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- PDB-8qdh: Engineered LmrR carrying a cyclic boronate ester formed between T... -

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Basic information

Entry
Database: PDB / ID: 8qdh
TitleEngineered LmrR carrying a cyclic boronate ester formed between Tris and p-boronophenylalanine at position 89
ComponentsTranscriptional regulator, PadR-like family
KeywordsTRANSCRIPTION / Artificial enzyme / boron catalysis / unnatural amino acid / 4-boronophenylalanine
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 / 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: 1.72 Å
AuthorsThunnissen, A.M.W.H. / Rozeboom, H.J. / Longwitz, L. / Leveson-Gower, R.B. / Roelfes, G.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)885396European Union
CitationJournal: Nature / Year: 2024
Title: Boron catalysis in a designer enzyme.
Authors: Longwitz, L. / Leveson-Gower, R.B. / Rozeboom, H.J. / Thunnissen, A.W.H. / Roelfes, G.
History
DepositionAug 29, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 1, 2024Provider: repository / Type: Initial release
Revision 1.1May 22, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jun 5, 2024Group: 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

Downloads & links

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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)30,8225
Polymers30,5462
Non-polymers2763
Water1,45981
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, light scattering
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4290 Å2
ΔGint-22 kcal/mol
Surface area12520 Å2
MethodPISA
Unit cell
Length a, b, c (Å)44.714, 53.691, 98.981
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: 15272.956 Da / Num. of mol.: 2
Mutation: M8H, A92L, F93E, M89 replaced with p-boronophenylalanine
Source method: isolated from a genetically manipulated source
Details: LmrR carrying a C-terminal strep-tag, with mutations M8H, A92L, F93E and with M89 replaced with para-boronophenylalanine. The para-boronophenylalanine at position 89 has reacted with Tris to ...Details: LmrR carrying a C-terminal strep-tag, with mutations M8H, A92L, F93E and with M89 replaced with para-boronophenylalanine. The para-boronophenylalanine at position 89 has reacted with Tris to form a cyclic boronate ester
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-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 81 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 1.93 Å3/Da / Density % sol: 36.27 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6
Details: Protein was concentrated to 6.5 mg/ml in 20 mM Tris-HCl, pH 8.0, 280 mM NaCl and 1 mM EDTA. Reservoir solution contained 0.2 M NaCl, 20% PEG 6000 in 0.1 M MES, pH 6.0. Crystal was cryo- ...Details: Protein was concentrated to 6.5 mg/ml in 20 mM Tris-HCl, pH 8.0, 280 mM NaCl and 1 mM EDTA. Reservoir solution contained 0.2 M NaCl, 20% PEG 6000 in 0.1 M MES, pH 6.0. Crystal was cryo-protected by addition of 25% glycerol.

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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 PILATUS3 6M / Detector: PIXEL / Date: Apr 23, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.96546 Å / Relative weight: 1
ReflectionResolution: 1.72→49.49 Å / Num. obs: 25538 / % possible obs: 98.2 % / Redundancy: 12.9 % / CC1/2: 0.999 / Rmerge(I) obs: 0.082 / Rpim(I) all: 0.024 / Rrim(I) all: 0.086 / Χ2: 0.93 / Net I/σ(I): 14.8 / Num. measured all: 329575
Reflection shellResolution: 1.72→1.75 Å / % possible obs: 97.6 % / Redundancy: 13.2 % / Rmerge(I) obs: 2.034 / Num. measured all: 17356 / Num. unique obs: 1311 / CC1/2: 0.628 / Rpim(I) all: 0.576 / Rrim(I) all: 2.116 / Χ2: 0.8 / Net I/σ(I) obs: 1.1

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Processing

Software
NameVersionClassification
PHENIX(1.20rc1_4395: ???)refinement
XDS20220110data reduction
Aimless0.7.9data scaling
PHASER2.8.3phasing
PDB_EXTRACTdata extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.72→47.19 Å / SU ML: 0.26 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 28.74 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.26 1256 4.93 %
Rwork0.2055 --
obs0.2083 25463 97.84 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.72→47.19 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1817 0 18 81 1916
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0151892
X-RAY DIFFRACTIONf_angle_d1.4752550
X-RAY DIFFRACTIONf_dihedral_angle_d13.7746
X-RAY DIFFRACTIONf_chiral_restr0.074269
X-RAY DIFFRACTIONf_plane_restr0.013320
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.72-1.790.36431250.3392632X-RAY DIFFRACTION96
1.79-1.870.30231210.28292608X-RAY DIFFRACTION97
1.87-1.970.30671440.24932625X-RAY DIFFRACTION98
1.97-2.090.30291300.2462684X-RAY DIFFRACTION98
2.09-2.250.28111460.21642645X-RAY DIFFRACTION97
2.25-2.480.28591490.20462665X-RAY DIFFRACTION98
2.48-2.840.24661530.21872686X-RAY DIFFRACTION98
2.84-3.580.26291560.2022750X-RAY DIFFRACTION99
3.58-47.190.23131320.17932912X-RAY DIFFRACTION99
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)
12.3913-0.0186-0.08020.7158-0.5231.0556-0.1067-0.07030.05710.0130.12360.13140.1863-0.13340.00010.2288-0.03390.01390.29150.01580.2523-2.590310.5024-6.3481
20.60750.13010.81560.7394-0.52381.9931-1.2745-0.0378-0.70870.0701-0.0698-0.30271.43981.117-0.57470.49460.12970.20230.5429-0.01850.60947.31476.2423-12.1851
30.5021-0.0614-0.13531.2668-0.75480.4764-0.08650.08680.0770.3406-0.08250.06480.08-0.359-0.0510.244-0.08730.02520.31760.01480.235411.808118.9547-12.9545
40.49580.67081.39450.80091.18890.4077-0.19920.13440.062-0.0862-0.00710.2161-0.14260.1569-0.02550.299-0.05010.00340.2847-0.01880.2719-12.86497.2615-18.6626
50.03870.4924-0.13942.0028-0.19190.59930.20880.10590.07770.3461-0.08720.12490.06980.08620.01030.3181-0.0228-0.00470.2011-0.01510.2589-19.0012-6.876-19.0969
60.10950.0887-0.28740.1507-0.4620.93180.28820.19590.2661-0.5863-0.7657-0.78691.31130.4498-0.0350.52880.1481-0.12010.5121-0.05230.7614-13.3808-17.021-15.6631
70.8065-0.6228-0.42120.7034-0.06640.37360.15340.1547-0.14550.6723-0.1213-0.0613-0.0099-0.1028-0.03310.3904-0.0219-0.06110.22880.02830.2529-25.275-24.1512-14.0528
80.82550.26740.95740.47721.21860.44240.2407-0.05160.09760.3006-0.2392-0.15920.2848-0.16650.00720.3439-0.08740.03090.32190.0060.2415-16.03051.7458-5.5957
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 5 through 46 )
2X-RAY DIFFRACTION2chain 'A' and (resid 47 through 59 )
3X-RAY DIFFRACTION3chain 'A' and (resid 60 through 76 )
4X-RAY DIFFRACTION4chain 'A' and (resid 77 through 112 )
5X-RAY DIFFRACTION5chain 'B' and (resid 2 through 46 )
6X-RAY DIFFRACTION6chain 'B' and (resid 47 through 59 )
7X-RAY DIFFRACTION7chain 'B' and (resid 60 through 76 )
8X-RAY DIFFRACTION8chain 'B' and (resid 77 through 112 )

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