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Yorodumi- PDB-8p23: Cryo-EM structure of the anaerobic ribonucleotide reductase from ... -
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-Basic information
Entry | Database: PDB / ID: 8p23 | |||||||||||||||
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Title | Cryo-EM structure of the anaerobic ribonucleotide reductase from Prevotella copri in its dimeric, ATP/CTP-bound state | |||||||||||||||
Components | Anaerobic ribonucleoside-triphosphate reductase | |||||||||||||||
Keywords | BIOSYNTHETIC PROTEIN / ribonucleotide reductase glycyl radical enzyme allosteric regulation nucleotide biosynthesis | |||||||||||||||
Function / homology | Function and homology information ribonucleoside-triphosphate reductase (thioredoxin) activity / DNA replication / ATP binding Similarity search - Function | |||||||||||||||
Biological species | Prevotella copri (bacteria) | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.17 Å | |||||||||||||||
Authors | Banerjee, I. / Bimai, O. / Sjoberg, B.M. / Logan, D.T. | |||||||||||||||
Funding support | Sweden, United States, 4items
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Citation | Journal: Elife / Year: 2024 Title: Nucleotide binding to the ATP-cone in anaerobic ribonucleotide reductases allosterically regulates activity by modulating substrate binding. Authors: Ornella Bimai / Ipsita Banerjee / Inna Rozman Grinberg / Ping Huang / Lucas Hultgren / Simon Ekström / Daniel Lundin / Britt-Marie Sjöberg / Derek T Logan / Abstract: A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine ...A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerisation and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical, and structural information on the effects of ATP and dATP binding to the anaerobic RNR from . The enzyme exists in a dimer-tetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP-cone and tetramers when two dATP molecules are bound. In the presence of ATP, NrdD is active and has a fully ordered glycyl radical domain (GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it cannot be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 Å away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation. #1: Journal: Elife / Year: 2023 Title: Activity modulation in anaerobic ribonucleotide reductase: nucleotide binding to the ATP-cone mediates long-range order-disorder transitions in the active site Authors: Bimai, O. / Banerjee, I. / Rozman Grinberg, I. / Huang, P. / Lundin, D. / Sjoberg, B.M. / Logan, D.T. | |||||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8p23.cif.gz | 259.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8p23.ent.gz | 206.8 KB | Display | PDB format |
PDBx/mmJSON format | 8p23.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8p23_validation.pdf.gz | 1.4 MB | Display | wwPDB validaton report |
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Full document | 8p23_full_validation.pdf.gz | 1.4 MB | Display | |
Data in XML | 8p23_validation.xml.gz | 54.7 KB | Display | |
Data in CIF | 8p23_validation.cif.gz | 79.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/p2/8p23 ftp://data.pdbj.org/pub/pdb/validation_reports/p2/8p23 | HTTPS FTP |
-Related structure data
Related structure data | 17357MC 8p27C 8p28C 8p2cC 8p2dC 8p2sC 8p39C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 84636.055 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Prevotella copri (bacteria) / Gene: BN510_01369 / Production host: Escherichia coli (E. coli) / References: UniProt: R6BY16 #2: Chemical | ChemComp-ATP / #3: Chemical | ChemComp-CTP / | #4: Chemical | ChemComp-ZN / | Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Anaerobic ribonucleotide reductase from Prevotella copri in its dimeric, ATP/CTP-bound state Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT | |||||||||||||||||||||||||||||||||||
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Molecular weight | Value: 0.1684 MDa / Experimental value: NO | |||||||||||||||||||||||||||||||||||
Source (natural) | Organism: Prevotella copri (bacteria) | |||||||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) | |||||||||||||||||||||||||||||||||||
Buffer solution | pH: 7.5 | |||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1 | |||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot force 1, 5s blot time |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 2.1 sec. / Electron dose: 48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 17033 |
EM imaging optics | Energyfilter name: GIF Bioquantum |
Image scans | Width: 5760 / Height: 4092 |
-Processing
EM software |
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CTF correction | Details: Patch CTF correction in cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 14132328 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 291231 / Num. of class averages: 8 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: correlation coefficient Details: Manual fitting was done using Coot and automatic real space refinement used phenix.refine | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Details: Half of a previously-built tetrameric form of the same enzyme Source name: Other / Type: experimental model | ||||||||||||||||||||||||||||||||||||||||
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