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- PDB-8p2c: Cryo-EM structure of the anaerobic ribonucleotide reductase from ... -

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Basic information

Entry
Database: PDB / ID: 8p2c
TitleCryo-EM structure of the anaerobic ribonucleotide reductase from Prevotella copri in its tetrameric state produced in the presence of dATP and CTP
ComponentsAnaerobic ribonucleoside-triphosphate reductase
KeywordsBIOSYNTHETIC PROTEIN / ribonucleotide reductase glycyl radical enzyme allosteric regulation nucleotide biosynthesis / OXIDOREDUCTASE
Function / homology
Function and homology information


ribonucleoside-triphosphate reductase (thioredoxin) activity / DNA replication / ATP binding
Similarity search - Function
Ribonucleoside-triphosphate reductase, anaerobic / Anaerobic ribonucleoside-triphosphate reductase / ATP-cone domain / ATP cone domain / ATP-cone domain profile.
Similarity search - Domain/homology
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / Anaerobic ribonucleoside-triphosphate reductase
Similarity search - Component
Biological speciesSegatella copri (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.59 Å
AuthorsBanerjee, I. / Bimai, O. / Sjoberg, B.M. / Logan, D.T.
Funding support Sweden, United States, 4items
OrganizationGrant numberCountry
Swedish Research Council2016-04855 Sweden
Swedish Research Council2019-01400 Sweden
CancerfondenCAN 20 1210 PjF Sweden
Wenner-Gren Foundation United States
CitationJournal: 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.
History
DepositionMay 15, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 13, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 25, 2024Group: Data collection / Database references / Refinement description
Category: citation / citation_author ...citation / citation_author / em_3d_fitting_list / em_admin
Item: _citation.journal_abbrev / _citation.journal_volume ..._citation.journal_abbrev / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_3d_fitting_list.initial_refinement_model_id / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Anaerobic ribonucleoside-triphosphate reductase
B: Anaerobic ribonucleoside-triphosphate reductase
C: Anaerobic ribonucleoside-triphosphate reductase
D: Anaerobic ribonucleoside-triphosphate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)344,53620
Polymers338,5444
Non-polymers5,99116
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Anaerobic ribonucleoside-triphosphate reductase


Mass: 84636.055 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Segatella copri (bacteria) / Gene: BN510_01369 / Production host: Escherichia coli (E. coli) / References: UniProt: R6BY16
#2: Chemical
ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE


Mass: 491.182 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H16N5O12P3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestY

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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: Anaerobic ribonucleotide reductase from Prevotella copri in its tetrameric, dATP/CTP-bound state
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.3368 MDa / Experimental value: NO
Source (natural)Organism: Prevotella copri (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPES bufferHEPES-NaOH1
2100 mMpotassium chlorideKCl1
30.5 mMtris(2-carboxyethyl)phosphineTCEP1
410 mMmagnesium chlorideMgCl21
50.5 mMdeoxyadenosine triphosphatedATP1
60.5 mMcytosine triphosphateCTP1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot force 1, 5s blot time

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 38 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11780
EM imaging opticsEnergyfilter name: GIF Bioquantum
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.2.0particle selection
2EPU2.8.1image acquisition
4cryoSPARC3.2.0CTF correction
7Coot0.9.8.7model fitting
9cryoSPARC3.3.2initial Euler assignment
10cryoSPARC4.1.2final Euler assignment
11cryoSPARC3.3.2classification
12cryoSPARC4.2.13D reconstruction
13PHENIX1.19.2-4158model refinement
CTF correctionDetails: Patch CTF correction in cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5762760
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1105348 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: 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 buildingPDB-ID: 8P27

8p27


Accession code: 8P27 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221604
ELECTRON MICROSCOPYf_angle_d0.48129194
ELECTRON MICROSCOPYf_dihedral_angle_d11.718032
ELECTRON MICROSCOPYf_chiral_restr0.0413134
ELECTRON MICROSCOPYf_plane_restr0.0033717

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