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- EMDB-13179: Streptomyces coelicolor dATP/ATP-loaded NrdR in complex with its ... -

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Basic information

Entry
Database: EMDB / ID: EMD-13179
TitleStreptomyces coelicolor dATP/ATP-loaded NrdR in complex with its cognate DNA
Map data
Sample
  • Complex: Tetrameric assembly of dATP/ATP-loaded NrdR in complex with its cognate DNA
    • Complex: Transcriptional repressor NrdR
      • Protein or peptide: Transcriptional repressor NrdR
    • Complex: DNA (50-MER)
      • DNA: DNA (50-MER)
      • DNA: DNA (50-MER)
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: ZINC ION
Function / homologyRibonucleotide reductase regulator NrdR-like / ATP-cone domain / ATP cone domain / ATP-cone domain profile. / negative regulation of DNA-templated transcription / DNA binding / zinc ion binding / ATP binding / Transcriptional repressor NrdR
Function and homology information
Biological speciesStreptomyces coelicolor A3(2) (bacteria) / Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.31 Å
AuthorsMartinez-Carranza M / Stenmark P
Funding support Sweden, 5 items
OrganizationGrant numberCountry
Swedish Research Council2018-03406 Sweden
Swedish Research Council2019-01400 Sweden
Wenner-Gren Foundation Sweden
Cancerfonden20 1287 PjF Sweden
Cancerfonden2018/820 Sweden
CitationJournal: Nat Commun / Year: 2022
Title: A nucleotide-sensing oligomerization mechanism that controls NrdR-dependent transcription of ribonucleotide reductases.
Authors: Inna Rozman Grinberg / Markel Martínez-Carranza / Ornella Bimai / Ghada Nouaïria / Saher Shahid / Daniel Lundin / Derek T Logan / Britt-Marie Sjöberg / Pål Stenmark /
Abstract: Ribonucleotide reductase (RNR) is an essential enzyme that catalyzes the synthesis of DNA building blocks in virtually all living cells. NrdR, an RNR-specific repressor, controls the transcription of ...Ribonucleotide reductase (RNR) is an essential enzyme that catalyzes the synthesis of DNA building blocks in virtually all living cells. NrdR, an RNR-specific repressor, controls the transcription of RNR genes and, often, its own, in most bacteria and some archaea. NrdR senses the concentration of nucleotides through its ATP-cone, an evolutionarily mobile domain that also regulates the enzymatic activity of many RNRs, while a Zn-ribbon domain mediates binding to NrdR boxes upstream of and overlapping the transcription start site of RNR genes. Here, we combine biochemical and cryo-EM studies of NrdR from Streptomyces coelicolor to show, at atomic resolution, how NrdR binds to DNA. The suggested mechanism involves an initial dodecamer loaded with two ATP molecules that cannot bind to DNA. When dATP concentrations increase, an octamer forms that is loaded with one molecule each of dATP and ATP per monomer. A tetramer derived from this octamer then binds to DNA and represses transcription of RNR. In many bacteria - including well-known pathogens such as Mycobacterium tuberculosis - NrdR simultaneously controls multiple RNRs and hence DNA synthesis, making it an excellent target for novel antibiotics development.
History
DepositionJul 7, 2021-
Header (metadata) releaseMay 11, 2022-
Map releaseMay 11, 2022-
UpdateJun 8, 2022-
Current statusJun 8, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13179.png

Downloads & links

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Map

FileDownload / File: emd_13179.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.67 Å
Density
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-3.0322328 - 4.968559
Average (Standard dev.)-0.0017668055 (±0.086916134)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 234.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_13179_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_13179_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Tetrameric assembly of dATP/ATP-loaded NrdR in complex with its c...

EntireName: Tetrameric assembly of dATP/ATP-loaded NrdR in complex with its cognate DNA
Components
  • Complex: Tetrameric assembly of dATP/ATP-loaded NrdR in complex with its cognate DNA
    • Complex: Transcriptional repressor NrdR
      • Protein or peptide: Transcriptional repressor NrdR
    • Complex: DNA (50-MER)
      • DNA: DNA (50-MER)
      • DNA: DNA (50-MER)
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: ZINC ION

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Supramolecule #1: Tetrameric assembly of dATP/ATP-loaded NrdR in complex with its c...

SupramoleculeName: Tetrameric assembly of dATP/ATP-loaded NrdR in complex with its cognate DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightExperimental: 125 KDa

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Supramolecule #2: Transcriptional repressor NrdR

SupramoleculeName: Transcriptional repressor NrdR / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Supramolecule #3: DNA (50-MER)

SupramoleculeName: DNA (50-MER) / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
Recombinant expressionOrganism: Synthetic construct (others)

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Macromolecule #1: Transcriptional repressor NrdR

MacromoleculeName: Transcriptional repressor NrdR / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) (bacteria)
Strain: ATCC BAA-471 / A3(2) / M145
Molecular weightTheoretical: 21.271629 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHCPFCRHPD SRVVDSRTTD DGTSIRRRRQ CPDCSRRFTT VETCSLMVVK RSGVTEPFSR TKVINGVRKA CQGRPVTEDA LAQLGQRVE EAVRATGSAE LTTHDVGLAI LGPLQELDLV AYLRFASVYR AFDSLEDFEA AIAELRETTG HPGEEDDTGA G SQENDRGP ...String:
MHCPFCRHPD SRVVDSRTTD DGTSIRRRRQ CPDCSRRFTT VETCSLMVVK RSGVTEPFSR TKVINGVRKA CQGRPVTEDA LAQLGQRVE EAVRATGSAE LTTHDVGLAI LGPLQELDLV AYLRFASVYR AFDSLEDFEA AIAELRETTG HPGEEDDTGA G SQENDRGP TGAGQVPEPA GAADKLAAAL EHHHHHH

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Macromolecule #2: DNA (50-MER)

MacromoleculeName: DNA (50-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
Molecular weightTheoretical: 17.507195 KDa
SequenceString: (DG)(DC)(DC)(DA)(DA)(DT)(DC)(DC)(DC)(DC) (DA)(DC)(DA)(DT)(DC)(DT)(DA)(DG)(DT)(DG) (DG)(DT)(DT)(DG)(DG)(DA)(DT)(DA)(DG) (DC)(DG)(DT)(DG)(DA)(DG)(DC)(DA)(DG)(DC) (DC) (DC)(DA)(DC)(DA)(DA)(DG) ...String:
(DG)(DC)(DC)(DA)(DA)(DT)(DC)(DC)(DC)(DC) (DA)(DC)(DA)(DT)(DC)(DT)(DA)(DG)(DT)(DG) (DG)(DT)(DT)(DG)(DG)(DA)(DT)(DA)(DG) (DC)(DG)(DT)(DG)(DA)(DG)(DC)(DA)(DG)(DC) (DC) (DC)(DA)(DC)(DA)(DA)(DG)(DT)(DT) (DG)(DT)(DG)(DG)(DT)(DC)(DC)(DC)(DC)

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Macromolecule #3: DNA (50-MER)

MacromoleculeName: DNA (50-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
Molecular weightTheoretical: 17.627266 KDa
SequenceString: (DG)(DG)(DG)(DG)(DA)(DC)(DC)(DA)(DC)(DA) (DA)(DC)(DT)(DT)(DG)(DT)(DG)(DG)(DG)(DC) (DT)(DG)(DC)(DT)(DC)(DA)(DC)(DG)(DC) (DT)(DA)(DT)(DC)(DC)(DA)(DA)(DC)(DC)(DA) (DC) (DT)(DA)(DG)(DA)(DT)(DG) ...String:
(DG)(DG)(DG)(DG)(DA)(DC)(DC)(DA)(DC)(DA) (DA)(DC)(DT)(DT)(DG)(DT)(DG)(DG)(DG)(DC) (DT)(DG)(DC)(DT)(DC)(DA)(DC)(DG)(DC) (DT)(DA)(DT)(DC)(DC)(DA)(DA)(DC)(DC)(DA) (DC) (DT)(DA)(DG)(DA)(DT)(DG)(DT)(DG) (DG)(DG)(DG)(DA)(DT)(DT)(DG)(DG)(DC)

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 4 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #5: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 4 / Formula: DTP
Molecular weightTheoretical: 491.182 Da
Chemical component information

ChemComp-DTP:
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / Deoxyadenosine triphosphate

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration0.2 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3562 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 3.1)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.31 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 445937
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7p37


Chain - Chain ID: A / Chain - Residue range: 1-147
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7p3f:
Streptomyces coelicolor dATP/ATP-loaded NrdR in complex with its cognate DNA

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