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- PDB-9gkr: Crystal structure of artificial enzyme LmrR_pAF variant RMH in cr... -

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Basic information

Entry
Database: PDB / ID: 9gkr
TitleCrystal structure of artificial enzyme LmrR_pAF variant RMH in crystal form 1
ComponentsTranscriptional regulator, PadR-like family
KeywordsDNA BINDING PROTEIN / Artificial enzyme / unnatural amino acid / 4-aminophenylalanine / directed evolution
Function / homology: / Transcription regulator PadR, N-terminal / Transcriptional regulator PadR-like family / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / metal ion binding / Transcriptional regulator, PadR-like family
Function and homology information
Biological speciesLactococcus cremoris subsp. cremoris MG1363 (lactic acid bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.55 Å
AuthorsThunnissen, A.M.W.H. / Leveson-Gower, R.B. / Rozeboom, H.J. / Roelfes, G.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)885396European Union
CitationJournal: Acs Catalysis / Year: 2025
Title: Evolutionary Specialization of a Promiscuous Designer Enzyme.
Authors: Leveson-Gower, R.B. / Tiessler-Sala, L. / Rozeboom, H.J. / Thunnissen, A.W.H. / Marechal, J.D. / Roelfes, G.
History
DepositionAug 26, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 26, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Transcriptional regulator, PadR-like family
B: Transcriptional regulator, PadR-like family


Theoretical massNumber of molelcules
Total (without water)30,3262
Polymers30,3262
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2910 Å2
ΔGint-18 kcal/mol
Surface area13950 Å2
MethodPISA
Unit cell
Length a, b, c (Å)107.026, 36.463, 69.116
Angle α, β, γ (deg.)90.00, 97.78, 90.00
Int Tables number5
Space group name H-MC121

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Components

#1: Protein Transcriptional regulator, PadR-like family


Mass: 15163.173 Da / Num. of mol.: 2 / Mutation: V15pAF, N19M, A92R, F93H
Source method: isolated from a genetically manipulated source
Details: LmrR carrying a C-terminal strep-tag, with V15 replaced by para-aminophenylalanine (pAF) and with mutations A92R, N19M, F93H (RMH)
Source: (gene. exp.) Lactococcus cremoris subsp. cremoris MG1363 (lactic acid bacteria)
Gene: llmg_0323 / Production host: Escherichia coli (E. coli) / References: UniProt: A2RI36
Has ligand of interestY
Has protein modificationY

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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: 2.2 Å3/Da / Density % sol: 44.17 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: Protein solution: 7-8 mg/ml in 20 mM Tris-HCl, pH 8.0, 280 mM NaCl and 1 mM EDTA. Crystallization solution: 10-13% PEG 2000 MME in 0.1 M Bis-Tris propane, pH 8.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: MASSIF-1 / Wavelength: 0.87313 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 4, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.87313 Å / Relative weight: 1
ReflectionResolution: 2.55→44.97 Å / Num. obs: 8855 / % possible obs: 99.8 % / Redundancy: 5.7 % / CC1/2: 0.996 / Rmerge(I) obs: 0.137 / Rpim(I) all: 0.063 / Rrim(I) all: 0.152 / Χ2: 1.06 / Net I/σ(I): 7.7 / Num. measured all: 50650
Reflection shellResolution: 2.55→2.66 Å / % possible obs: 99.9 % / Redundancy: 6 % / Rmerge(I) obs: 1.75 / Num. measured all: 6308 / Num. unique obs: 1059 / CC1/2: 0.409 / Rpim(I) all: 0.788 / Rrim(I) all: 1.924 / Χ2: 1.03 / Net I/σ(I) obs: 1

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Processing

Software
NameVersionClassification
XDSdata reduction
Aimlessdata scaling
PHASERphasing
PHENIX1.20rc1_4395refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.55→39.42 Å / SU ML: 0.57 / Cross valid method: FREE R-VALUE / σ(F): 0.08 / Phase error: 40.26 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3238 799 4.81 %
Rwork0.2593 --
obs0.2626 8400 98.35 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.55→39.42 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1773 0 0 0 1773
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0051799
X-RAY DIFFRACTIONf_angle_d0.792407
X-RAY DIFFRACTIONf_dihedral_angle_d13.008699
X-RAY DIFFRACTIONf_chiral_restr0.041256
X-RAY DIFFRACTIONf_plane_restr0.006304
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.55-2.710.44031450.40052650X-RAY DIFFRACTION99
2.71-2.920.44681130.3732634X-RAY DIFFRACTION99
2.92-3.210.42421260.36942644X-RAY DIFFRACTION98
3.21-3.680.40251360.28922658X-RAY DIFFRACTION99
3.68-4.630.2721520.22992601X-RAY DIFFRACTION97
4.63-39.420.25861270.18972614X-RAY DIFFRACTION98
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)
11.9774-0.2306-0.96591.94751.36852.3003-0.08230.17090.04560.3140.0494-0.18610.448-0.1388-0.0250.4114-0.0173-0.01840.3070.0150.52117.092-4.6569.835
20.7835-0.7403-0.2209-1.00191.35191.8905-0.2135-0.3426-0.31160.29530.07440.07591.0089-0.3678-0.00120.8894-0.01910.01130.8040.00140.48568.178-0.67532.767
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1( CHAIN B AND RESID 4:110 )B4 - 110
2X-RAY DIFFRACTION2( CHAIN A AND RESID 4:110 )A4 - 110

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