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- EMDB-13119: S. agalactiae BusR in complex with its busA-promotor DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-13119
TitleS. agalactiae BusR in complex with its busA-promotor DNA
Map data
Sample
  • Complex: transcriptional repressor BusR bound to target DNA
    • Protein or peptide: GntR family transcriptional regulator
    • DNA: pBusA_rev
    • DNA: pBusA_for
  • Ligand: (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
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
GntR family transcriptional regulator
Similarity search - Component
Biological speciesStreptococcus agalactiae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.46 Å
AuthorsBandera AM / Witte G
Funding support Germany, 2 items
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, 2021-
Header (metadata) releaseAug 11, 2021-
Map releaseAug 11, 2021-
UpdateOct 13, 2021-
Current statusOct 13, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7oz3
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13119.png

Downloads & links

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Map

FileDownload / File: emd_13119.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.046 Å
Density
Contour LevelBy AUTHOR: 0.019 / Movie #1: 0.019
Minimum - Maximum-0.037420247 - 0.07822399
Average (Standard dev.)0.00017319388 (±0.0020986376)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 267.776 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0461.0461.046
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z267.776267.776267.776
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0370.0780.000

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Supplemental data

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Mask #1

Fileemd_13119_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_13119_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_13119_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : transcriptional repressor BusR bound to target DNA

EntireName: transcriptional repressor BusR bound to target DNA
Components
  • Complex: transcriptional repressor BusR bound to target DNA
    • Protein or peptide: GntR family transcriptional regulator
    • DNA: pBusA_rev
    • DNA: pBusA_for
  • Ligand: (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

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Supramolecule #1: transcriptional repressor BusR bound to target DNA

SupramoleculeName: transcriptional repressor BusR bound to target DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Streptococcus agalactiae (bacteria)
Molecular weightTheoretical: 122 KDa

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Macromolecule #1: GntR family transcriptional regulator

MacromoleculeName: GntR family transcriptional regulator / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus agalactiae (bacteria)
Molecular weightTheoretical: 23.88016 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPMVSEQSEI VTSKYQKIAV AVAQRIANGD YEVGEKLKSR TTIASTFNVS PETARKGLNI LADLQILTLK HGSGAIILSK EKAIEFLNQ YETSHSVAIL KGKIRDNIKA QQQEMEELAT LVDDFLLQTR AVSKQYPLAP YEIIVSEDSE HLGKSIGELN V WHQTGATI ...String:
GPMVSEQSEI VTSKYQKIAV AVAQRIANGD YEVGEKLKSR TTIASTFNVS PETARKGLNI LADLQILTLK HGSGAIILSK EKAIEFLNQ YETSHSVAIL KGKIRDNIKA QQQEMEELAT LVDDFLLQTR AVSKQYPLAP YEIIVSEDSE HLGKSIGELN V WHQTGATI VAIEHEGKFI VSPGPFSVIE QGDHIFFVGD EDVYARMKTY FNLRMGL

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Macromolecule #2: pBusA_rev

MacromoleculeName: pBusA_rev / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Streptococcus agalactiae (bacteria)
Molecular weightTheoretical: 46.903086 KDa
SequenceString: (DA)(DT)(DA)(DG)(DG)(DA)(DT)(DC)(DC)(DG) (DT)(DG)(DT)(DC)(DT)(DT)(DA)(DT)(DC)(DT) (DA)(DT)(DA)(DA)(DA)(DG)(DT)(DG)(DA) (DC)(DG)(DT)(DT)(DG)(DA)(DG)(DT)(DT)(DA) (DT) (DC)(DG)(DT)(DA)(DA)(DA) ...String:
(DA)(DT)(DA)(DG)(DG)(DA)(DT)(DC)(DC)(DG) (DT)(DG)(DT)(DC)(DT)(DT)(DA)(DT)(DC)(DT) (DA)(DT)(DA)(DA)(DA)(DG)(DT)(DG)(DA) (DC)(DG)(DT)(DT)(DG)(DA)(DG)(DT)(DT)(DA) (DT) (DC)(DG)(DT)(DA)(DA)(DA)(DA)(DG) (DG)(DG)(DT)(DA)(DG)(DT)(DC)(DA)(DC)(DT) (DT)(DT) (DT)(DT)(DT)(DA)(DC)(DT)(DC) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DT)(DA)(DA) (DA)(DC)(DT) (DG)(DC)(DC)(DG)(DA)(DG) (DA)(DC)(DA)(DG)(DA)(DC)(DC)(DA)(DT)(DA) (DA)(DC)(DT)(DA) (DT)(DA)(DA)(DT)(DA) (DT)(DC)(DA)(DT)(DG)(DT)(DA)(DT)(DG)(DG) (DT)(DC)(DT)(DC)(DT) (DT)(DT)(DA)(DT) (DG)(DT)(DC)(DA)(DA)(DC)(DA)(DC)(DC)(DT) (DT)(DG)(DC)(DT)(DT)(DA) (DG)(DA)(DG) (DA)(DA)(DG)(DC)(DT)(DT)(DT)(DA)(DT)

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Macromolecule #3: pBusA_for

MacromoleculeName: pBusA_for / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Streptococcus agalactiae (bacteria)
Molecular weightTheoretical: 46.903062 KDa
SequenceString: (DA)(DT)(DA)(DA)(DA)(DG)(DC)(DT)(DT)(DC) (DT)(DC)(DT)(DA)(DA)(DG)(DC)(DA)(DA)(DG) (DG)(DT)(DG)(DT)(DT)(DG)(DA)(DC)(DA) (DT)(DA)(DA)(DA)(DG)(DA)(DG)(DA)(DC)(DC) (DA) (DT)(DA)(DC)(DA)(DT)(DG) ...String:
(DA)(DT)(DA)(DA)(DA)(DG)(DC)(DT)(DT)(DC) (DT)(DC)(DT)(DA)(DA)(DG)(DC)(DA)(DA)(DG) (DG)(DT)(DG)(DT)(DT)(DG)(DA)(DC)(DA) (DT)(DA)(DA)(DA)(DG)(DA)(DG)(DA)(DC)(DC) (DA) (DT)(DA)(DC)(DA)(DT)(DG)(DA)(DT) (DA)(DT)(DT)(DA)(DT)(DA)(DG)(DT)(DT)(DA) (DT)(DG) (DG)(DT)(DC)(DT)(DG)(DT)(DC) (DT)(DC)(DG)(DG)(DC)(DA)(DG)(DT)(DT)(DT) (DA)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DG) (DA)(DG)(DT)(DA)(DA)(DA)(DA)(DA)(DA)(DG) (DT)(DG)(DA)(DC) (DT)(DA)(DC)(DC)(DC) (DT)(DT)(DT)(DT)(DA)(DC)(DG)(DA)(DT)(DA) (DA)(DC)(DT)(DC)(DA) (DA)(DC)(DG)(DT) (DC)(DA)(DC)(DT)(DT)(DT)(DA)(DT)(DA)(DG) (DA)(DT)(DA)(DA)(DG)(DA) (DC)(DA)(DC) (DG)(DG)(DA)(DT)(DC)(DC)(DT)(DA)(DT)

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Macromolecule #4: (2R,3R,3aS,5R,7aR,9R,10R,10aS,12R,14aR)-2,9-bis(6-amino-9H-purin-...

MacromoleculeName: (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
type: ligand / ID: 4 / Number of copies: 2 / Formula: 2BA
Molecular weightTheoretical: 658.412 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.6 mg/mL
BufferpH: 6.5
Component:
ConcentrationNameFormula
20.0 mMHepes
100.0 mMsodium chlorideNaClSodium chloride

Details: degassed, filtered
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K
Details: 0.05% beta-octyl glycoside added prior to plunge freezing.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: RELION (ver. 3.1.2)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7b5t


Details: structure of ligand-free full-length BusR
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.46 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.2) / Number images used: 112451
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7b5t

residue_range: 1-127

7b5u

residue_range: 136-200
DetailsRCK_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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 177
Output model

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

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