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- PDB-6o1h: Tryptophan synthase Q114A mutant in complex with N-(4'-trifluorom... -

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Basic information

Entry
Database: PDB / ID: 6o1h
TitleTryptophan synthase Q114A mutant in complex with N-(4'-trifluoromethoxybenzenesulfonyl)-2-amino-1-ethylphosphate (F9F) at the enzyme alpha-site, cesium ion at the metal coordination site, and 2-aminophenol quinonoid at the enzyme beta site
Components(Tryptophan synthase ...) x 2
KeywordsLYASE / Tryptophan synthase / mutant Q114A / lyase-inhibitor / protein complex / Salmonella
Function / homology
Function and homology information


tryptophan synthase / tryptophan synthase activity / tryptophan biosynthetic process / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Tryptophan synthase, alpha chain / Tryptophan synthase, alpha chain, active site / Tryptophan synthase alpha chain / Tryptophan synthase alpha chain signature. / Tryptophan synthase, beta chain, conserved site / Tryptophan synthase, beta chain / Tryptophan synthase beta chain/beta chain-like / Tryptophan synthase beta chain pyridoxal-phosphate attachment site. / Pyridoxal-phosphate dependent enzyme / Tryptophan synthase beta subunit-like PLP-dependent enzyme ...Tryptophan synthase, alpha chain / Tryptophan synthase, alpha chain, active site / Tryptophan synthase alpha chain / Tryptophan synthase alpha chain signature. / Tryptophan synthase, beta chain, conserved site / Tryptophan synthase, beta chain / Tryptophan synthase beta chain/beta chain-like / Tryptophan synthase beta chain pyridoxal-phosphate attachment site. / Pyridoxal-phosphate dependent enzyme / Tryptophan synthase beta subunit-like PLP-dependent enzyme / Pyridoxal-phosphate dependent enzyme / Ribulose-phosphate binding barrel / Aldolase class I / Aldolase-type TIM barrel / TIM Barrel / Alpha-Beta Barrel / Alpha Beta
Similarity search - Domain/homology
Chem-1D0 / : / Chem-F9F / pyridin-2-amine / Tryptophan synthase alpha chain / Tryptophan synthase alpha chain / Tryptophan synthase beta chain
Similarity search - Component
Biological speciesSalmonella typhimurium (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.64 Å
AuthorsHilario, E. / Dunn, M.F. / Mueller, L.J. / Fan, L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)2R01GM097569-06A1 United States
CitationJournal: To be Published
Title: Crystal structure of Salmonella typhimurium Tryptophan Synthase mutant beta-Q114A with 2-({[4-(trifluoromethoxy)phenyl]sulfonyl}amino)ethyl dihydrogen phosphate (F9F) at the alpha-site, Cesium ...Title: Crystal structure of Salmonella typhimurium Tryptophan Synthase mutant beta-Q114A with 2-({[4-(trifluoromethoxy)phenyl]sulfonyl}amino)ethyl dihydrogen phosphate (F9F) at the alpha-site, Cesium ion at the metal coordination site, and [3-hydroxy-2-methyl-5-phosphonooxymethyl-pyridin-4-ylmethyl]-serine (PLS) at the beta-site.
Authors: Hilario, E. / Dunn, M.F. / Mueller, L.J. / Fan, L.
History
DepositionFeb 19, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 19, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 11, 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 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_alt_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_alt_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_ptnr2_label_alt_id / _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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Tryptophan synthase alpha chain
B: Tryptophan synthase beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)73,79232
Polymers71,3162
Non-polymers2,47630
Water12,773709
1
A: Tryptophan synthase alpha chain
B: Tryptophan synthase beta chain
hetero molecules

A: Tryptophan synthase alpha chain
B: Tryptophan synthase beta chain
hetero molecules


  • defined by author&software
  • Evidence: gel filtration, To verify the oligomeric size of Tryptophan synthase (TS) mutant beta-Q114A in buffered solution, we have used a HiPrep 16/60 Sephacryl S-100 High Resolution column (GE ...Evidence: gel filtration, To verify the oligomeric size of Tryptophan synthase (TS) mutant beta-Q114A in buffered solution, we have used a HiPrep 16/60 Sephacryl S-100 High Resolution column (GE Healthcare) in 50mM bicine:CsCl, pH 7.80, containing 50mM CsCl and 0.01mM PLP at 10 degrees Celsius. 1mg of pure TS mutant beta-Q114A was loaded at a flowrate of 30mL/h and 2mL fraction size. Marker proteins were used for column calibration: ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and lactalbumin (14 kDa). The effluent was monitored by absorbance at 280nm and the protein sample from the peak fraction was loaded in a 15% SDS-PAGE gel. Tryptophan synthase is a tetrameter composed of two alpha-chains and two beta-chains (Alpha2-Beta2).
  • 148 kDa, 4 polymers
  • Search similar-shape structures of this assembly by Omokage search (details)
Theoretical massNumber of molelcules
Total (without water)147,58464
Polymers142,6334
Non-polymers4,95160
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
Buried area18630 Å2
ΔGint-435 kcal/mol
Surface area41480 Å2
MethodPISA
Unit cell
Length a, b, c (Å)183.749, 60.720, 67.210
Angle α, β, γ (deg.)90.000, 94.630, 90.000
Int Tables number5
Space group name H-MC121

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Components

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Tryptophan synthase ... , 2 types, 2 molecules AB

#1: Protein Tryptophan synthase alpha chain


Mass: 28698.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella typhimurium (bacteria) / Gene: trpA, DD95_04145 / Plasmid: pEBA-10 / Details (production host): mutant Q114A-beta / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Rosetta(DE3)pLysS
References: UniProt: A0A0D6FWC1, UniProt: P00929*PLUS, tryptophan synthase
#2: Protein Tryptophan synthase beta chain


Mass: 42617.480 Da / Num. of mol.: 1 / Mutation: Q114A
Source method: isolated from a genetically manipulated source
Details: Beta-chain contains mutation Q114A / Source: (gene. exp.) Salmonella typhimurium (bacteria) / Gene: trpB, STM1726 / Plasmid: pEBA-10 / Details (production host): mutation Q114A / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Rosetta(DE3)pLysS / References: UniProt: P0A2K1, tryptophan synthase

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Non-polymers , 7 types, 739 molecules

#3: Chemical
ChemComp-DMS / DIMETHYL SULFOXIDE / Dimethyl sulfoxide


Mass: 78.133 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#4: Chemical ChemComp-F9F / 2-({[4-(TRIFLUOROMETHOXY)PHENYL]SULFONYL}AMINO)ETHYL DIHYDROGEN PHOSPHATE / N-(4'-TRIFLUOROMETHOXYBENZENESULFONYL)-2-AMINO-1-ETHYLPHOSPHATE, F9


Mass: 365.220 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H11F3NO7PS
#5: Chemical ChemComp-CS / CESIUM ION


Mass: 132.905 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cs
#6: Chemical
ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Cl
#7: Chemical ChemComp-HVK / pyridin-2-amine / 2-Aminopyridine


Mass: 94.115 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H6N2
#8: Chemical ChemComp-1D0 / (2E)-2-[({3-hydroxy-2-methyl-5-[(phosphonooxy)methyl]pyridin-4-yl}methyl)imino]-3-[(2-hydroxyphenyl)amino]propanoic acid


Mass: 425.330 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H20N3O8P
#9: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 709 / 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: 2.62 Å3/Da / Density % sol: 53.06 % / Description: Large plate-like or rectangular-like crystals
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.8
Details: 100 mM Bicine-CsOH, pH 7.8, 5-10% PEG8000, 2 mM F9F, 0.01 mM PLP
PH range: 7.7-8.0 / Temp details: room temperature

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: Oxford Cobra Cryosystem / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.5418 Å
DetectorType: RIGAKU RAXIS IV++ / Detector: IMAGE PLATE / Date: Oct 2, 2018 / Details: Varimax Confocal Max-Flux
RadiationMonochromator: Osmic Varimax HF ArcSec / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.64→40 Å / Num. obs: 90369 / % possible obs: 99.9 % / Redundancy: 3.5 % / Biso Wilson estimate: 16.7 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.051 / Rpim(I) all: 0.031 / Rrim(I) all: 0.06 / Rsym value: 0.051 / Net I/av σ(I): 9.6 / Net I/σ(I): 11.5
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsRpim(I) allRrim(I) allRsym value% possible all
1.64-1.733.40.3062.4130330.1950.3640.30699.5
1.73-1.833.40.2033.6124130.1280.2410.20399.8
1.83-1.963.50.1484.7117270.0920.1750.148100
1.96-2.123.50.17.1108760.0620.1170.1100
2.12-2.323.60.0769100770.0460.0890.076100
2.32-2.593.60.06310.690950.0380.0730.063100
2.59-2.993.70.05312.280210.0320.0610.053100
2.99-3.673.70.04314.468290.0260.050.043100
3.67-5.193.70.03120.253220.0190.0360.031100
5.19-39.3083.60.03118.329760.0190.0370.03199.8

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation6.58 Å19.77 Å
Phasing dmMethod: Solvent flattening and Histogram matching / Reflection: 90010
Phasing dm shell
Resolution (Å)Delta phi finalFOM Reflection
8.89-10032.60.904613
6.29-8.8939.10.8951082
5.13-6.2936.40.9011370
4.44-5.1326.60.9271621
3.98-4.4425.30.931839
3.63-3.9823.20.9362022
3.36-3.6322.60.9352176
3.14-3.3622.50.9342362
2.96-3.1421.80.932496
2.81-2.9622.70.9192618
2.68-2.8121.40.9182752
2.57-2.6820.70.932880
2.47-2.5720.60.9183003
2.38-2.4719.70.9223099
2.3-2.3819.10.9223236
2.22-2.3190.9233321
2.16-2.2219.40.9253404
2.1-2.1617.90.9263557
2.04-2.119.50.9183652
1.99-2.0420.80.9153763
1.94-1.9921.10.9173818
1.9-1.9421.60.9043899
1.85-1.922.80.9014005
1.81-1.8523.30.9074069
1.78-1.81240.9044174
1.74-1.7825.50.8874288
1.71-1.7427.20.8884339
1.68-1.7129.60.8714365
1.64-1.6838.70.7836187

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Processing

Software
NameVersionClassification
CrystalClear2data collection
iMOSFLM7.2.2data reduction
SCALA3.3.22data scaling
PHASER2.8.2phasing
DM7.0.067phasing
REFMAC5.8.0238refinement
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 4HPJ

4hpj


Resolution: 1.64→39.31 Å / Cor.coef. Fo:Fc: 0.974 / Cor.coef. Fo:Fc free: 0.96 / SU B: 5.049 / SU ML: 0.073 / SU R Cruickshank DPI: 0.1101 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.11 / ESU R Free: 0.088 / Details: U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.199 4448 4.9 %RANDOM
Rwork0.1538 ---
obs0.156 85508 99.42 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 86.28 Å2 / Biso mean: 29.599 Å2 / Biso min: 15.51 Å2
Baniso -1Baniso -2Baniso -3
1-0.88 Å2-0 Å20.14 Å2
2---0.81 Å20 Å2
3----0.09 Å2
Refinement stepCycle: final / Resolution: 1.64→39.31 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4950 0 119 743 5812
Biso mean--36.06 43.48 -
Num. residues----663
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.0125249
X-RAY DIFFRACTIONr_angle_refined_deg1.241.6317127
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.3095686
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.80521.882255
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.59315838
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.6091533
X-RAY DIFFRACTIONr_chiral_restr0.0940.2684
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.024056
X-RAY DIFFRACTIONr_rigid_bond_restr7.95735249
LS refinement shellResolution: 1.64→1.683 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.349 321 -
Rwork0.329 6194 -
all-6515 -
obs--98.85 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.0007-0.00040.00050.0051-0.0060.0083-0.001-0.0039-0.0067-0.0036-0.0008-0.00580.01230.00010.00180.05980.0015-0.01550.1414-0.00280.110235.971518.20629.6259
20.0068-0.0020.00410.0007-0.00120.0025-0.0033-0.01080.0028-0.00130.00030.0008-0.0009-0.00580.0030.061-0.0001-0.01480.1352-0.00270.116438.9472.38796.7884
30.0020.00950.00290.22060.07120.0232-0.0008-0.00360.01170.01530.0121-0.02460.0051-0.0003-0.01120.05080.0052-0.01110.1482-0.00030.109843.5567-0.671619.4761
43.4961-1.5812-1.07071.03960.41760.7041-0.0264-0.292-0.0137-0.02880.058-0.167-0.05450.1575-0.03150.0575-0.0016-0.00550.19180.02350.144250.4999-0.569226.8745
50.01360.01710.00280.02570.00350.00090.00070.0001-0.0026-0.00550.0025-0.00070.0034-0.0022-0.00330.05590.0029-0.01160.1468-0.00130.11246.323417.268621.5957
60.0006-0.0021-0.00020.00850.00070.0001-0.00160.00260.00230.00360.0010.00050.0005-0.00040.00050.0613-0.0004-0.01260.1323-0.00060.115112.4066-14.05424.881
70.067-0.04890.08050.0385-0.06240.1022-0.0122-0.00740.00050.00120.0029-0.01310.0003-0.00370.00930.05750.0027-0.01580.1351-0.00350.117213.37153.054432.0102
80.00010.000100.00380.00010.0001-0.00020.0008-0.0022-0.00080.002-0.00060.0017-0.0011-0.00180.0624-0.0004-0.0130.132-0.00030.1158.1027-6.057414.7972
90.00480.0001-0.00160.00140.00020.00060.00140.0003-0.0151-0.0075-0.0065-0.0006-0.0019-0.00310.00510.05120.007-0.0210.142-0.00180.1207-6.5498-1.074818.5754
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A30 - 91
2X-RAY DIFFRACTION2A92 - 159
3X-RAY DIFFRACTION3A160 - 187
4X-RAY DIFFRACTION4A188 - 202
5X-RAY DIFFRACTION5A203 - 268
6X-RAY DIFFRACTION6B2 - 100
7X-RAY DIFFRACTION7B101 - 165
8X-RAY DIFFRACTION8B166 - 365
9X-RAY DIFFRACTION9B366 - 396

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