rhodopsin, N-terminal / Amino terminal of the G-protein receptor rhodopsin / Rhodopsin / Opsin / Visual pigments (opsins) retinal binding site / Visual pigments (opsins) retinal binding site. / Rhopdopsin 7-helix transmembrane proteins / Rhodopsin 7-helix transmembrane proteins / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / 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 / P-loop containing nucleoside triphosphate hydrolase / Mainly Alpha
Similarity search - Domain/homology
ACETATE ION / PALMITIC ACID / RETINAL / Rhodopsin / Guanine nucleotide-binding protein G(t) subunit alpha-1 / Guanine nucleotide-binding protein G(t) subunit alpha-3
Similarity search - Component
Biological speciesBOS TAURUS (cattle)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.3 Å
AuthorsDeupi, X. / Edwards, P. / Singhal, A. / Nickle, B. / Oprian, D.D. / Schertler, G.F.X. / Standfuss, J.
Citation
Journal: Proc.Natl.Acad.Sci.USA / Year: 2012
Title: Stabilized G Protein Binding Site in the Structure of Constitutively Active Metarhodopsin-II.
Authors: Deupi, X. / Edwards, P. / Singhal, A. / Nickle, B. / Oprian, D. / Schertler, G. / Standfuss, J.
#1: Journal: J.Mol.Biol. / Year: 2007
Title: Crystal Structure of a Thermally Stable Rhodopsin Mutant.
Authors: Standfuss, J. / Xie, G. / Edwards, P.C. / Burghammer, M. / Oprian, D.D. / Schertler, G.F.X.
#2: Journal: Nature / Year: 2011
Title: The Structural Basis of Agonist-Induced Activation in Constitutively Active Rhodopsin.
Authors: Standfuss, J. / Edwards, P.C. / D'Antona, A. / Fransen, M. / Xie, G. / Oprian, D.D. / Schertler, G.F.X.
History
DepositionOct 17, 2011Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 25, 2012Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2019Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other
Category: pdbx_database_status / struct_conn / struct_ref_seq_dif
Item: _pdbx_database_status.status_code_sf / _struct_conn.pdbx_leaving_atom_flag / _struct_ref_seq_dif.details
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.src_method / _entity.type / _pdbx_struct_assembly_gen.asym_id_list / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Dec 20, 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: RHODOPSIN
B: GUANINE NUCLEOTIDE-BINDING PROTEIN G(T) SUBUNIT ALPHA-3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)41,6497
Polymers40,3322
Non-polymers1,3175
Water1629
1
A: RHODOPSIN
B: GUANINE NUCLEOTIDE-BINDING PROTEIN G(T) SUBUNIT ALPHA-3
hetero molecules

A: RHODOPSIN
B: GUANINE NUCLEOTIDE-BINDING PROTEIN G(T) SUBUNIT ALPHA-3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)83,29714
Polymers80,6644
Non-polymers2,63310
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_556y,x,-z+11
Buried area9720 Å2
ΔGint-22.9 kcal/mol
Surface area29330 Å2
MethodPISA
Unit cell
Length a, b, c (Å)242.190, 242.190, 109.829
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number155
Space group name H-MH32

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Components

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Protein / Protein/peptide , 2 types, 2 molecules AB

#1: Protein RHODOPSIN / / CONSTITUTIVELY ACTIVE RHODOPSIN MUTANT


Mass: 39066.562 Da / Num. of mol.: 1 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) BOS TAURUS (cattle) / Tissue: RETINA / Cell: ROD PHOTORECEPTOR / Organ: EYE / Cell line (production host): HEK293S GNTI- / Production host: HOMO SAPIENS (human) / References: UniProt: P02699
#2: Protein/peptide GUANINE NUCLEOTIDE-BINDING PROTEIN G(T) SUBUNIT ALPHA-3 / GACT PEPTIDE / GUSTDUCIN ALPHA-3 CHAIN


Mass: 1265.499 Da / Num. of mol.: 1 / Fragment: RESIDUES 344-354 / Mutation: YES / Source method: obtained synthetically / Source: (synth.) BOS TAURUS (cattle) / References: UniProt: P0C7Q4, UniProt: P04695*PLUS

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Sugars , 2 types, 2 molecules

#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#6: Sugar ChemComp-BOG / octyl beta-D-glucopyranoside / Beta-Octylglucoside / octyl beta-D-glucoside / octyl D-glucoside / octyl glucoside / Octyl glucoside


Type: D-saccharide / Mass: 292.369 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C14H28O6 / Comment: detergent*YM
IdentifierTypeProgram
b-octylglucosideIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0

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Non-polymers , 4 types, 12 molecules

#4: Chemical ChemComp-RET / RETINAL / Retinal


Mass: 284.436 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H28O
#5: Chemical ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H3O2
#7: Chemical ChemComp-PLM / PALMITIC ACID / Palmitic acid


Mass: 256.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H32O2
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Formula: H2O

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Details

Compound detailsENGINEERED RESIDUE IN CHAIN A, ASN 2 TO CYS ENGINEERED RESIDUE IN CHAIN A, MET 257 TO TYR ...ENGINEERED RESIDUE IN CHAIN A, ASN 2 TO CYS ENGINEERED RESIDUE IN CHAIN A, MET 257 TO TYR ENGINEERED RESIDUE IN CHAIN A, ASP 282 TO CYS ENGINEERED RESIDUE IN CHAIN B, LYS 345 TO LEU

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 3

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Sample preparation

CrystalDensity Matthews: 7.96 Å3/Da / Density % sol: 84.43 % / Description: NONE
Crystal growpH: 4.5
Details: 3.0-3.4 M AMMONIUM SULPHATE, 100 MM SODIUM ACETATE PH 4.5

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06SA / Wavelength: 1
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.3→45 Å / Num. obs: 17649 / % possible obs: 94.8 % / Observed criterion σ(I): -3 / Redundancy: 5.7 % / Biso Wilson estimate: 75.56 Å2 / Rmerge(I) obs: 0.18 / Net I/σ(I): 8.5
Reflection shellResolution: 3.3→3.48 Å / Redundancy: 2.9 % / Rmerge(I) obs: 0.66 / Mean I/σ(I) obs: 1.8 / % possible all: 65.4

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Processing

Software
NameVersionClassification
PHENIX(PHENIX.REFINE: 1.6.1_357)refinement
XDSdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 2X72

2x72


Resolution: 3.3→45.77 Å / SU ML: 0.42 / σ(F): 1.35 / Phase error: 26.86 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2618 901 5.1 %
Rwork0.2157 --
obs0.218 17632 94.64 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 40.896 Å2 / ksol: 0.279 e/Å3
Displacement parameters
Baniso -1Baniso -2Baniso -3
1--19.6526 Å20 Å20 Å2
2---19.6526 Å20 Å2
3---39.3052 Å2
Refinement stepCycle: LAST / Resolution: 3.3→45.77 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2683 0 89 9 2781
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0112859
X-RAY DIFFRACTIONf_angle_d1.453881
X-RAY DIFFRACTIONf_dihedral_angle_d17.6691015
X-RAY DIFFRACTIONf_chiral_restr0.084434
X-RAY DIFFRACTIONf_plane_restr0.01476
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.3-3.50670.3731120.30112006X-RAY DIFFRACTION69
3.5067-3.77730.27851690.25092905X-RAY DIFFRACTION100
3.7773-4.15720.24951670.19422917X-RAY DIFFRACTION100
4.1572-4.75820.20181580.162928X-RAY DIFFRACTION100
4.7582-5.99280.22351440.18272969X-RAY DIFFRACTION100
5.9928-45.7740.28781510.23463006X-RAY DIFFRACTION99

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