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- PDB-5lrs: The Transcriptional Regulator PrfA from Listeria Monocytogenes in... -

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Basic information

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


positive regulation of single-species biofilm formation on inanimate substrate / 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
GLUTATHIONE / DNA / DNA (> 10) / Listeriolysin regulatory protein / Positive regulatory factor A
Similarity search - Component
Biological speciesListeria monocytogenes (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.9 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
DepositionAug 19, 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 17, 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 positive regulatory factor A
B: Listeriolysin positive regulatory factor A
C: DNA (30-MER)
D: DNA (30-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)73,7156
Polymers73,1014
Non-polymers6152
Water27015
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11340 Å2
ΔGint-100 kcal/mol
Surface area28010 Å2
MethodPISA
Unit cell
Length a, b, c (Å)78.961, 78.961, 265.224
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 positive regulatory factor A / Listeriolysin positive regulatory protein / Listeriolysin regulatory protein / Pleitrophic ...Listeriolysin positive regulatory protein / Listeriolysin regulatory protein / Pleitrophic regulatory factor A / Positive regulatory factor A / PrfA


Mass: 27329.391 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Listeria monocytogenes (bacteria) / Gene: prfA, M643_11230 / Production host: Escherichia coli (E. coli) / References: UniProt: Q4TVQ0, UniProt: P22262*PLUS
#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: Chemical ChemComp-GSH / GLUTATHIONE / Glutathione


Mass: 307.323 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N3O6S
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 15 / 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: 2.83 Å3/Da / Density % sol: 56.5 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 4.6
Details: Prior to the crystallization setup GSH and DTT were added to the protein solution to final concentrations of 5 mM and 1 mM, respectively. Protein and duplex DNA were incubated together at a ...Details: Prior to the crystallization setup GSH and DTT were added to the protein solution to final concentrations of 5 mM and 1 mM, respectively. 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 PrfAWT-DNA crystals were soaked in a reservoir solution containing 30% glycerol and 100 mM GSH for 24 h.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 1.073 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Jul 6, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.073 Å / Relative weight: 1
ReflectionResolution: 2.9→58.9 Å / Num. obs: 19427 / % possible obs: 99.5 % / Redundancy: 25.6 % / Rmerge(I) obs: 0.176 / Net I/σ(I): 16
Reflection shellResolution: 2.9→3.08 Å / Redundancy: 26.2 % / Rmerge(I) obs: 1.218 / Mean I/σ(I) obs: 3.1 / % possible all: 99.8

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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: 2.9→54.636 Å / SU ML: 0.42 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 28.97
RfactorNum. reflection% reflection
Rfree0.2792 933 4.82 %
Rwork0.2481 --
obs0.2495 19374 99.19 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.9→54.636 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3840 1224 40 15 5119
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0035338
X-RAY DIFFRACTIONf_angle_d0.537470
X-RAY DIFFRACTIONf_dihedral_angle_d17.642924
X-RAY DIFFRACTIONf_chiral_restr0.039826
X-RAY DIFFRACTIONf_plane_restr0.002728
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.9-3.05290.35381370.33912579X-RAY DIFFRACTION100
3.0529-3.24410.3471110.30422557X-RAY DIFFRACTION98
3.2441-3.49460.31371400.28112554X-RAY DIFFRACTION98
3.4946-3.84620.32391370.2552591X-RAY DIFFRACTION99
3.8462-4.40250.26821320.2382637X-RAY DIFFRACTION100
4.4025-5.54580.26821300.22912685X-RAY DIFFRACTION100
5.5458-54.6460.23351460.22232838X-RAY DIFFRACTION99

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