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- PDB-6wg7: Coordinates of NanR dimer fitted in Hexameric NanR-DNA hetero-com... -

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Basic information

Entry
Database: PDB / ID: 6wg7
TitleCoordinates of NanR dimer fitted in Hexameric NanR-DNA hetero-complex cryo-EM map
Components
  • (DNA (35-MER)) x 2
  • HTH-type transcriptional repressor NanR
KeywordsGENE REGULATION / NanR dimer-DNA hetero-complex / transcriptional regulator / GntR superfamily / sialic acid / Neu5Ac / cooperativity / Gene regulation.
Function / homology
Function and homology information


DNA-binding transcription factor activity / negative regulation of DNA-templated transcription / DNA binding
Similarity search - Function
HTH-type transcriptional repressor NanR / FCD / GntR, C-terminal / FCD domain / Transcription regulator FadR/GntR, C-terminal / Transcription regulator HTH, GntR / Bacterial regulatory proteins, gntR family / GntR-type HTH domain profile. / helix_turn_helix gluconate operon transcriptional repressor / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / HTH-type transcriptional repressor NanR
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.3 Å
AuthorsHariprasad, V. / Horne, C. / Santosh, P. / Amy, H. / Emre, B. / Rachel, N. / Michael, G. / Georg, R. / Borries, D. / Renwick, D.
Funding support New Zealand, 1items
OrganizationGrant numberCountry
Marsden FundUOC1506 New Zealand
CitationJournal: Nat Commun / Year: 2021
Title: Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism.
Authors: Christopher R Horne / Hariprasad Venugopal / Santosh Panjikar / David M Wood / Amy Henrickson / Emre Brookes / Rachel A North / James M Murphy / Rosmarie Friemann / Michael D W Griffin / ...Authors: Christopher R Horne / Hariprasad Venugopal / Santosh Panjikar / David M Wood / Amy Henrickson / Emre Brookes / Rachel A North / James M Murphy / Rosmarie Friemann / Michael D W Griffin / Georg Ramm / Borries Demeler / Renwick C J Dobson /
Abstract: Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and ...Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.
History
DepositionApr 5, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 10, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 14, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-21661
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: DNA (35-MER)
B: DNA (35-MER)
C: HTH-type transcriptional repressor NanR
D: HTH-type transcriptional repressor NanR
E: HTH-type transcriptional repressor NanR
G: HTH-type transcriptional repressor NanR
F: HTH-type transcriptional repressor NanR
H: HTH-type transcriptional repressor NanR


Theoretical massNumber of molelcules
Total (without water)198,9288
Polymers198,9288
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: equilibrium centrifugation
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: DNA chain DNA (35-MER)


Mass: 10856.016 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: DNA chain DNA (35-MER)


Mass: 10673.923 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Protein
HTH-type transcriptional repressor NanR


Mass: 29566.354 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: yhcK, glcC_1, glcC_2, lutR_1, lutR_2, nanR, nanR_2
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: J7QHT8

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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: Hexameric NanR-DNA hetero-complex / Type: COMPLEX / Details: Hexameric NanR-DNA hetero-complex / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.1985 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8 / Details: 20mM Tris-HCL,50mM NaCl, pH 8.0
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Blot force: -3 Blot time: 3 sec Drain time: 0

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 51.54 sec. / Electron dose: 45 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2287

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4cryoSPARCv2.14.2CTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 8.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48931 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building

3D fitting-ID: 1 / Accession code: 6WFQ / Initial refinement model-ID: 1 / PDB-ID: 6WFQ

6wfq

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1C
2D
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 247.92 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004312444
ELECTRON MICROSCOPYf_angle_d0.761717105
ELECTRON MICROSCOPYf_chiral_restr0.03491897
ELECTRON MICROSCOPYf_plane_restr0.00292032
ELECTRON MICROSCOPYf_dihedral_angle_d25.74974798

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