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- PDB-7b5y: S. agalactiae BusR in complex with its busAB-promotor DNA -

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Basic information

Entry
Database: PDB / ID: 7b5y
TitleS. agalactiae BusR in complex with its busAB-promotor DNA
Components
  • BusR binding site in the busAB promotor. strand1
  • BusR binding site in the busAB promotor. strand2
  • GntR family transcriptional regulator
KeywordsDNA BINDING PROTEIN / Repressor / complex / GntR
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 / DNA / DNA (> 10) / GntR family transcriptional regulator
Similarity search - Component
Biological speciesStreptococcus agalactiae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.1 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 13, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info
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 / _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code_mod

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Structure visualization

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Assembly

Deposited unit
C: GntR family transcriptional regulator
A: GntR family transcriptional regulator
B: GntR family transcriptional regulator
D: GntR family transcriptional regulator
E: BusR binding site in the busAB promotor. strand1
F: BusR binding site in the busAB promotor. strand2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)125,1678
Polymers123,8506
Non-polymers1,3172
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS, SAS data of this complex in solution are fully compatible with the cryoEM structure (CRYSOL fit chi2 is 5)
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25050 Å2
ΔGint-165 kcal/mol
Surface area52500 Å2
MethodPISA

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Components

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


Mass: 23880.160 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: N-terminal residues GP result from purification 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: DNA chain BusR binding site in the busAB promotor. strand1


Mass: 14309.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus agalactiae (bacteria)
#3: DNA chain BusR binding site in the busAB promotor. strand2


Mass: 14020.027 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus agalactiae (bacteria)
#4: 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.: 2 / Source method: obtained synthetically / Formula: C20H24N10O12P2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1transcriptional repressor BusR bound to DNACOMPLEX#1-#30MULTIPLE SOURCES
2GntR family transcriptional regulatorCOMPLEX#11RECOMBINANT
3BusR binding site in the busAB promotor DNACOMPLEX#2-#31RECOMBINANT
Molecular weightValue: 0.123 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Streptococcus agalactiae (bacteria)1311
23Streptococcus agalactiae (bacteria)1311
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli BL21(DE3) (bacteria)469008
23synthetic construct (others)32630
Buffer solutionpH: 6.5 / Details: degassed, filtered
Buffer componentConc.: 20 mM / Name: hepes / Formula: HEPES
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: homogeneous and monodisperse sample
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R2/2
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K
Details: 0.05% beta-octyl glycoside added prior to plunge freezing

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 45 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv2.15particle selection
2RELIONv3.1particle selection
3EPUimage acquisition
5cryoSPARC2.15CTF correction
8Coot0.9.2model fitting
10PHENIX1.18.2-3874model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 370421 / Details: CryoSparc blob-picker
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 20342 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 7B5T

7b5t

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