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- PDB-3ln6: Crystal structure of a bifunctional glutathione synthetase from S... -

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Basic information

Entry
Database: PDB / ID: 3ln6
TitleCrystal structure of a bifunctional glutathione synthetase from Streptococcus agalactiae
ComponentsGlutathione biosynthesis bifunctional protein gshAB
KeywordsLIGASE / gamma-glutamyl cysteine ligase domain / ATP-grasp domain / hybrid enzyme / ATP-binding / Glutathione biosynthesis / Magnesium / Manganese / Metal-binding / Multifunctional enzyme / Nucleotide-binding
Function / homology
Function and homology information


glutamate-cysteine ligase / glutamate-cysteine ligase activity / glutathione synthase / glutathione synthase activity / ATP binding / metal ion binding
Similarity search - Function
Glutathione biosynthesis bifunctional protein GshAB / GshAB, ATP-grasp-like domain / ATP-grasp-like domain / Glutamate--cysteine ligase, monofunctional / Glutamate--cysteine ligase / Glutamate-cysteine ligase / Creatine Kinase; Chain A, domain 2 - #20 / Phosphoribosylglycinamide synthetase, ATP-grasp (A) domain / Phosphoribosylglycinamide synthetase, ATP-grasp (A) domain / Creatine Kinase; Chain A, domain 2 ...Glutathione biosynthesis bifunctional protein GshAB / GshAB, ATP-grasp-like domain / ATP-grasp-like domain / Glutamate--cysteine ligase, monofunctional / Glutamate--cysteine ligase / Glutamate-cysteine ligase / Creatine Kinase; Chain A, domain 2 - #20 / Phosphoribosylglycinamide synthetase, ATP-grasp (A) domain / Phosphoribosylglycinamide synthetase, ATP-grasp (A) domain / Creatine Kinase; Chain A, domain 2 / Glutamine synthetase/guanido kinase, catalytic domain / ATP-grasp fold / ATP-grasp fold profile. / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Glutathione biosynthesis bifunctional protein GshAB
Similarity search - Component
Biological speciesStreptococcus agalactiae serogroup V (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SIRAS, molecular replacement / SIRAS / Resolution: 2.95 Å
AuthorsStout, J. / Vergauwen, B. / Savvides, S.N.
CitationJournal: To be Published
Title: Structures of two bifunctional gamma-Glutamate-cysteine Ligase/Glutathione synthetases (GshF) reveal a novel hybrid ATP-grasp fold
Authors: Stout, J. / De Vos, D. / Vergauwen, B. / Savvides, S.N.
History
DepositionFeb 2, 2010Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Apr 13, 2011Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Nov 1, 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 / diffrn_source / pdbx_initial_refinement_model / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _diffrn_source.pdbx_synchrotron_site / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Glutathione biosynthesis bifunctional protein gshAB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)85,7902
Polymers85,6941
Non-polymers961
Water63135
1
A: Glutathione biosynthesis bifunctional protein gshAB
hetero molecules

A: Glutathione biosynthesis bifunctional protein gshAB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)171,5804
Polymers171,3882
Non-polymers1922
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation10_655-x+1,-y,z1
Buried area2220 Å2
ΔGint-13 kcal/mol
Surface area61850 Å2
MethodPISA
Unit cell
Length a, b, c (Å)143.020, 143.020, 211.230
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number98
Space group name H-MI4122
Components on special symmetry positions
IDModelComponents
11A-785-

HOH

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Components

#1: Protein Glutathione biosynthesis bifunctional protein gshAB / Gamma-GCS-GS / GCS-GS / Glutamate-cysteine ligase / Gamma-glutamylcysteine synthetase / Gamma-ECS / ...Gamma-GCS-GS / GCS-GS / Glutamate-cysteine ligase / Gamma-glutamylcysteine synthetase / Gamma-ECS / GCS / Glutathione synthetase / Glutathione synthase / GSH synthetase / GSH-S / GSHase / GS


Mass: 85693.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus agalactiae serogroup V (bacteria)
Strain: BAA-811 / Gene: gshAB, gshF, SAG1821 / Plasmid: pET11a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: Q8DXM9, glutamate-cysteine ligase, glutathione synthase
#2: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 35 / Source method: isolated from a natural source / Formula: H2O

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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: 3.15 Å3/Da / Density % sol: 60.97 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 1.6M (NH4)2SO4, 100mM Tris-HCl pH 8.5, 12% glycerol, VAPOR DIFFUSION, SITTING DROP, temperature 298K

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Data collection

Diffraction
IDMean temperature (K)Crystal-ID
11001
21001
Diffraction source
SourceSiteBeamlineIDWavelength (Å)
SYNCHROTRONEMBL/DESY, HAMBURG BW7A11.072
SYNCHROTRONESRF ID14-220.933
Detector
TypeIDDetectorDate
MAR CCD 165 mm1CCDFeb 23, 2007
ADSC QUANTUM 210r2CCDJul 12, 2008
Radiation
IDProtocolMonochromatic (M) / Laue (L)Scattering typeWavelength-ID
1SINGLE WAVELENGTHMx-ray1
2SINGLE WAVELENGTHMx-ray1
Radiation wavelength
IDWavelength (Å)Relative weight
11.0721
20.9331
ReflectionNumber: 86169 / Rmerge(I) obs: 0.101 / D res high: 4 Å / Num. obs: 17647 / % possible obs: 99.7
Diffraction reflection shell
Highest resolution (Å)Lowest resolution (Å)Num. obs% possible obs (%)IDRmerge(I) obs
12.6417.8836499.710.03
10.3212.6447010010.034
8.9410.3255610010.036
88.9462999.510.045
7.3868510010.055
6.767.376599.710.08
6.326.7681110010.098
5.966.3285310010.119
5.655.9691810010.149
5.395.6597499.910.157
5.165.39100210010.154
4.965.16105410010.156
4.784.96107199.710.155
4.624.78111199.710.184
4.474.62117610010.206
4.344.47121210010.249
4.214.34123710010.292
4.14.21127610010.348
44.1130398.610.392
ReflectionResolution: 2.9→20 Å / Num. obs: 24531 / % possible obs: 99.6 % / Observed criterion σ(I): -3 / Redundancy: 8.4 % / Biso Wilson estimate: 76.731 Å2 / Rmerge(I) obs: 0.077 / Net I/σ(I): 23.9
Reflection shellResolution: 2.9→2.98 Å / Redundancy: 8.1 % / Rmerge(I) obs: 0.132 / Mean I/σ(I) obs: 1.8 / Num. measured obs: 14292 / Num. unique all: 1765 / Num. unique obs: 1765 / % possible all: 98.1

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Phasing

PhasingMethod: SIRAS
Phasing MADD res high: 4.5 Å / D res low: 15 Å / FOM : 0.55 / Reflection: 6310
Phasing MAD set site
IDAtom type symbolB isoFract xFract yFract zOccupancy
1HV1600.90.1410.1270.42
2HV1600.1760.3170.0720.472
3HV1600.4870.0320.1190.505
Phasing MAD shell
Resolution (Å)FOM Reflection
11.6-150.56465
8.78-11.60.63593
7.34-8.780.64693
6.43-7.340.61773
5.8-6.430.57846
5.32-5.80.52915
4.94-5.320.49981
4.63-4.940.451044

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Processing

Software
NameVersionClassificationNB
XSCALEdata processing
SOLVE2.13phasing
PHENIXrefinement
PDB_EXTRACT3.005data extraction
XDSdata reduction
XSCALEdata scaling
MOLREPphasing
PHASERphasing
RefinementMethod to determine structure: SIRAS, molecular replacement
Starting model: 1V4G, 1UC8

1v4g

1uc8


Resolution: 2.95→20 Å / Occupancy max: 1 / Occupancy min: 0.25 / Isotropic thermal model: anisotropic (TLS) / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber / Details: mlhl maximum likelihood refinement procedure
RfactorNum. reflection% reflectionSelection details
Rfree0.286 1178 -RANDOM
Rwork0.253 ---
obs-23309 99.9 %-
Displacement parametersBiso max: 494.08 Å2 / Biso mean: 95.45 Å2 / Biso min: 26.3 Å2
Baniso -1Baniso -2Baniso -3
1--1.03 Å20 Å20 Å2
2---1.03 Å20 Å2
3---2.07 Å2
Refinement stepCycle: LAST / Resolution: 2.95→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5467 0 5 35 5507
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONf_bond_d0.014
X-RAY DIFFRACTIONf_angle_d1.512
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 6 / % reflection obs: 1 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection obs
2.95-3.080.4281530.3532717
3.08-3.250.4181450.3222732
3.25-3.450.3551420.32718
3.45-3.710.2731420.2742739
3.71-4.080.3161480.2472747
4.08-4.670.261450.2072757

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