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- PDB-9h88: Crystal structure of LmrR variant V15aY-RNYW with Val15 replaced ... -

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Basic information

Entry
Database: PDB / ID: 9h88
TitleCrystal structure of LmrR variant V15aY-RNYW with Val15 replaced by 3-aminotyrosine and evolved as Friedel-Crafts alkylase
ComponentsTranscriptional regulator, PadR-like family
KeywordsDNA BINDING PROTEIN / Artificial enzyme / Friedel-Crafts alkylase / unnatural amino acid / 3-aminotyrosine / 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 / NITRATE ION / 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: 1.2 Å
AuthorsThunnissen, A.M.W.H. / Brouwer, B. / Roelfes, G.
Funding supportEuropean Union, Netherlands, 2items
OrganizationGrant numberCountry
European Research Council (ERC)885396European Union
Netherlands Organisation for Scientific Research (NWO)OCENW.KLEIN.143 Netherlands
CitationJournal: Chem Sci / Year: 2025
Title: Genetically encoded 3-aminotyrosine as catalytic residue in a designer Friedel-Crafts alkylase.
Authors: Brouwer, B. / Della-Felice, F. / Thunnissen, A.W.H. / Roelfes, G.
History
DepositionOct 28, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 30, 2025Provider: repository / Type: Initial release
Revision 1.1May 7, 2025Group: Database references / Category: citation / citation_author
Item: _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI ..._citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jun 4, 2025Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)30,7385
Polymers30,5522
Non-polymers1863
Water3,747208
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3630 Å2
ΔGint-24 kcal/mol
Surface area13150 Å2
MethodPISA
Unit cell
Length a, b, c (Å)45.700, 58.533, 74.000
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Transcriptional regulator, PadR-like family


Mass: 15276.136 Da / Num. of mol.: 2 / Mutation: V15(3-amino-Y),L18R,N19W,M89N,A92N
Source method: isolated from a genetically manipulated source
Details: LmrR containing a C-terminal strep-tag, with Val15 replaced by 3-amino-tyrosine and with mutations L18R,N19W,M89N,A92N
Source: (gene. exp.) Lactococcus cremoris subsp. cremoris MG1363 (lactic acid bacteria)
Gene: llmg_0323 / Production host: Escherichia coli (E. coli) / References: UniProt: A2RI36
#2: Chemical ChemComp-NO3 / NITRATE ION


Mass: 62.005 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: NO3
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 208 / 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 % sol: 24.06 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: The protein solution contained 16 mg/ml protein in 20 mM HEPES, pH 7, 280 mM NaCl. The reservoir solution contained 0.2 M Na-citrate, 0.1 M Bis-Tris propane, pH 8.5, 20% (w/v) PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: MASSIF-1 / Wavelength: 0.965459 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Apr 27, 2024
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.965459 Å / Relative weight: 1
ReflectionResolution: 1.2→74 Å / Num. obs: 62694 / % possible obs: 99.8 % / Redundancy: 12.3 % / CC1/2: 1 / Rmerge(I) obs: 0.062 / Rpim(I) all: 0.018 / Rrim(I) all: 0.065 / Χ2: 1.04 / Net I/σ(I): 17.4 / Num. measured all: 770092
Reflection shellResolution: 1.2→1.22 Å / % possible obs: 96 % / Redundancy: 8.1 % / Rmerge(I) obs: 2.211 / Num. measured all: 23880 / Num. unique obs: 2937 / CC1/2: 0.409 / Rpim(I) all: 0.803 / Rrim(I) all: 2.36 / Χ2: 1.07 / Net I/σ(I) obs: 1

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Processing

Software
NameVersionClassification
XDSdata reduction
Aimlessdata scaling
PHASERphasing
REFMAC5.8.0425refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.2→45.95 Å / Cor.coef. Fo:Fc: 0.968 / Cor.coef. Fo:Fc free: 0.958 / SU B: 3.231 / SU ML: 0.058 / Cross valid method: THROUGHOUT / ESU R: 0.049 / ESU R Free: 0.049 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflectionSelection details
Rfree0.21268 3173 5.1 %RANDOM
Rwork0.17425 ---
obs0.17618 59446 99.77 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 20.756 Å2
Baniso -1Baniso -2Baniso -3
1--1.88 Å20 Å2-0 Å2
2--4.21 Å2-0 Å2
3----2.33 Å2
Refinement stepCycle: 1 / Resolution: 1.2→45.95 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1831 0 12 208 2051
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0121944
X-RAY DIFFRACTIONr_bond_other_d0.0010.0161853
X-RAY DIFFRACTIONr_angle_refined_deg1.7211.8562620
X-RAY DIFFRACTIONr_angle_other_deg0.6171.8064254
X-RAY DIFFRACTIONr_dihedral_angle_1_deg4.7685235
X-RAY DIFFRACTIONr_dihedral_angle_2_deg7.213517
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.10110377
X-RAY DIFFRACTIONr_dihedral_angle_4_deg
X-RAY DIFFRACTIONr_chiral_restr0.0910.2265
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.022366
X-RAY DIFFRACTIONr_gen_planes_other0.0030.02492
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it4.5321.751898
X-RAY DIFFRACTIONr_mcbond_other4.5321.754899
X-RAY DIFFRACTIONr_mcangle_it6.5933.1491126
X-RAY DIFFRACTIONr_mcangle_other6.6023.1521127
X-RAY DIFFRACTIONr_scbond_it7.0622.1471046
X-RAY DIFFRACTIONr_scbond_other7.0362.151038
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other10.1343.7851479
X-RAY DIFFRACTIONr_long_range_B_refined13.37621.352367
X-RAY DIFFRACTIONr_long_range_B_other12.85720.062327
X-RAY DIFFRACTIONr_rigid_bond_restr6.07833797
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.2→1.231 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.394 226 -
Rwork0.331 4206 -
obs--96.79 %

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