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- PDB-7b5u: RCK_C domain dimer of S.agalactiae BusR in complex with c-di-AMP -

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Basic information

Entry
Database: PDB / ID: 7b5u
TitleRCK_C domain dimer of S.agalactiae BusR in complex with c-di-AMP
ComponentsGntR family transcriptional regulator
KeywordsDNA BINDING PROTEIN / effector binding domain / RCK_C
Function / homology
Function and homology information


monoatomic cation transmembrane transporter activity / potassium ion transport / DNA-binding transcription factor activity
Similarity search - Function
GntR-type HTH domain profile. / helix_turn_helix gluconate operon transcriptional repressor / Transcription regulator HTH, GntR / Bacterial regulatory proteins, gntR family / Regulator of K+ conductance, C-terminal / Regulator of K+ conductance, C-terminal domain superfamily / TrkA-C domain / RCK C-terminal domain profile. / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
Chem-2BA / GntR family transcriptional regulator
Similarity search - Component
Biological speciesStreptococcus agalactiae (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.2 Å
AuthorsBandera, A.M. / Witte, G.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)WI3717/3-1 Germany
German Research Foundation (DFG)GRK1721 Germany
CitationJournal: Nucleic Acids Res / Year: 2021
Title: BusR senses bipartite DNA binding motifs by a unique molecular ruler architecture.
Authors: Adrian M Bandera / Joseph Bartho / Katja Lammens / David Jan Drexler / Jasmin Kleinschwärzer / Karl-Peter Hopfner / Gregor Witte /
Abstract: The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with various direct interactions with ...The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with various direct interactions with proteins such as potassium channels, the second messenger also specifically binds to transcription factors, thereby altering the processes in the cell on the transcriptional level. We here describe the structural and biochemical characterization of BusR from the human pathogen Streptococcus agalactiae. BusR is a member of a yet structurally uncharacterized subfamily of the GntR family of transcription factors that downregulates transcription of the genes for the BusA (OpuA) glycine-betaine transporter upon c-di-AMP binding. We report crystal structures of full-length BusR, its apo and c-di-AMP bound effector domain, as well as cryo-EM structures of BusR bound to its operator DNA. Our structural data, supported by biochemical and biophysical data, reveal that BusR utilizes a unique domain assembly with a tetrameric coiled-coil in between the binding platforms, serving as a molecular ruler to specifically recognize a 22 bp separated bipartite binding motif. Binding of c-di-AMP to BusR induces a shift in equilibrium from an inactivated towards an activated state that allows BusR to bind the target DNA, leading to transcriptional repression.
History
DepositionDec 7, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 6, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_PDB_rev / database_PDB_rev_record / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Oct 13, 2021Group: Data collection / Category: pdbx_database_proc
Revision 1.3Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: GntR family transcriptional regulator
B: GntR family transcriptional regulator
hetero molecules


Theoretical massNumber of molelcules
Total (without water)18,6873
Polymers18,0282
Non-polymers6581
Water4,990277
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS, SAXS data are only compatible with a dimeric assembly of the RCK_C domain
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1770 Å2
ΔGint-16 kcal/mol
Surface area8010 Å2
MethodPISA
Unit cell
Length a, b, c (Å)33.164, 60.947, 35.714
Angle α, β, γ (deg.)90.000, 101.730, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein GntR family transcriptional regulator / Transcriptional regulator / GntR family


Mass: 9014.149 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: G133 results from N-terminal tag / Source: (gene. exp.) Streptococcus agalactiae (bacteria)
Gene: BM110_ORF1201, AX245_01365, C6N10_09995, F5043_05515, GD434_05225, RDF_1124
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: K0JNC6
#2: Chemical ChemComp-2BA / (2R,3R,3aS,5R,7aR,9R,10R,10aS,12R,14aR)-2,9-bis(6-amino-9H-purin-9-yl)octahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8 ]tetraoxadiphosphacyclododecine-3,5,10,12-tetrol 5,12-dioxide / bis-(3',5')-cyclic-dimeric-Adenosine-monophosphate


Mass: 658.412 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H24N10O12P2 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 277 / 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: 1.96 Å3/Da / Density % sol: 37.25 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 4.6 / Details: 100 mM Na-acetate, 3% (w/v) PEG4000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 0.9763 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jun 29, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 1.2→17.67 Å / Num. obs: 41497 / % possible obs: 95.4 % / Redundancy: 6.9 % / Biso Wilson estimate: 11.49 Å2 / CC1/2: 0.99 / Rrim(I) all: 0.034 / Net I/σ(I): 33.3
Reflection shellResolution: 1.2→1.23 Å / Mean I/σ(I) obs: 14.86 / Num. unique obs: 2859 / CC1/2: 0.993 / Rrim(I) all: 0.11

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Processing

Software
NameVersionClassification
XSCALEdata scaling
PHENIX1.18.2_3874refinement
PDB_EXTRACT3.27data extraction
XDSdata reduction
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7B5T

7b5t


Resolution: 1.2→17.67 Å / SU ML: 0.07 / Cross valid method: THROUGHOUT / σ(F): 1.43 / Phase error: 14.94 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1553 1923 4.63 %
Rwork0.1308 39574 -
obs0.132 41497 95.47 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 58.48 Å2 / Biso mean: 12.8787 Å2 / Biso min: 4.2 Å2
Refinement stepCycle: final / Resolution: 1.2→17.67 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1241 0 44 278 1563
Biso mean--5.36 25.37 -
Num. residues----157
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.2-1.230.13961320.11412619275188
1.23-1.260.16931410.11362732287394
1.26-1.30.16941430.11492733287693
1.3-1.340.15971350.11442759289493
1.34-1.390.15191280.11862847297595
1.39-1.450.15391190.11342860297996
1.45-1.510.15361350.11462826296196
1.51-1.590.14451250.11712837296296
1.59-1.690.15421460.1242864301097
1.69-1.820.15291250.13022852297796
1.82-20.14181280.13282897302598
2-2.290.15981500.13452898304898
2.29-2.890.16481580.14562920307898
2.89-17.670.1531580.14312930308898

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