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- EMDB-18864: Central glycolytic genes regulator (CggR) bound to DNA operator -

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Basic information

Entry
Database: EMDB / ID: EMD-18864
TitleCentral glycolytic genes regulator (CggR) bound to DNA operator
Map data
Sample
  • Complex: A complex of Central glycolytic genes regulator (CggR) from Bacillus subtilis and the DNA operator (OLR).
    • Protein or peptide: Central glycolytic genes regulator
    • DNA: operator DNA
    • DNA: operator DNA
KeywordsBacillus subtilis / transcription repressor / glucose catabolism / SorC family / DNA BINDING PROTEIN
Function / homology
Function and homology information


regulation of DNA-templated transcription initiation / cis-regulatory region sequence-specific DNA binding / carbohydrate binding
Similarity search - Function
: / CggR N-terminal DNA binding domain / Sugar-binding domain, putative / : / Putative sugar-binding domain / NagB/RpiA transferase-like / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
Central glycolytic genes regulator
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsSkerlova J / Soltysova M / Rezacova P / Skubnik K
Funding support Czech Republic, European Union, 2 items
OrganizationGrant numberCountry
Ministry of Education, Youth and Sports of the Czech RepublicCIISB, Instruct-CZ Centre of Instruct-ERIC EU consortium, funded by MEYS CR infrastructure project LM2018127 Czech Republic
European Regional Development FundOP RDE Project No. CZ.02.1.01/0.0/0.0/16_019/0000729European Union
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Structural characterization of two prototypical repressors of SorC family reveals tetrameric assemblies on DNA and mechanism of function.
Authors: Markéta Šoltysová / Jana Škerlová / Petr Pachl / Karel Škubník / Milan Fábry / Irena Sieglová / Martina Farolfi / Irina Grishkovskaya / Michal Babiak / Jiří Nováček / Libor ...Authors: Markéta Šoltysová / Jana Škerlová / Petr Pachl / Karel Škubník / Milan Fábry / Irena Sieglová / Martina Farolfi / Irina Grishkovskaya / Michal Babiak / Jiří Nováček / Libor Krásný / Pavlína Řezáčová /
Abstract: The SorC family of transcriptional regulators plays a crucial role in controlling the carbohydrate metabolism and quorum sensing. We employed an integrative approach combining X-ray crystallography ...The SorC family of transcriptional regulators plays a crucial role in controlling the carbohydrate metabolism and quorum sensing. We employed an integrative approach combining X-ray crystallography and cryo-electron microscopy to investigate architecture and functional mechanism of two prototypical representatives of two sub-classes of the SorC family: DeoR and CggR from Bacillus subtilis. Despite possessing distinct DNA-binding domains, both proteins form similar tetrameric assemblies when bound to their respective DNA operators. Structural analysis elucidates the process by which the CggR-regulated gapA operon is derepressed through the action of two effectors: fructose-1,6-bisphosphate and newly confirmed dihydroxyacetone phosphate. Our findings provide the first comprehensive understanding of the DNA binding mechanism of the SorC-family proteins, shedding new light on their functional characteristics.
History
DepositionNov 9, 2023-
Header (metadata) releaseJun 19, 2024-
Map releaseJun 19, 2024-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18864.png

Downloads & links

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Map

FileDownload / File: emd_18864.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 352 pix.
= 289.344 Å		0.82 Å/pix.
x 352 pix.
= 289.344 Å		0.82 Å/pix.
x 352 pix.
= 289.344 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.822 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.162
Minimum - Maximum-0.19824444 - 0.5428169
Average (Standard dev.)0.0012862254 (±0.014502233)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 289.344 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18864_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18864_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18864_half_map_2.map
Projections & Slices
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Sample components

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Entire : A complex of Central glycolytic genes regulator (CggR) from Bacil...

EntireName: A complex of Central glycolytic genes regulator (CggR) from Bacillus subtilis and the DNA operator (OLR).
Components
  • Complex: A complex of Central glycolytic genes regulator (CggR) from Bacillus subtilis and the DNA operator (OLR).
    • Protein or peptide: Central glycolytic genes regulator
    • DNA: operator DNA
    • DNA: operator DNA

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Supramolecule #1: A complex of Central glycolytic genes regulator (CggR) from Bacil...

SupramoleculeName: A complex of Central glycolytic genes regulator (CggR) from Bacillus subtilis and the DNA operator (OLR).
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: A complex of full-length CggR with its 45bp DNA operator.
Source (natural)Organism: Bacillus subtilis (bacteria) / Location in cell: cytoplasm

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Macromolecule #1: Central glycolytic genes regulator

MacromoleculeName: Central glycolytic genes regulator / type: protein_or_peptide / ID: 1
Details: N-terminal amino-acid sequence GIDPFT remains a part of the protein upon TEV cleavage as a cloning artefact.
Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis (bacteria) / Cell: Bacterium
Molecular weightTheoretical: 38.623496 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GIDPFT(MSE)NQL IQAQKKLLPD LLLV(MSE)QKRFE ILQYIRLTEP IGRRSLSASL GISERVLRGE VQFLKEQNLV DI KTNG(MSE)TL TEEGYELLSV LEDT(MSE)KDVLG LTLLEKTLKE RLNLKDAIIV SGDSDQSPWV KKE(MSE)GRAAVA C (MSE)KKRFSGK ...String:
GIDPFT(MSE)NQL IQAQKKLLPD LLLV(MSE)QKRFE ILQYIRLTEP IGRRSLSASL GISERVLRGE VQFLKEQNLV DI KTNG(MSE)TL TEEGYELLSV LEDT(MSE)KDVLG LTLLEKTLKE RLNLKDAIIV SGDSDQSPWV KKE(MSE)GRAAVA C (MSE)KKRFSGK NIVAVTGGTT IEAVAE(MSE)(MSE)TP DSKNRELLFV PARGGLGEDV KNQANTICAH (MSE)AEKAS GTY RLLFVPGQLS QGAYSSIIEE PSVKEVLNTI KSAS(MSE)LVHGI GEAKT(MSE)AQRR NTPLEDLKKI DDNDAVTEA FGYYFNADGE VVHKVHSVG(MSE) QLDDIDAIPD IIAVAGGSSK AEAIEAYFKK PRNTVLVTDE GAAKKLLRDE

UniProtKB: Central glycolytic genes regulator

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Macromolecule #2: operator DNA

MacromoleculeName: operator DNA / type: dna / ID: 2
Details: Only 41bp were modelled into the electron density map (two base pairs at both ends are missing).
Number of copies: 1 / Classification: DNA
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 13.78087 KDa
SequenceString:
(DT)(DT)(DG)(DC)(DT)(DG)(DG)(DA)(DC)(DA) (DT)(DT)(DA)(DT)(DA)(DT)(DG)(DT)(DC)(DC) (DC)(DG)(DC)(DT)(DA)(DT)(DG)(DA)(DC) (DA)(DA)(DA)(DA)(DA)(DA)(DC)(DG)(DT)(DC) (DC) (DC)(DG)(DT)(DC)(DA)

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Macromolecule #3: operator DNA

MacromoleculeName: operator DNA / type: dna / ID: 3
Details: Only 41bp were modelled into the electron density map (two base pairs at both sides are missing).
Number of copies: 1 / Classification: DNA
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 13.931952 KDa
SequenceString:
(DT)(DG)(DA)(DC)(DG)(DG)(DG)(DA)(DC)(DG) (DT)(DT)(DT)(DT)(DT)(DT)(DG)(DT)(DC)(DA) (DT)(DA)(DG)(DC)(DG)(DG)(DG)(DA)(DC) (DA)(DT)(DA)(DT)(DA)(DA)(DT)(DG)(DT)(DC) (DC) (DA)(DG)(DC)(DA)(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

Concentration0.86 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMTris hydrochloride
100.0 mMsodium chlorideNaCl
0.02 % (v/v)beta-mercaptoethanol

Details: 20 mM Tris-HCl, pH 7.5, 100 mM NaCl and 0.02% (v/v) beta-mercaptoethanol
GridModel: C-flat / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 30.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab-initio model generation
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 220146
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7oyk

source_name: PDB, initial_model_type: experimental model

2okg

source_name: PDB, initial_model_type: experimental model
DetailsRigid body fitting of starting PDB models in Phenix and Coot was combined with manual rebuilding of certain model regions in Coot.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 24.32
Output model

PDB-8r3g:
Central glycolytic genes regulator (CggR) bound to DNA operator

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