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- PDB-7urg: cryo-EM structure of ribonucleotide reductase from Synechococcus ... -

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Basic information

Entry
Database: PDB / ID: 7urg
Titlecryo-EM structure of ribonucleotide reductase from Synechococcus phage S-CBP4 bound with TTP
ComponentsRibonucleotide reductase
KeywordsOXIDOREDUCTASE / ribonucleotide reductase / Synechoccus phage / TTP
Function / homologyRibonucleotide reductase large subunit, C-terminal / Ribonucleotide reductase, barrel domain / DNA replication / THYMIDINE-5'-TRIPHOSPHATE / Ribonucleotide reductase domain-containing protein
Function and homology information
Biological speciesSynechococcus phage S-CBP4 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsXu, D. / Burnim, A.A. / Ando, N.
Funding support United States, 2items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)MCB-1942668 United States
National Science Foundation (NSF, United States)DMR-1719875 United States
CitationJournal: Elife / Year: 2022
Title: Comprehensive phylogenetic analysis of the ribonucleotide reductase family reveals an ancestral clade.
Authors: Audrey A Burnim / Matthew A Spence / Da Xu / Colin J Jackson / Nozomi Ando /
Abstract: Ribonucleotide reductases (RNRs) are used by all free-living organisms and many viruses to catalyze an essential step in the de novo biosynthesis of DNA precursors. RNRs are remarkably diverse by ...Ribonucleotide reductases (RNRs) are used by all free-living organisms and many viruses to catalyze an essential step in the de novo biosynthesis of DNA precursors. RNRs are remarkably diverse by primary sequence and cofactor requirement, while sharing a conserved fold and radical-based mechanism for nucleotide reduction. Here, we structurally aligned the diverse RNR family by the conserved catalytic barrel to reconstruct the first large-scale phylogeny consisting of 6779 sequences that unites all extant classes of the RNR family and performed evo-velocity analysis to independently validate our evolutionary model. With a robust phylogeny in-hand, we uncovered a novel, phylogenetically distinct clade that is placed as ancestral to the classes I and II RNRs, which we have termed clade Ø. We employed small-angle X-ray scattering (SAXS), cryogenic-electron microscopy (cryo-EM), and AlphaFold2 to investigate a member of this clade from phage S-CBP4 and report the most minimal RNR architecture to-date. Based on our analyses, we propose an evolutionary model of diversification in the RNR family and delineate how our phylogeny can be used as a roadmap for targeted future study.
History
DepositionApr 21, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 7, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 14, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Oct 5, 2022Group: Structure summary / Category: struct / Item: _struct.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Ribonucleotide reductase
B: Ribonucleotide reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)103,8084
Polymers102,8432
Non-polymers9642
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS, SAXS data best fit to a dimer model, electron microscopy, Observed dimer with the EM map
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Ribonucleotide reductase


Mass: 51421.672 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus phage S-CBP4 (virus) / Gene: SVPG_00036 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: M1PRZ0
#2: Chemical ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE


Mass: 482.168 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N2O14P3 / 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: Ribonucleotide reductase from Synechoccus phage S-CBP4 bound with TTP
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Synechococcus phage S-CBP4 (virus)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.6
Details: 50 mM HEPES, 150 mM NaCl, 1% v/v glycerol, 7.55 mM MgCl2
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
31 % (w/v)glycerolC3H8O31
47.55 mMmagnesium chlorideMgCl21
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 4 uM ribonucleotide reductase from Synechococcus phage S-CBP4 with 200 uM TTP, 200 uM GDP
Specimen supportDetails: glow discharged on a PELCO easiGlow system for 45 s with 15 mA current
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 4 seconds before plunging

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Details: Data was collected on a Thermo Fisher Talos Arcica Cryo-TEM with a Gatan K3 camera and BioQuantum energy filter.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 79000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingAverage exposure time: 2.164 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 856
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.3.1particle selectionBlot picker and template picker were used to pick the particles.
2SerialEM3.8image acquisition
4cryoSPARC3.3.1CTF correctioncryoSPARC patchCTF was used to estimate CTF parameters.
7PHENIX1.20.1model fittingPHENIX was used to dock the alpha fold model into the EM map.
9cryoSPARC3.3.1initial Euler assignmentfrom cryoSPARC ab initio reconstruction
10cryoSPARC3.3.1final Euler assignmentfrom cryoSPARC non-uniform refinement
12cryoSPARC3.3.13D reconstruction
19PHENIX1.20.1model refinementphenix.real_space_refine was used to refine the docked structure.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 581884
Details: 46 high quality micrographs were then selected, from which the blob picker routine was used to pick particles. The resulting 99k particles were extracted and subjected to 2D classification, ...Details: 46 high quality micrographs were then selected, from which the blob picker routine was used to pick particles. The resulting 99k particles were extracted and subjected to 2D classification, and the top four unique 2D classes were selected and used as templates for template picking on the entire dataset. Due to the large variance in ice conditions in many of our micrographs, masks were manually defined for every micrograph, and particle picks outside the ideal ice region were excluded.
3D reconstructionResolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 107885 / Symmetry type: POINT
Atomic model buildingB value: 74.37 / Protocol: FLEXIBLE FIT / Space: REAL
Details: The sequence for the ribonucleotide reductase from Synechococcus phage S-CBP4 was retrieved from UniProt with accession number M1PRZ0. The sequence was used as input for AlphaFold2 ...Details: The sequence for the ribonucleotide reductase from Synechococcus phage S-CBP4 was retrieved from UniProt with accession number M1PRZ0. The sequence was used as input for AlphaFold2 prediction with the five default model parameters and a template date cutoff of 2020-05-14. As the five models were largely identical in the core region and differing only in the location of the C-terminal tail, the structure predicted with the first model parameter was used in the subsequent process. The predicted structure was first processed and docked into the unsharpened map in phenix. The 25 N-terminal residues and 45 C-terminal residues were then manually removed due to lack of cryo-EM density, and residues 26-426 were retained in the model. We observed unmodeled density at the specificity site, and based on solution composition, we modeled a TTP molecule. The TTP molecule with magnesium ion from the crystal structure of Bacillus subtilis RNR (pdb: 6mt9) was extracted and rigid body fit into the unmodeled density in Coot. The combined model was refined with the unsharpened and sharpened maps using phenix.real_space_refine, with a constraint applied on the magnesium ion coordinated by the triphosphate in TTP according to the original configuration. Residue and loop conformations in the resulting structure were manually adjusted in Coot to maximize fit to map and input for an additional round of real-space refinement in phenix with an additional restraint for the disulfide bond between C30 and C196. Due to poor density of the magnesium ion, it was removed when deposited into PDB.

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