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- PDB-6i8n: Crystal structure of LmrR with V15 replaced by unnatural amino ac... -

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Basic information

Entry
Database: PDB / ID: 6i8n
TitleCrystal structure of LmrR with V15 replaced by unnatural amino acid 4-amino-L-phenylalanine
ComponentsTranscriptional regulator, PadR-like family
KeywordsDNA BINDING PROTEIN / PadR family / transcriptional regulator / engineered variant / artificial enzyme / unnatural amino acid
Function / homology
Function and homology information


: / Transcription regulator PadR, N-terminal / Transcriptional regulator PadR-like family / Winged helix-like DNA-binding domain superfamily/Winged helix DNA-binding domain / Arc Repressor Mutant, subunit A / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Transcriptional regulator, PadR-like family
Similarity search - Component
Biological speciesLactococcus lactis subsp. cremoris (lactic acid bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.79 Å
AuthorsReddem, R. / Thunnissen, A.M.W.H.
Funding support Netherlands, 1items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research724.013.003 Netherlands
CitationJournal: Angew. Chem. Int. Ed. Engl. / Year: 2019
Title: Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid.
Authors: Mayer, C. / Dulson, C. / Reddem, E. / Thunnissen, A.W.H. / Roelfes, G.
History
DepositionNov 20, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 2, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 13, 2019Group: Data collection / Database references / Category: citation / citation_author / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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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)29,8653
Polymers29,6562
Non-polymers2091
Water4,990277
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, light scattering
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4070 Å2
ΔGint-18 kcal/mol
Surface area13270 Å2
MethodPISA
Unit cell
Length a, b, c (Å)35.727, 55.149, 145.085
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid 2 through 75 or resid 77 through 94 or resid 96 or resid 98 through 111))
21(chain B and (resid 2 through 69 or resid 74...

NCS domain segments:

Ens-ID: 1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ALAALAARGARG(chain A and (resid 2 through 75 or resid 77 through 94 or resid 96 or resid 98 through 111))AA2 - 752 - 75
12LYSLYSGLUGLU(chain A and (resid 2 through 75 or resid 77 through 94 or resid 96 or resid 98 through 111))AA77 - 9477 - 94
13TRPTRPTRPTRP(chain A and (resid 2 through 75 or resid 77 through 94 or resid 96 or resid 98 through 111))AA9696
14ARGARGLYSLYS(chain A and (resid 2 through 75 or resid 77 through 94 or resid 96 or resid 98 through 111))AA98 - 11198 - 111
21ALAALAASPASP(chain B and (resid 2 through 69 or resid 74...BB2 - 692 - 69
22GLYGLYARGARG(chain B and (resid 2 through 69 or resid 74...BB74 - 7574 - 75
23LYSLYSGLUGLU(chain B and (resid 2 through 69 or resid 74...BB77 - 9477 - 94
24TRPTRPTRPTRP(chain B and (resid 2 through 69 or resid 74...BB9696
25ARGARGLYSLYS(chain B and (resid 2 through 69 or resid 74...BB98 - 11198 - 111

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Components

#1: Protein Transcriptional regulator, PadR-like family


Mass: 14827.846 Da / Num. of mol.: 2 / Mutation: V15 replaced with 4-amino-phenylalanine (HOX)
Source method: isolated from a genetically manipulated source
Details: LmrR carrying a C-terminal strep-tag and with valine 15 replaced with 4-amino-phenylalanine
Source: (gene. exp.) Lactococcus lactis subsp. cremoris (strain MG1363) (lactic acid bacteria)
Strain: MG1363 / Gene: llmg_0323 / Production host: Escherichia coli (E. coli) / References: UniProt: A2RI36
#2: Chemical ChemComp-MPO / 3[N-MORPHOLINO]PROPANE SULFONIC ACID


Mass: 209.263 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C7H15NO4S / Comment: pH buffer*YM
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 277 / Source method: isolated from a natural source / Formula: H2O

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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: 2.69 Å3/Da / Density % sol: 54.31 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 0.1 M sodium propionate/cacodylate/bis-tris propane buffer, pH 7.0, 25% PEG 1500

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, DESY / Beamline: P11 / Wavelength: 1.0332 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Sep 17, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0332 Å / Relative weight: 1
ReflectionResolution: 1.79→48.36 Å / Num. obs: 27893 / % possible obs: 99.6 % / Redundancy: 12.6 % / CC1/2: 0.999 / Rmerge(I) obs: 0.067 / Rpim(I) all: 0.02 / Rrim(I) all: 0.07 / Net I/σ(I): 22.2 / Num. measured all: 352691 / Scaling rejects: 11
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
1.79-1.8212.20.6341751114350.9260.1850.6613.792.8
9.12-48.3610.70.03728102620.9990.0120.0395099.1

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Processing

Software
NameVersionClassification
PHENIX1.14rc1_3161refinement
XDSdata reduction
Aimless0.7.2data scaling
PDB_EXTRACT3.24data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3F8C

3f8c


Resolution: 1.79→43.903 Å / SU ML: 0.18 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 18.97
RfactorNum. reflection% reflection
Rfree0.1948 1368 4.92 %
Rwork0.1723 --
obs0.1734 27818 99.66 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 106.15 Å2 / Biso mean: 31.0977 Å2 / Biso min: 13.78 Å2
Refinement stepCycle: final / Resolution: 1.79→43.903 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1818 0 13 277 2108
Biso mean--32.68 36.29 -
Num. residues----221
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A920X-RAY DIFFRACTION9.84TORSIONAL
12B920X-RAY DIFFRACTION9.84TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 10

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.79-1.8540.25181380.24912511264997
1.854-1.92820.23851380.217125902728100
1.9282-2.0160.19771320.188526162748100
2.016-2.12230.21181260.171526362762100
2.1223-2.25520.21771240.172626342758100
2.2552-2.42940.17961520.174126052757100
2.4294-2.67380.21491290.174826332762100
2.6738-3.06060.18571410.17226852826100
3.0606-3.85570.18471450.156326942839100
3.8557-43.91590.18431430.16728462989100

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