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- PDB-7k1a: TtgR quadruple mutant (C137I I141W M167L F168Y) -

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Basic information

Entry
Database: PDB / ID: 7k1a
TitleTtgR quadruple mutant (C137I I141W M167L F168Y)
ComponentsHTH-type transcriptional regulator TtgR
KeywordsTRANSCRIPTION / TtgR / TetR family / epistasis
Function / homology
Function and homology information


DNA-binding transcription repressor activity / protein-DNA complex / transcription cis-regulatory region binding
Similarity search - Function
Transcription regulator MAATS, C-terminal / MAATS-type transcriptional repressor, C-terminal region / DNA-binding HTH domain, TetR-type, conserved site / TetR-type HTH domain signature. / : / Tetracyclin repressor-like, C-terminal domain superfamily / Bacterial regulatory proteins, tetR family / DNA-binding HTH domain, TetR-type / TetR-type HTH domain profile. / Homeobox-like domain superfamily
Similarity search - Domain/homology
HTH-type transcriptional regulator TtgR
Similarity search - Component
Biological speciesPseudomonas putida (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.75 Å
AuthorsBingman, C.A. / Nishikawa, K.K. / Smith, R.W. / Raman, S.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM07215 United States
Department of Defense (DOD, United States)W911NF-17-1-0043 United States
CitationJournal: Nat Commun / Year: 2021
Title: Epistasis shapes the fitness landscape of an allosteric specificity switch.
Authors: Nishikawa, K.K. / Hoppe, N. / Smith, R. / Bingman, C. / Raman, S.
History
DepositionSep 7, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 6, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: HTH-type transcriptional regulator TtgR
B: HTH-type transcriptional regulator TtgR
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,1664
Polymers48,1172
Non-polymers492
Water5,026279
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4490 Å2
ΔGint-37 kcal/mol
Surface area18630 Å2
MethodPISA
Unit cell
Length a, b, c (Å)57.920, 64.280, 223.870
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Space group name HallC2c2
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y,-z+1/2
#4: -x,-y,z+1/2
#5: x+1/2,y+1/2,z
#6: x+1/2,-y+1/2,-z
#7: -x+1/2,y+1/2,-z+1/2
#8: -x+1/2,-y+1/2,z+1/2

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Components

#1: Protein HTH-type transcriptional regulator TtgR / Toluene efflux pump ttgABC operon repressor


Mass: 24058.510 Da / Num. of mol.: 2 / Mutation: C137I, I141W, M167L, F168Y
Source method: isolated from a genetically manipulated source
Details: Quadruple mutant (C137I I141W M167L F168Y) / Source: (gene. exp.) Pseudomonas putida (bacteria) / Strain: DOT-T1E / Gene: ttgR, T1E_0244 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q9AIU0
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 279 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 2.17 Å3/Da / Density % sol: 43.19 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 200 nL of protein at 9.7 mg/mL in 5mM HEPES pH 7.5, 50 mM NaCl, 0.3 mM TCEP was equilibrated against 150 nL 20% MEPEG, 0.2M MgCl2, 0.1M bistris HCl pH 6.5 in a SD2 plate using a Mosquito ...Details: 200 nL of protein at 9.7 mg/mL in 5mM HEPES pH 7.5, 50 mM NaCl, 0.3 mM TCEP was equilibrated against 150 nL 20% MEPEG, 0.2M MgCl2, 0.1M bistris HCl pH 6.5 in a SD2 plate using a Mosquito crystallization robot. Samples were cryoprotected with reservoir solution supplemented to 35% MEPEG 2000. Samples looped in Mitegen micro mounts were flash cooled by immersion in liquid nitrogen

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-G / Wavelength: 0.97856 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Dec 16, 2018 / Details: 3.0G undulator, diamond (111)
RadiationMonochromator: diamond 111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97856 Å / Relative weight: 1
ReflectionResolution: 1.75→32.14 Å / Num. obs: 42585 / % possible obs: 99.71 % / Redundancy: 13.6 % / Biso Wilson estimate: 35.18 Å2 / CC1/2: 0.987 / Rmerge(I) obs: 0.09432 / Rpim(I) all: 0.02647 / Rrim(I) all: 0.09805 / Net I/σ(I): 16.2
Reflection shellResolution: 1.75→1.813 Å / Redundancy: 10.1 % / Rmerge(I) obs: 1.303 / Mean I/σ(I) obs: 1.29 / Num. unique obs: 4137 / CC1/2: 0.578 / CC star: 0.856 / Rpim(I) all: 0.4257 / Rrim(I) all: 1.373 / % possible all: 97.73

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2UXU

2uxu


Resolution: 1.75→32.14 Å / SU ML: 0.2732 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 24.1075
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2401 1979 4.65 %
Rwork0.1967 40587 -
obs0.1987 42566 99.73 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 39.41 Å2
Refinement stepCycle: LAST / Resolution: 1.75→32.14 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3303 0 2 279 3584
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00333375
X-RAY DIFFRACTIONf_angle_d0.47764570
X-RAY DIFFRACTIONf_chiral_restr0.0317519
X-RAY DIFFRACTIONf_plane_restr0.0022598
X-RAY DIFFRACTIONf_dihedral_angle_d20.35751268
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.75-1.790.3885980.41522831X-RAY DIFFRACTION96.83
1.79-1.840.38061980.33932763X-RAY DIFFRACTION100
1.84-1.90.372990.27882961X-RAY DIFFRACTION100
1.9-1.960.27061980.2312794X-RAY DIFFRACTION100
1.96-2.030.2912990.21952888X-RAY DIFFRACTION99.97
2.03-2.110.2518990.21382919X-RAY DIFFRACTION100
2.11-2.20.24431980.19972857X-RAY DIFFRACTION100
2.2-2.320.242990.19242920X-RAY DIFFRACTION100
2.32-2.470.22241980.19152825X-RAY DIFFRACTION100
2.47-2.660.2502990.19772960X-RAY DIFFRACTION100
2.66-2.920.24061980.19472876X-RAY DIFFRACTION100
2.92-3.350.2464990.19772969X-RAY DIFFRACTION100
3.35-4.210.2311150.17912991X-RAY DIFFRACTION100
4.21-32.140.21831820.18213033X-RAY DIFFRACTION99.41
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)
12.412149144160.3496326109131.77759674863.232341559430.8822700522973.98388291115-0.000869527989615-0.04982663395910.05655029222950.2083516216570.13935455342-0.387898082224-0.01589641448920.3774180371037.66890687945E-50.3802823128060.0521535163698-0.0191781410640.333010675581-0.02703557114110.384866286426-13.0710614422-2.1928402504643.9120522526
22.21345385141-0.430903498819-0.2790906038350.998735404377-0.2474290164881.207287810120.0875779195328-0.00493789480176-0.0216220997980.07495725193920.0596265371152-0.113408803346-0.03107639162660.604334424972-2.33908318953E-50.3186030816970.00887434330546-0.02751186130510.370591878097-0.01445952470990.275274991269-17.5782818662-3.7078096654922.5394197094
32.97999531812-0.4747995395220.2537239518582.37348761849-0.6391631201710.75031028624-0.0937983506502-0.1429995813640.2171078338850.09102121421890.109030555033-0.0925291105622-0.5402489090390.8224357872320.0003383677568530.234051535508-0.0521923213659-0.0155526640940.2347294315310.01346100539940.295975604662-19.64288545728.8981076145619.679064759
42.74534150430.594322726924-0.2137431153051.56903431644-0.2684180308192.88828060592-0.07980774069460.28436734177-0.0956833515535-0.1614431145020.1775856767450.1783219924410.0975607005883-0.1466758040981.69185209402E-50.241713582963-0.00310648844386-0.02469431573730.216650074108-0.01368296117960.243852941063-28.7675538801-2.8830524191215.9030249077
50.5912858574040.200703068417-0.185672212351.471481488561.049633011871.442941105040.385539208663-0.07265657815290.536660671408-0.0048824671125-0.04886455577270.186998153203-0.15874483554-0.193735881806-9.1034430305E-70.3934768803140.06116497470540.06681310761390.3642304304510.05321612484290.358849446781-48.567368693311.739871865844.596786101
61.95813338481-0.62088930514-1.325852941440.521818072466-0.01305924124521.47169376610.312763534619-1.543813080060.4672425063580.4443232736660.108329544173-0.368254239819-0.822080060881.175525656910.05799765551740.3203988431180.05623200134140.02369018807880.363523120274-0.03623116509680.305461947604-38.9772962358.9576342880150.2017453532
70.7473677562691.02288306711-0.1987450623731.71605359564-0.3627218852411.16625715778-0.03933169875180.151353093346-0.148931273134-0.00413573443621-0.03576994890150.0395205597070.420005414119-0.707075338653-0.0008734900130160.3524044015490.03892869499090.03251680359290.3297048574640.04785495902450.403447302811-47.52429824793.7150773525946.4146698787
81.01874105483-0.1983205512980.4481579241091.383584350.3720752053181.136872749450.020396001946-0.1569562877450.1354551481620.181251192171-0.02963229935640.6841382233230.196284770455-0.615823388319-0.0009694697768250.29089302972-0.0410583733557-0.04240635913230.4216917762810.09589230389710.438759490867-49.5385582014.8684811583817.8637364458
90.3889087537360.4803697646280.3656712789770.8601529701390.07191860105310.7919260308840.124558613312-0.2094059880880.1176274409870.1091403939130.06655176447660.369348709799-0.315688899650.0480477535747-2.00213990936E-50.3199959012830.07018934668390.01167527554150.3137329686590.0004225469255250.387728203302-38.713310308110.254028769333.1043887219
101.42120203781-0.8938068559040.9431494401582.081275591990.2012845918541.13958961579-0.388308615246-1.93963706419-0.6241657214531.358998705920.6713567099241.333743991410.110874552267-1.404310331760.01341633657750.439222974747-0.008480861350820.04126900936160.5627774305720.1173639885060.58093815928-33.8731564453-3.1319789280142.2832280974
111.81414447920.04608725220090.04657031891662.395552748320.2667014847491.01114997566-0.0854319850342-0.178468518907-0.04269658523570.01897300357740.04818993007480.4522067252160.69331441338-0.350720464775-0.0003286369761220.303100954484-0.0602717963647-0.001907138985930.2456732902060.08134888408130.41155632099-44.3593344948-6.7264367313919.5311309558
121.22470401556-0.707645974686-0.6270196239290.959686757487-0.4813497909562.07164656545-0.06245895809040.0910667514247-0.01521969877960.3095515245550.2554901634410.1336570616830.271770051497-0.008527455261732.85438079148E-50.2782484804880.0562055042168-0.03225562673050.2767863683620.04653393979670.253677130033-34.77315734381.5693923569221.011324693
130.049960369587-0.04285912353110.1013466163070.0172773542472-0.06552922186050.129908114453-0.6940028384150.434908042250.665325827860.388630526956-0.1937962963710.244091129351-0.9668942056070.219198386167-0.000146552088710.387438126496-0.000530264212497-0.1247765226910.3368509244170.08508430979010.397726834345-32.022068774216.216679095619.9639281916
141.51579925468-0.761225665228-0.2083020084740.3602495964420.4174461212131.70488629276-0.1029354876040.3066368500760.0547769207052-0.3276044146850.2475141012560.0619560082770.07796175749-0.0917197822992-5.09011720252E-50.369937873683-0.0439620803485-0.04610122655720.3279034128990.07464267761440.269309236723-36.66757840881.719543025368.94211686163
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 6 through 68 )AA6 - 681 - 64
22chain 'A' and (resid 69 through 121 )AA69 - 12165 - 117
33chain 'A' and (resid 122 through 160 )AA122 - 160118 - 156
44chain 'A' and (resid 161 through 211 )AA161 - 211157 - 207
55chain 'B' and (resid 6 through 28 )BB6 - 282 - 24
66chain 'B' and (resid 29 through 42 )BB29 - 4225 - 38
77chain 'B' and (resid 43 through 67 )BB43 - 6739 - 63
88chain 'B' and (resid 68 through 103 )BB68 - 10364 - 99
99chain 'B' and (resid 104 through 116 )BB104 - 116100 - 112
1010chain 'B' and (resid 117 through 125 )BB117 - 125113 - 121
1111chain 'B' and (resid 126 through 160 )BB126 - 160122 - 156
1212chain 'B' and (resid 161 through 181 )BB161 - 181157 - 177
1313chain 'B' and (resid 182 through 191 )BB182 - 191178 - 187
1414chain 'B' and (resid 192 through 211 )BB192 - 211188 - 208

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