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- PDB-8p23: Cryo-EM structure of the anaerobic ribonucleotide reductase from ... -

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Basic information

Entry
Database: PDB / ID: 8p23
TitleCryo-EM structure of the anaerobic ribonucleotide reductase from Prevotella copri in its dimeric, ATP/CTP-bound state
ComponentsAnaerobic ribonucleoside-triphosphate reductase
KeywordsBIOSYNTHETIC 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
Ribonucleoside-triphosphate reductase, anaerobic / Anaerobic ribonucleoside-triphosphate reductase / ATP-cone domain / ATP cone domain / ATP-cone domain profile.
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / CYTIDINE-5'-TRIPHOSPHATE / Anaerobic ribonucleoside-triphosphate reductase
Similarity search - Component
Biological speciesPrevotella copri (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.17 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
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
DepositionMay 14, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 30, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 25, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)171,8498
Polymers169,2722
Non-polymers2,5776
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Anaerobic ribonucleoside-triphosphate reductase


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 / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#3: Chemical ChemComp-CTP / CYTIDINE-5'-TRIPHOSPHATE


Mass: 483.156 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H16N3O14P3 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
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 dimeric, ATP/CTP-bound state
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.1684 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 triphosphateATP1
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: 2400 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: 2.1 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 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
9PHENIX1.19.2-4158model refinement
10cryoSPARC3.3.2initial Euler assignment
11cryoSPARC4.1.2final Euler assignment
12cryoSPARC3.3.2classification
13cryoSPARC3D reconstruction
CTF correctionDetails: Patch CTF correction in cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 14132328
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 291231 / Num. of class averages: 8 / 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 buildingDetails: Half of a previously-built tetrameric form of the same enzyme
Source name: Other / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00211623
ELECTRON MICROSCOPYf_angle_d0.50215711
ELECTRON MICROSCOPYf_dihedral_angle_d10.8344317
ELECTRON MICROSCOPYf_chiral_restr0.0461706
ELECTRON MICROSCOPYf_plane_restr0.0042013

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