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- PDB-6wfq: NanR dimer-DNA hetero-complex -

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Basic information

Entry
Database: PDB / ID: 6wfq
TitleNanR dimer-DNA hetero-complex
Components
  • DNA (5'-D(P*GP*GP*TP*AP*TP*AP*AP*CP*AP*GP*GP*TP*AP*TP*A)-3')
  • DNA (5'-D(P*TP*AP*TP*AP*CP*CP*TP*GP*TP*TP*AP*TP*AP*CP*C)-3')
  • HTH-type transcriptional repressor NanR
KeywordsGENE REGULATION / NanR dimer-DNA hetero-complex / transcriptional regulator / GntR superfamily / sialic acid / Neu5Ac / cooperativity.
Function / homology
Function and homology information


DNA-binding transcription factor activity / negative regulation of transcription, DNA-templated / DNA binding
Transcription regulator HTH, GntR / Transcription regulator FadR/GntR, C-terminal / GntR, C-terminal / HTH-type transcriptional repressor NanR / Winged helix-like DNA-binding domain superfamily / Winged helix DNA-binding domain superfamily
HTH-type transcriptional repressor NanR
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionApr 3, 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

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
C: HTH-type transcriptional repressor NanR
D: HTH-type transcriptional repressor NanR
A: DNA (5'-D(P*GP*GP*TP*AP*TP*AP*AP*CP*AP*GP*GP*TP*AP*TP*A)-3')
B: DNA (5'-D(P*TP*AP*TP*AP*CP*CP*TP*GP*TP*TP*AP*TP*AP*CP*C)-3')


Theoretical massNumber of molelcules
Total (without water)68,3094
Polymers68,3094
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein HTH-type transcriptional repressor NanR


Mass: 29566.354 Da / Num. of mol.: 2
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
#2: DNA chain DNA (5'-D(P*GP*GP*TP*AP*TP*AP*AP*CP*AP*GP*GP*TP*AP*TP*A)-3')


Mass: 4657.060 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(P*TP*AP*TP*AP*CP*CP*TP*GP*TP*TP*AP*TP*AP*CP*C)-3')


Mass: 4518.959 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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: NanR-DNA hetero-complex / Type: COMPLEX / Details: Dimeric NanR-DNA hetero-complex / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.0728 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Details: 20mM Tris-HCL, 150mM NaCl, 100 microM ZnCl2, pH 8.0
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
250 mMsodium chlorideNaClSodium chloride1
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: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: blotting conditions: 3 sec blotting time and -3 blot force.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 215000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 12.8 sec. / Electron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3465
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 32 / Used frames/image: 1-20

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Processing

EM software
IDNameVersionCategoryDetails
1Gautomatch0.53particle selection
2EPUimage acquisition
4Gctf1.06CTF correctionCTF determination
5RELION3CTF correctionCTF correction
8PHENIX1.18model fittingcryo_fit for flexible fitting
9ISOLDE1.0b3model fittingFlexible fitting
11PHENIX1.18model refinement
12RELION3.1_betainitial Euler assignment
13RELION3.1_betafinal Euler assignment
15RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 695465
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 141663 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 290.52 Å2
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00194300
ELECTRON MICROSCOPYf_angle_d0.46515933
ELECTRON MICROSCOPYf_chiral_restr0.0315659
ELECTRON MICROSCOPYf_plane_restr0.0026684
ELECTRON MICROSCOPYf_dihedral_angle_d24.37541662

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