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- PDB-5lek: The Transcriptional Regulator PrfA-G145S mutant from Listeria Mon... -

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Basic information

Entry
Database: PDB / ID: 5lek
TitleThe Transcriptional Regulator PrfA-G145S mutant from Listeria Monocytogenes in complex with a 30-bp operator PrfA-box motif
Components
  • (DNA (30-MER)) x 2
  • Listeriolysin regulatory protein
KeywordsTRANSCRIPTION / Transcription regulator / DNA binding / activation / Listeria monocytogenes
Function / homology
Function and homology information


DNA-binding transcription factor activity / DNA binding / cytosol
Similarity search - Function
Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / Crp-type HTH domain profile. / Crp-like helix-turn-helix domain / Crp-type HTH domain / Cyclic nucleotide-binding domain superfamily / Jelly Rolls / RmlC-like jelly roll fold / Winged helix-like DNA-binding domain superfamily/Winged helix DNA-binding domain / Arc Repressor Mutant, subunit A ...Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / Crp-type HTH domain profile. / Crp-like helix-turn-helix domain / Crp-type HTH domain / Cyclic nucleotide-binding domain superfamily / Jelly Rolls / RmlC-like jelly roll fold / Winged helix-like DNA-binding domain superfamily/Winged helix DNA-binding domain / Arc Repressor Mutant, subunit A / Winged helix DNA-binding domain superfamily / Jelly Rolls / Winged helix-like DNA-binding domain superfamily / Sandwich / Orthogonal Bundle / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
DNA / DNA (> 10) / Listeriolysin regulatory protein
Similarity search - Component
Biological speciesListeria monocytogenes serovar 1/2a (bacteria)
Listeria monocytogenes (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.8 Å
AuthorsHall, M. / Grundstrom, C. / Begum, A. / Lindberg, M. / Sauer, U.H. / Almqvist, F. / Johansson, J. / Sauer-Eriksson, A.E.
Funding support Sweden, 2items
OrganizationGrant numberCountry
Swedish Research Council Sweden
Knut and Alice Wallenberg Foundation Sweden
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2016
Title: Structural basis for glutathione-mediated activation of the virulence regulatory protein PrfA in Listeria.
Authors: Hall, M. / Grundstrom, C. / Begum, A. / Lindberg, M.J. / Sauer, U.H. / Almqvist, F. / Johansson, J. / Sauer-Eriksson, A.E.
History
DepositionJun 29, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 7, 2016Provider: repository / Type: Initial release
Revision 1.1Dec 21, 2016Group: Database references
Revision 1.2Jan 11, 2017Group: Database references
Revision 1.3Sep 13, 2017Group: Author supporting evidence / Derived calculations / Category: pdbx_audit_support / struct_conn / Item: _pdbx_audit_support.funding_organization
Revision 1.4Jan 10, 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
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Listeriolysin regulatory protein
B: Listeriolysin regulatory protein
C: DNA (30-MER)
D: DNA (30-MER)


Theoretical massNumber of molelcules
Total (without water)73,1614
Polymers73,1614
Non-polymers00
Water543
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9980 Å2
ΔGint-83 kcal/mol
Surface area28620 Å2
MethodPISA
Unit cell
Length a, b, c (Å)79.081, 79.081, 265.978
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212

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Components

#1: Protein Listeriolysin regulatory protein


Mass: 27359.418 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e) (bacteria)
Gene: prfA, lmo0200 / Production host: Escherichia coli (E. coli) / References: UniProt: P22262
#2: DNA chain DNA (30-MER)


Mass: 9261.000 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Listeria monocytogenes (bacteria)
#3: DNA chain DNA (30-MER)


Mass: 9180.953 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Listeria monocytogenes (bacteria)
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3 / 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: 2.84 Å3/Da / Density % sol: 56.72 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 5.5
Details: Protein and duplex DNA were incubated together at a ratio of 1:1.3 (PrfA dimer:hly DNA) at final concentrations of 50 microM and 70 microM respectively in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, ...Details: Protein and duplex DNA were incubated together at a ratio of 1:1.3 (PrfA dimer:hly DNA) at final concentrations of 50 microM and 70 microM respectively in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT for 60 min at room temperature, before being used for crystal setups. Crystals were obtained after 24 h by mixing 4 microL protein-DNA solution with 2 microL reservoir solution consisting of 8% PEG 8000, 100 mM sodium acetate pH 4.6, 100 mM magnesium acetate, 20% glycerol. Prior to vitrification the soaking of PrfAG145S-DNA crystals were soaked in a reservoir solution containing 30% glycerol for 24 h

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: BESSY / Beamline: 14.1 / Wavelength: 0.8729 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Jan 22, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.8729 Å / Relative weight: 1
ReflectionResolution: 2.8→47.3 Å / Num. obs: 21616 / % possible obs: 99.4 % / Redundancy: 7 % / Rmerge(I) obs: 0.091 / Net I/σ(I): 14
Reflection shellResolution: 2.8→2.95 Å / Redundancy: 6.9 % / Rmerge(I) obs: 1.281 / Mean I/σ(I) obs: 1.7 / % possible all: 99.2

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155: ???)refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2bgc

2bgc


Resolution: 2.8→47.297 Å / SU ML: 0.5 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 30.24
RfactorNum. reflection% reflection
Rfree0.2666 1088 5.03 %
Rwork0.2313 --
obs0.233 21616 99.21 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.8→47.297 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3844 1224 0 3 5071
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0045312
X-RAY DIFFRACTIONf_angle_d0.7617438
X-RAY DIFFRACTIONf_dihedral_angle_d17.0322928
X-RAY DIFFRACTIONf_chiral_restr0.05825
X-RAY DIFFRACTIONf_plane_restr0.004721
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.7982-2.92560.44551410.36872499X-RAY DIFFRACTION99
2.9256-3.07980.38561430.34072479X-RAY DIFFRACTION99
3.0798-3.27270.32451270.29592507X-RAY DIFFRACTION98
3.2727-3.52530.28041370.25442498X-RAY DIFFRACTION99
3.5253-3.87990.31451330.24262544X-RAY DIFFRACTION99
3.8799-4.4410.22881470.22072574X-RAY DIFFRACTION100
4.441-5.59370.22071230.20672646X-RAY DIFFRACTION100
5.5937-47.30380.22951370.18952781X-RAY DIFFRACTION99

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