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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


negative regulation of cyclic-nucleotide phosphodiesterase activity / Adenylate cyclase inhibitory pathway / Opsins / VxPx cargo-targeting to cilium / sensory perception of bitter taste / sperm head plasma membrane / rod bipolar cell differentiation / absorption of visible light / opsin binding / The canonical retinoid cycle in rods (twilight vision) ...negative regulation of cyclic-nucleotide phosphodiesterase activity / Adenylate cyclase inhibitory pathway / Opsins / VxPx cargo-targeting to cilium / sensory perception of bitter taste / sperm head plasma membrane / rod bipolar cell differentiation / absorption of visible light / opsin binding / The canonical retinoid cycle in rods (twilight vision) / G protein-coupled opsin signaling pathway / photoreceptor inner segment membrane / podosome assembly / Sensory perception of sweet, bitter, and umami (glutamate) taste / 11-cis retinal binding / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / G protein-coupled photoreceptor activity / sensory perception of umami taste / rod photoreceptor outer segment / cellular response to light stimulus / detection of light stimulus involved in visual perception / sensory perception of sweet taste / G protein-coupled receptor complex / Inactivation, recovery and regulation of the phototransduction cascade / thermotaxis / Activation of the phototransduction cascade / outer membrane / detection of temperature stimulus involved in thermoception / response to light intensity / photoreceptor cell maintenance / arrestin family protein binding / ADP signalling through P2Y purinoceptor 12 / Extra-nuclear estrogen signaling / photoreceptor outer segment membrane / G alpha (i) signalling events / acyl binding / response to light stimulus / phototransduction, visible light / G-protein alpha-subunit binding / phototransduction / photoreceptor outer segment / sperm midpiece / photoreceptor inner segment / visual perception / guanyl-nucleotide exchange factor activity / G protein-coupled receptor binding / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein beta/gamma-subunit complex binding / microtubule cytoskeleton organization / photoreceptor disc membrane / GDP binding / cell-cell junction / heterotrimeric G-protein complex / gene expression / G protein-coupled receptor signaling pathway / Golgi membrane / GTPase activity / protein kinase binding / GTP binding / zinc ion binding / metal ion binding / identical protein binding / membrane / 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 ...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 / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / 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 (domestic 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
Revision 2.2Oct 16, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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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 (domestic 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 (domestic 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


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


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


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


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

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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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