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

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Basic information

Entry
Database: PDB / ID: 8r3g
TitleCentral glycolytic genes regulator (CggR) bound to DNA operator
Components
  • (operator DNA) x 2
  • Central glycolytic genes regulator
KeywordsDNA BINDING PROTEIN / Bacillus subtilis / transcription repressor / glucose catabolism / SorC family
Function / homology
Function and homology information


carbohydrate binding / DNA 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
DNA / DNA (> 10) / Central glycolytic genes regulator
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsSkerlova, J. / Soltysova, M. / Rezacova, P. / Skubnik, K.
Funding support Czech Republic, European Union, 2items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 19, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Central glycolytic genes regulator
A: Central glycolytic genes regulator
F: operator DNA
E: operator DNA
B: Central glycolytic genes regulator
D: Central glycolytic genes regulator


Theoretical massNumber of molelcules
Total (without water)182,2076
Polymers182,2076
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: scanning transmission electron microscopy, SAXS, Chaix, D., Ferguson, M. L., Atmanene, C., Van Dorsselaer, A., Sanglier-Cianferani, S., Royer, C. A., Declerck, N. Physical Basis of the ...Evidence: scanning transmission electron microscopy, SAXS, Chaix, D., Ferguson, M. L., Atmanene, C., Van Dorsselaer, A., Sanglier-Cianferani, S., Royer, C. A., Declerck, N. Physical Basis of the Inducer-Dependent Cooperativity of the Central Glycolytic Genes Repressor-DNA Complex. Nucleic Acids Res 2010, 38 (17)., gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17010 Å2
ΔGint-106 kcal/mol
Surface area69750 Å2

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Components

#1: Protein
Central glycolytic genes regulator


Mass: 38623.496 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: N-terminal amino-acid sequence GIDPFT remains a part of the protein upon TEV cleavage as a cloning artefact.
Source: (gene. exp.) Bacillus subtilis (bacteria) / Cell: Bacterium / Gene: cggR, yvbQ, BSU33950 / Plasmid: pET151 / Details (production host): ampicillin resistance / Cell (production host): Bacterium / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O32253
#2: DNA chain operator DNA


Mass: 13780.870 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Only 41bp were modelled into the electron density map (two base pairs at both ends are missing).
Source: (synth.) Bacillus subtilis (bacteria)
#3: DNA chain operator DNA


Mass: 13931.952 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Only 41bp were modelled into the electron density map (two base pairs at both sides are missing).
Source: (synth.) Bacillus subtilis (bacteria)
Has ligand of interestN

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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: A complex of Central glycolytic genes regulator (CggR) from Bacillus subtilis and the DNA operator (OLR).
Type: COMPLEX
Details: A complex of full-length CggR with its 45bp DNA operator.
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bacillus subtilis (bacteria) / Cellular location: cytoplasm
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3) / Plasmid: pET151
Buffer solutionpH: 7.5
Details: 20 mM Tris-HCl, pH 7.5, 100 mM NaCl and 0.02% (v/v) beta-mercaptoethanol
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris hydrochloride1
2100 mMsodium chlorideNaCl1
30.02 % (v/v)beta-mercaptoethanol1
SpecimenConc.: 0.86 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 / Nominal magnification: 165000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 30 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
7PHENIXmodel fitting
8Cootmodel fitting
14Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 220146 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 24.32 / Protocol: RIGID BODY FIT / Space: REAL
Details: Rigid body fitting of starting PDB models in Phenix and Coot was combined with manual rebuilding of certain model regions in Coot.
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
17oyk

7oyk

17oyk1PDBexperimental model
22okg

2okg

12okg2PDBexperimental model

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