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- PDB-5ia9: The structure of microsomal glutathione transferase 1 in complex ... -

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Basic information

Entry
Database: PDB / ID: 5ia9
TitleThe structure of microsomal glutathione transferase 1 in complex with Meisenheimer complex
ComponentsMicrosomal glutathione S-transferase 1
KeywordsTRANSFERASE / Membrane / Enzyme / Meisenheimer complex
Function / homology
Function and homology information


cellular response to lipid hydroperoxide / Aflatoxin activation and detoxification / glutathione transport / Glutathione conjugation / glutathione binding / Leydig cell differentiation / glutathione peroxidase activity / peroxisomal membrane / Neutrophil degranulation / glutathione transferase ...cellular response to lipid hydroperoxide / Aflatoxin activation and detoxification / glutathione transport / Glutathione conjugation / glutathione binding / Leydig cell differentiation / glutathione peroxidase activity / peroxisomal membrane / Neutrophil degranulation / glutathione transferase / glutathione transferase activity / glutathione metabolic process / response to organonitrogen compound / apical part of cell / mitochondrial outer membrane / response to lipopolysaccharide / response to xenobiotic stimulus / endoplasmic reticulum membrane / endoplasmic reticulum / mitochondrion / membrane / identical protein binding
Similarity search - Function
Microsomal glutathione S-transferase 1-like / Membrane associated eicosanoid/glutathione metabolism-like domain / Membrane-associated, eicosanoid/glutathione metabolism (MAPEG) protein / Membrane associated eicosanoid/glutathione metabolism-like domain superfamily / MAPEG family / Four Helix Bundle (Hemerythrin (Met), subunit A) / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
Chem-GTD / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / PALMITIC ACID / Microsomal glutathione S-transferase 1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 3.5 Å
AuthorsKuang, Q. / Purhonen, P. / Jegerschold, C. / Morgenstern, R. / Hebert, H.
Funding support Sweden, 2items
OrganizationGrant numberCountry
VR Sweden
CIMED Sweden
CitationJournal: Sci Rep / Year: 2017
Title: Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation.
Authors: Qie Kuang / Pasi Purhonen / Johan Ålander / Richard Svensson / Veronika Hoogland / Jens Winerdal / Linda Spahiu / Astrid Ottosson-Wadlund / Caroline Jegerschöld / Ralf Morgenstern / Hans Hebert /
Abstract: Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used ...Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two aliphatic chains and one glutathione (GSH) molecule. The functional unit is a homotrimer centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer supported by new in vitro mutagenesis data. Mutation of Arginine 130 to alanine resulted in complete loss of activity consistent with a role for Arginine 130 in stabilizing the strongly nucleophilic GSH thiolate required for catalysis. Based on the new model and an electron diffraction data set from crystals soaked with trinitrobenzene, that forms a dead-end Meisenheimer complex with GSH, a difference map was calculated. The map reveals side chain movements opening a cavity that defines the second substrate site.
History
DepositionFeb 21, 2016Deposition site: RCSB / Processing site: PDBE
Revision 1.0Jul 12, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 23, 2017Group: Data collection / Database references / Category: citation / citation_author / em_image_scans
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name

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Structure visualization

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Assembly

Deposited unit
A: Microsomal glutathione S-transferase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)20,1066
Polymers17,4921
Non-polymers2,6145
Water0
1
A: Microsomal glutathione S-transferase 1
hetero molecules

A: Microsomal glutathione S-transferase 1
hetero molecules

A: Microsomal glutathione S-transferase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)60,31818
Polymers52,4773
Non-polymers7,84115
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_655-y+1,x-y,z1
crystal symmetry operation3_665-x+y+1,-x+1,z1
Buried area15790 Å2
ΔGint-91 kcal/mol
Surface area20160 Å2
MethodPISA
Unit cell
Length a, b, c (Å)81.800, 81.800, 100.000
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number168
Space group name H-MP6

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Components

#1: Protein Microsomal glutathione S-transferase 1 / / Microsomal GST-1 / Microsomal GST-I


Mass: 17492.488 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Mgst1, Gst12 / Production host: Escherichia coli (E. coli) / References: UniProt: P08011, glutathione transferase
#2: Chemical ChemComp-GTD / 1-(S-GLUTATHIONYL)-2,4,6-TRINITROCYCLOHEXA-2,5-DIENE / (S)-2-AMINO-5-((R)-1-(CARBOXYMETHYLAMINO)-1-OXO-3-(2,4,6-TRINITROCYCLOHEXA-2,5-DIENYLTHIO)PROPAN-2-YLAMINO)-5-OXOPENTANOIC ACID


Mass: 520.428 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H20N6O12S
#3: Chemical ChemComp-PC1 / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / 3-SN-PHOSPHATIDYLCHOLINE / Phosphatidylcholine


Mass: 790.145 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C44H88NO8P / Comment: phospholipid*YM
#4: Chemical ChemComp-PLM / PALMITIC ACID / Palmitic acid


Mass: 256.424 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C16H32O2

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography
Crystal symmetry∠γ: 120 ° / A: 81.8 Å / B: 81.8 Å / C: 100 Å / Space group name H-M: P6

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Sample preparation

ComponentName: The structure of microsomal glutathione transferase 1 in complex with the Meisenheimer complex
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.54357 MDa / Experimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pSP19T7LT
EM crystal formationDetails: dialysis / Lipid mixture: bovine liver lecithin / Lipid protein ratio: 3 / Temperature: 303 K / Time: 7
Buffer solutionpH: 7.4
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
EM embeddingMaterial: trehalose
VitrificationCryogen name: NITROGEN

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Data collection

MicroscopyModel: JEOL 2100F
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Image recordingElectron dose: 1 e/Å2 / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k)
EM diffractionCamera length: 200 mm
EM diffraction statsFourier space coverage: 72.4 % / High resolution: 3.5 Å / Num. of intensities measured: 43603 / Num. of structure factors: 3063 / Phase error: 0.0001 ° / Phase residual: 0.0001 ° / Phase error rejection criteria: 0 / Rmerge: 34.3 / Rsym: 12
Reflection twin
Crystal-IDIDOperatorDomain-IDFraction
11H, K, L10.5
11-H-K, K, -L20.5

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Processing

SoftwareName: REFMAC / Version: 5.5.0110 / Classification: refinement
EM softwareName: REFMAC / Version: 5 / Category: model refinement
Crystal symmetry∠γ: 120 ° / A: 81.8 Å / B: 81.8 Å / C: 100 Å / Space group name H-M: P6
CTF correctionType: NONE
3D reconstructionResolution: 3.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 2D CRYSTAL
RefinementResolution: 3.5→10 Å / Cor.coef. Fo:Fc: 0.71 / Cor.coef. Fo:Fc free: 0.398 / Cross valid method: FREE R-VALUE / ESU R Free: 0.162 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.31055 175 5.4 %RANDOM
Rwork0.28144 ---
obs0.28287 5539 72.4 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å
Displacement parametersBiso mean: 19.369 Å2
Baniso -1Baniso -2Baniso -3
1--34.57 Å20 Å20 Å2
2---34.57 Å20 Å2
3---69.13 Å2
Refinement stepCycle: 1 / Total: 1147
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0250.0221164
ELECTRON CRYSTALLOGRAPHYr_bond_other_d
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg3.0842.1021546
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg10.7935121
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg34.78520.95242
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg22.52515171
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg25.9461510
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.250.2167
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0110.021792
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other
ELECTRON CRYSTALLOGRAPHYr_nbd_refined
ELECTRON CRYSTALLOGRAPHYr_nbd_other
ELECTRON CRYSTALLOGRAPHYr_nbtor_refined
ELECTRON CRYSTALLOGRAPHYr_nbtor_other
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_refined
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_other
ELECTRON CRYSTALLOGRAPHYr_metal_ion_refined
ELECTRON CRYSTALLOGRAPHYr_metal_ion_other
ELECTRON CRYSTALLOGRAPHYr_symmetry_vdw_refined
ELECTRON CRYSTALLOGRAPHYr_symmetry_vdw_other
ELECTRON CRYSTALLOGRAPHYr_symmetry_hbond_refined
ELECTRON CRYSTALLOGRAPHYr_symmetry_hbond_other
ELECTRON CRYSTALLOGRAPHYr_symmetry_metal_ion_refined
ELECTRON CRYSTALLOGRAPHYr_symmetry_metal_ion_other
ELECTRON CRYSTALLOGRAPHYr_mcbond_it0.6961.5619
ELECTRON CRYSTALLOGRAPHYr_mcbond_other
ELECTRON CRYSTALLOGRAPHYr_mcangle_it0.9212986
ELECTRON CRYSTALLOGRAPHYr_mcangle_other
ELECTRON CRYSTALLOGRAPHYr_scbond_it0.4123545
ELECTRON CRYSTALLOGRAPHYr_scbond_other
ELECTRON CRYSTALLOGRAPHYr_scangle_it0.6284.5560
ELECTRON CRYSTALLOGRAPHYr_scangle_other
ELECTRON CRYSTALLOGRAPHYr_long_range_B_refined
ELECTRON CRYSTALLOGRAPHYr_long_range_B_other
ELECTRON CRYSTALLOGRAPHYr_rigid_bond_restr
ELECTRON CRYSTALLOGRAPHYr_sphericity_free
ELECTRON CRYSTALLOGRAPHYr_sphericity_bonded
LS refinement shellResolution: 3.498→3.577 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.257 7 -
Rwork0.257 172 -
obs--58.5 %

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