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- EMDB-50819: Cryo-EM structure of E. coli transcription factor NrdR in complex... -

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Basic information

Entry
Database: EMDB / ID: EMD-50819
TitleCryo-EM structure of E. coli transcription factor NrdR in complex with DNA
Map dataSharpened map from non-uniform refinement in cryoSPARC
Sample
  • Complex: E. coli NrdR/ATP/dATP/DNA
    • Protein or peptide: E. coli NrdR
    • DNA: DNA 57-mer from E. coli with two NrdR boxes
Keywordstranscription factor / ribonucleotide reductase / repressor / ATP-cone / Zn-ribbon / DNA BINDING PROTEIN
Function / homology
Function and homology information


double-stranded DNA binding / negative regulation of DNA-templated transcription / zinc ion binding / ATP binding
Similarity search - Function
Ribonucleotide reductase regulator NrdR-like / : / Transcriptional repressor NrdR-like, N-terminal domain / ATP-cone domain / ATP cone domain / ATP-cone domain profile.
Similarity search - Domain/homology
Transcriptional repressor NrdR
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsBanerjee I / Bimai O / Martinez-Carranza M / Stenmark P / Sjoberg BM / Rozman Grinberg I / Logan DT
Funding support Sweden, United States, 6 items
OrganizationGrant numberCountry
Carl Trygger FoundationCTS 20:361 Sweden
Cancerfonden201210PJF Sweden
Wenner-Gren Foundation United States
Swedish Research Council2016-04855 Sweden
Swedish Research Council2019-03681 Sweden
Swedish Research Council2023-05074 Sweden
CitationJournal: FEBS J / Year: 2025
Title: Bacterial transcriptional repressor NrdR - a flexible multifactorial nucleotide sensor.
Authors: Inna Rozman Grinberg / Ornella Bimaï / Saher Shahid / Lena Philipp / Markel Martínez-Carranza / Ipsita Banerjee / Daniel Lundin / Pål Stenmark / Britt-Marie Sjöberg / Derek T Logan /
Abstract: NrdR is a bacterial transcriptional repressor consisting of a zinc (Zn)-ribbon domain followed by an ATP-cone domain. Understanding its mechanism of action could aid the design of novel ...NrdR is a bacterial transcriptional repressor consisting of a zinc (Zn)-ribbon domain followed by an ATP-cone domain. Understanding its mechanism of action could aid the design of novel antibacterials. NrdR binds specifically to two "NrdR boxes" upstream of ribonucleotide reductase operons, of which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, in the last of which we identified a new box. We show that E. coli NrdR (EcoNrdR) has similar binding strength to all three sites when loaded with ATP plus deoxyadenosine triphosphate (dATP) or equivalent diphosphate combinations. No other combination of adenine nucleotides promotes binding to DNA. We present crystal structures of EcoNrdR-ATP-dATP and EcoNrdR-ADP-dATP, which are the first high-resolution crystal structures of an NrdR. We have also determined cryo-electron microscopy structures of DNA-bound EcoNrdR-ATP-dATP and novel filaments of EcoNrdR-ATP. Tetrameric forms of EcoNrdR involve alternating interactions between pairs of Zn-ribbon domains and ATP-cones. The structures reveal considerable flexibility in relative orientation of ATP-cones vs Zn-ribbon domains. The structure of DNA-bound EcoNrdR-ATP-dATP shows that significant conformational rearrangements between ATP-cones and Zn-ribbons accompany DNA binding while the ATP-cones retain the same relative orientation. In contrast, ATP-loaded EcoNrdR filaments show rearrangements of the ATP-cone pairs and sequester the DNA-binding residues of NrdR such that they are unable to bind to DNA. Our results, in combination with a previous structural and biochemical study, point to highly flexible EcoNrdR structures that, when loaded with the correct nucleotides, adapt to an optimal promoter-binding conformation.
History
DepositionJun 28, 2024-
Header (metadata) releaseApr 9, 2025-
Map releaseApr 9, 2025-
UpdateApr 9, 2025-
Current statusApr 9, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50819.png

Downloads & links

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Map

FileDownload / File: emd_50819.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map from non-uniform refinement in cryoSPARC
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.26 Å/pix.
x 256 pix.
= 322.56 Å		1.26 Å/pix.
x 256 pix.
= 322.56 Å		1.26 Å/pix.
x 256 pix.
= 322.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.26 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08
Minimum - Maximum-0.34972045 - 0.8023758
Average (Standard dev.)-0.00052494986 (±0.014394364)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 322.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Locally sharpened map from DeepEMhancer

Fileemd_50819_additional_1.map
AnnotationLocally sharpened map from DeepEMhancer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B

Fileemd_50819_half_map_1.map
AnnotationHalf-map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A

Fileemd_50819_half_map_2.map
AnnotationHalf-map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : E. coli NrdR/ATP/dATP/DNA

EntireName: E. coli NrdR/ATP/dATP/DNA
Components
  • Complex: E. coli NrdR/ATP/dATP/DNA
    • Protein or peptide: E. coli NrdR
    • DNA: DNA 57-mer from E. coli with two NrdR boxes

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Supramolecule #1: E. coli NrdR/ATP/dATP/DNA

SupramoleculeName: E. coli NrdR/ATP/dATP/DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Tetrameric transcription factor NrdR from E. coli in complex with ATP, dATP and a DNA 57-mer containing two NrdR boxes.
Molecular weightTheoretical: 128 KDa

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Macromolecule #1: E. coli NrdR

MacromoleculeName: E. coli NrdR / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString:
MHCPFCFAVD TKVIDSRLVG EGSSVRRRRQ CLVCNERFTT FEVAELVMPR VVKSNDVREP FNEEKLRSGM LRALEKRPV SSDDVEMAIN HIKSQLRATG EREVPSKMIG NLVMEQLKKL DKVAYIRFAS VYRSFEDIKD F GEEIARLE DKLAAALEHH HHHH

UniProtKB: Transcriptional repressor NrdR

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Macromolecule #2: DNA 57-mer from E. coli with two NrdR boxes

MacromoleculeName: DNA 57-mer from E. coli with two NrdR boxes / type: dna / ID: 2 / Details: DNA 57-mer from E. coli with two NrdR boxes / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString:
GCACTATTTG CTATATATTG TGTGGTTGAA TCTTTTTTCA ACTACATCTA GTATCTC

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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

Concentration0.31 mg/mL
BufferpH: 8.5
Component:
ConcentrationNameFormula
25.0 mMTris-HCl
100.0 mMNaCl
5.0 mMMgCl2MgCl2
1.0 mMTCEP
0.5 mMdATP
0.5 mMATP
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK IV
Details16 uM monomeric protein, 6 uM oligonucleotide

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 14789 / Average electron dose: 58.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.2 µm
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

Detailspixel size 0.63 A
Particle selectionNumber selected: 3400000
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 178716
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC (ver. 4.2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7p3f

chain_id: E, source_name: PDB, initial_model_type: experimental model

7p3f

chain_id: F, source_name: PDB, initial_model_type: experimental model
DetailsThree models were used to fit the best cryo-EM volume: DNA from the S. coelicor NrdR-dATP-ATP-DNA complex, and pairs of ATP-cones and Zn-ribbons from the EcNrdR-ATP-dAMPPNP complex. These were fitted to the volume using Molrep in the order DNA:ATP-cones:Zn ribbons. Domain-wise rigid body real space refinement was then carried out using phenix.refine.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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