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

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Basic information

Entry
Database: PDB / ID: 9gks
TitleCrystal structure of artificial enzyme LmrR_pAF variant RMH in crystal form 2
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: 2.24 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
Water39622
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3150 Å2
ΔGint-22 kcal/mol
Surface area12810 Å2
MethodPISA
Unit cell
Length a, b, c (Å)104.927, 35.303, 68.180
Angle α, β, γ (deg.)90.00, 97.27, 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, 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
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 22 / Source method: isolated from a natural source / Formula: H2O
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: 2.07 Å3/Da / Density % sol: 40.44 %
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: 0.96546 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Oct 3, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.96546 Å / Relative weight: 1
ReflectionResolution: 2.24→52.04 Å / Num. obs: 11930 / % possible obs: 97.7 % / Redundancy: 2.9 % / CC1/2: 0.999 / Rmerge(I) obs: 0.042 / Rpim(I) all: 0.029 / Rrim(I) all: 0.051 / Χ2: 0.5 / Net I/σ(I): 9.9 / Num. measured all: 34336
Reflection shellResolution: 2.24→2.31 Å / % possible obs: 86.2 % / Redundancy: 2.8 % / Rmerge(I) obs: 0.57 / Num. measured all: 2720 / Num. unique obs: 959 / CC1/2: 0.467 / Rpim(I) all: 0.403 / Rrim(I) all: 0.702 / Χ2: 0.19 / Net I/σ(I) obs: 1

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Processing

Software
NameVersionClassification
DIALSdata reduction
Aimlessdata scaling
PHASERphasing
PHENIX1.20rc1_4395refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.24→52.04 Å / SU ML: 0.35 / Cross valid method: FREE R-VALUE / σ(F): 0.12 / Phase error: 36.03 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2945 1081 5.03 %
Rwork0.2268 --
obs0.2302 10740 91.91 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.24→52.04 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1734 0 0 22 1756
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0141760
X-RAY DIFFRACTIONf_angle_d1.3692354
X-RAY DIFFRACTIONf_dihedral_angle_d13.287686
X-RAY DIFFRACTIONf_chiral_restr0.059251
X-RAY DIFFRACTIONf_plane_restr0.009295
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.24-2.340.35021110.30852336X-RAY DIFFRACTION83
2.34-2.460.33641230.26292402X-RAY DIFFRACTION88
2.46-2.620.26671210.28722691X-RAY DIFFRACTION96
2.62-2.820.37131330.28752679X-RAY DIFFRACTION96
2.82-3.110.30231560.28142649X-RAY DIFFRACTION95
3.11-3.550.33921540.24982425X-RAY DIFFRACTION89
3.55-4.480.28721360.20732640X-RAY DIFFRACTION95
4.48-52.040.25731470.18452578X-RAY DIFFRACTION93
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)
14.35410.0316-3.02712.5730.96064.0413-0.1652-0.0999-0.18910.2121-0.0872-0.21270.275-0.1590.16710.3565-0.007-0.01870.2841-0.0090.458216.326-1.56110.245
22.8467-0.60181.37291.08511.3134.9872-0.2848-0.75-0.1110.45460.432-0.18330.0873-1.1614-0.08250.78550.075-0.03840.90620.01720.55768.7612.42131.243
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1( CHAIN A AND RESID 4:111 )A4 - 111
2X-RAY DIFFRACTION2( CHAIN B AND RESID 2:111 )B2 - 111

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