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- PDB-7oz3: S. agalactiae BusR in complex with its busA-promotor DNA -

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Basic information

Entry
Database: PDB / ID: 7oz3
TitleS. agalactiae BusR in complex with its busA-promotor DNA
Components
  • GntR family transcriptional regulator
  • pBusA_for
  • pBusA_rev
KeywordsDNA BINDING PROTEIN / Repressor / complex / GntR / Transcription
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) / DNA (> 100) / GntR family transcriptional regulator
Similarity search - Component
Biological speciesStreptococcus agalactiae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.46 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
DepositionJun 25, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Data collection / Database references / Source and taxonomy
Category: citation / citation_author ...citation / citation_author / database_PDB_rev / database_PDB_rev_record / em_admin / em_entity_assembly_naturalsource / 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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Structure viewerMolecule:
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Assembly

Deposited unit
A: GntR family transcriptional regulator
B: GntR family transcriptional regulator
C: GntR family transcriptional regulator
D: GntR family transcriptional regulator
F: pBusA_rev
G: pBusA_for
hetero molecules


Theoretical massNumber of molelcules
Total (without water)190,6448
Polymers189,3276
Non-polymers1,3172
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area26090 Å2
ΔGint-174 kcal/mol
Surface area49530 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
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 pBusA_rev


Mass: 46903.086 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Streptococcus agalactiae (bacteria)
#3: DNA chain pBusA_for


Mass: 46903.062 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) 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

ComponentName: transcriptional repressor BusR bound to target DNA / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.122 MDa
Source (natural)Organism: Streptococcus agalactiae (bacteria)
Buffer solutionpH: 6.5 / Details: degassed, filtered
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepes1
2100 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationCryogen 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 FIELDBright-field microscopy
Image recordingElectron dose: 45 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4RELION3.1.2CTF correction
7Coot0.9.5model fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELION3.1.2classification
12RELION3.1.23D reconstruction
13PHENIX1.19model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 112451 / Symmetry type: POINT
Atomic model buildingB value: 177 / Protocol: FLEXIBLE FIT / Space: REAL
Details: RCK_C domains were build by rigid body fit of the model 7B5U. The flexible linker between RCK_C domain and coiled-coil domain were build de novo.
Atomic model building
IDPDB-ID 3D fitting-IDPdb chain residue range
17B5T

7b5t

11-127
27B5U

7b5u

1136-200
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 134.6 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00868547
ELECTRON MICROSCOPYf_angle_d1.353511955
ELECTRON MICROSCOPYf_chiral_restr0.07681371
ELECTRON MICROSCOPYf_plane_restr0.0051203
ELECTRON MICROSCOPYf_dihedral_angle_d23.14393257

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