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- EMDB-21652: NanR dimer-DNA hetero-complex -

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Basic information

Entry
Database: EMDB / ID: EMD-21652
TitleNanR dimer-DNA hetero-complex
Map dataRelion local resolution filtered map. Sharpening B-factor -100
Sample
  • Complex: NanR-DNA hetero-complex
    • Protein or peptide: HTH-type transcriptional repressor NanR
    • DNA: DNA (5'-D(P*GP*GP*TP*AP*TP*AP*AP*CP*AP*GP*GP*TP*AP*TP*A)-3')
    • DNA: DNA (5'-D(P*TP*AP*TP*AP*CP*CP*TP*GP*TP*TP*AP*TP*AP*CP*C)-3')
KeywordsNanR 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 / GntR-type HTH domain profile. / helix_turn_helix gluconate operon transcriptional repressor / Transcription regulator HTH, GntR / Bacterial regulatory proteins, gntR family / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
HTH-type transcriptional repressor NanR
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsHariprasad V / Horne C
Funding support New Zealand, 1 items
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 3, 2020-
Header (metadata) releaseMar 10, 2021-
Map releaseMar 10, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wfq
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21652.png

Downloads & links

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Map

FileDownload / File: emd_21652.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRelion local resolution filtered map. Sharpening B-factor -100
Voxel sizeX=Y=Z: 0.68 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.019632306 - 0.046468157
Average (Standard dev.)0.000044114655 (±0.0021020272)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 152.32 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.680.680.68
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z152.320152.320152.320
α/β/γ90.00090.00090.000
start NX/NY/NZ-170-170-170
NX/NY/NZ340340340
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.0200.0460.000

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Supplemental data

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Mask #1

Fileemd_21652_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #2

Fileemd_21652_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_21652_additional_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_21652_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_21652_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NanR-DNA hetero-complex

EntireName: NanR-DNA hetero-complex
Components
  • Complex: NanR-DNA hetero-complex
    • Protein or peptide: HTH-type transcriptional repressor NanR
    • DNA: DNA (5'-D(P*GP*GP*TP*AP*TP*AP*AP*CP*AP*GP*GP*TP*AP*TP*A)-3')
    • DNA: DNA (5'-D(P*TP*AP*TP*AP*CP*CP*TP*GP*TP*TP*AP*TP*AP*CP*C)-3')

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Supramolecule #1: NanR-DNA hetero-complex

SupramoleculeName: NanR-DNA hetero-complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Dimeric NanR-DNA hetero-complex
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 72.8 KDa

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Macromolecule #1: HTH-type transcriptional repressor NanR

MacromoleculeName: HTH-type transcriptional repressor NanR / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 29.566354 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MGLMNAFDSQ TEDSSPAIGR NLRSRPLARK KLSEMVEEEL EQMIRRREFG EGEQLPSERE LMAFFNVGRP SVREALAALK RKGLVQINN GERARVSRPS ADTIIGELSG MAKDFLSHPG GIAHFEQLRL FFESSLVRYA AEHATDEQID LLAKALEINS Q SLDNNAAF ...String:
MGLMNAFDSQ TEDSSPAIGR NLRSRPLARK KLSEMVEEEL EQMIRRREFG EGEQLPSERE LMAFFNVGRP SVREALAALK RKGLVQINN GERARVSRPS ADTIIGELSG MAKDFLSHPG GIAHFEQLRL FFESSLVRYA AEHATDEQID LLAKALEINS Q SLDNNAAF IRSDVDFHRV LAEIPGNPIF MAIHVALLDW LIAARPTVTD QALHEHNNVS YQQHIAIVDA IRRHDPDEAD RA LQSHLNS VSATWHAFGQ TTNKKK

UniProtKB: HTH-type transcriptional repressor NanR

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Macromolecule #2: DNA (5'-D(P*GP*GP*TP*AP*TP*AP*AP*CP*AP*GP*GP*TP*AP*TP*A)-3')

MacromoleculeName: DNA (5'-D(P*GP*GP*TP*AP*TP*AP*AP*CP*AP*GP*GP*TP*AP*TP*A)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.65706 KDa
SequenceString:
(DG)(DG)(DT)(DA)(DT)(DA)(DA)(DC)(DA)(DG) (DG)(DT)(DA)(DT)(DA)

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Macromolecule #3: DNA (5'-D(P*TP*AP*TP*AP*CP*CP*TP*GP*TP*TP*AP*TP*AP*CP*C)-3')

MacromoleculeName: DNA (5'-D(P*TP*AP*TP*AP*CP*CP*TP*GP*TP*TP*AP*TP*AP*CP*C)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.518959 KDa
SequenceString:
(DT)(DA)(DT)(DA)(DC)(DC)(DT)(DG)(DT)(DT) (DA)(DT)(DA)(DC)(DC)

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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

Concentration0.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
50.0 mMNaClSodium chloridesodium chloride

Details: 20mM Tris-HCL, 150mM NaCl, 100 microM ZnCl2, pH 8.0
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK III
Details: blotting conditions: 3 sec blotting time and -3 blot force..

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 215000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-20 / Number grids imaged: 1 / Number real images: 3465 / Average exposure time: 12.8 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 695465
Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1_beta)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1_beta)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 141663
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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