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Yorodumi- PDB-7urg: cryo-EM structure of ribonucleotide reductase from Synechococcus ... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7urg | |||||||||
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Title | cryo-EM structure of ribonucleotide reductase from Synechococcus phage S-CBP4 bound with TTP | |||||||||
Components | Ribonucleotide reductase | |||||||||
Keywords | OXIDOREDUCTASE / ribonucleotide reductase / Synechoccus phage / TTP | |||||||||
Function / homology | Ribonucleotide reductase large subunit, C-terminal / Ribonucleotide reductase, barrel domain / DNA replication / THYMIDINE-5'-TRIPHOSPHATE / Ribonucleotide reductase domain-containing protein Function and homology information | |||||||||
Biological species | Synechococcus phage S-CBP4 (virus) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.46 Å | |||||||||
Authors | Xu, D. / Burnim, A.A. / Ando, N. | |||||||||
Funding support | United States, 2items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7urg.cif.gz | 161.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7urg.ent.gz | 128.7 KB | Display | PDB format |
PDBx/mmJSON format | 7urg.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7urg_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 7urg_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 7urg_validation.xml.gz | 38.3 KB | Display | |
Data in CIF | 7urg_validation.cif.gz | 52.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ur/7urg ftp://data.pdbj.org/pub/pdb/validation_reports/ur/7urg | HTTPS FTP |
-Related structure data
Related structure data | 26712MC 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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1 |
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-Components
#1: Protein | 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 | 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: Ribonucleotide reductase from Synechoccus phage S-CBP4 bound with TTP Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT | |||||||||||||||||||||||||
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Source (natural) | Organism: Synechococcus phage S-CBP4 (virus) | |||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) | |||||||||||||||||||||||||
Buffer solution | pH: 7.6 Details: 50 mM HEPES, 150 mM NaCl, 1% v/v glycerol, 7.55 mM MgCl2 | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 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 support | Details: 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 | |||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 4 seconds before plunging |
-Electron microscopy imaging
Microscopy | Model: TFS TALOS Details: Data was collected on a Thermo Fisher Talos Arcica Cryo-TEM with a Gatan K3 camera and BioQuantum energy filter. |
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Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: 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 holder | Cryogen: NITROGEN / Specimen holder model: OTHER |
Image recording | Average 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 optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. 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 reconstruction | Resolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 107885 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B 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. |