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- EMDB-26712: cryo-EM structure of ribonucleotide reductase from Synechococcus ... -

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Basic information

Entry
Database: EMDB / ID: EMD-26712
Titlecryo-EM structure of ribonucleotide reductase from Synechococcus phage S-CBP4 bound with TTP
Map dataSharpened map from the reconstruction with C2 symmetry
Sample
  • Complex: Ribonucleotide reductase from Synechoccus phage S-CBP4 bound with TTP
    • Protein or peptide: Ribonucleotide reductase
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
Function / homologyRibonucleotide reductase large subunit, C-terminal / Ribonucleotide reductase, barrel domain / DNA replication / Ribonucleotide reductase domain-containing protein
Function and homology information
Biological speciesSynechococcus phage S-CBP4 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsXu D / Burnim AA / Ando N
Funding support United States, 2 items
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, 2022-
Header (metadata) releaseSep 7, 2022-
Map releaseSep 7, 2022-
UpdateOct 5, 2022-
Current statusOct 5, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26712.png

Downloads & links

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Map

FileDownload / File: emd_26712.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map from the reconstruction with C2 symmetry
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 2.3
Minimum - Maximum-15.769749 - 20.068428
Average (Standard dev.)-0.019359138 (±0.30192015)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A from the reconstruction with C2 symmetry

Fileemd_26712_half_map_1.map
AnnotationHalf map A from the reconstruction with C2 symmetry
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B from the reconstruction with C2 symmetry

Fileemd_26712_half_map_2.map
AnnotationHalf map B from the reconstruction with C2 symmetry
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ribonucleotide reductase from Synechoccus phage S-CBP4 bound with TTP

EntireName: Ribonucleotide reductase from Synechoccus phage S-CBP4 bound with TTP
Components
  • Complex: Ribonucleotide reductase from Synechoccus phage S-CBP4 bound with TTP
    • Protein or peptide: Ribonucleotide reductase
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE

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Supramolecule #1: Ribonucleotide reductase from Synechoccus phage S-CBP4 bound with TTP

SupramoleculeName: Ribonucleotide reductase from Synechoccus phage S-CBP4 bound with TTP
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Synechococcus phage S-CBP4 (virus)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: Ribonucleotide reductase

MacromoleculeName: Ribonucleotide reductase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Synechococcus phage S-CBP4 (virus)
Molecular weightTheoretical: 51.421672 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSKPPKELIA RTGRVQSWID DPTSRLPVSC TVFVVEDTME GENGIEASWR FVSHALRYGA GVAVHLSKLR PKGAENGKGL VASGPVSFA KIYSTLNEIL RRGGVYKNGA VVCHLDLSHP DVLEFITASR SELPWVKRCV NINDHWWKEA TPTVKNALLE G IKRGDIWL ...String:
MSKPPKELIA RTGRVQSWID DPTSRLPVSC TVFVVEDTME GENGIEASWR FVSHALRYGA GVAVHLSKLR PKGAENGKGL VASGPVSFA KIYSTLNEIL RRGGVYKNGA VVCHLDLSHP DVLEFITASR SELPWVKRCV NINDHWWKEA TPTVKNALLE G IKRGDIWL NKTKVDRNGN RIRGNVCLEV YLPSRGTCLL QHVNLGGCEL DEIRGAFAQG MSELCELHGK TNVGESGEYL PS ETDRQVG LGMLGLANLL RTQGVTYNDF GRALEALNSG RPYPSTPGYV IAQELKAGIQ AAAEIAKANK MERAFAIAPT ASC SYRYTD LDGYTTCPEI APPIARQVDR DSGTFGVQSF DYGPVEIASE VGWESYKRVV DGIIRLLDST GLLHGYSFNS WSDV VTYDE QFIEDWLASP QTSLYYSLQV MGDVQDKSDA YAALDDGDVT AYLESLLNDP VGASPPLAPD CNCGE

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Macromolecule #2: THYMIDINE-5'-TRIPHOSPHATE

MacromoleculeName: THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 2 / Formula: TTP
Molecular weightTheoretical: 482.168 Da
Chemical component information

ChemComp-TTP:
THYMIDINE-5'-TRIPHOSPHATE / Thymidine triphosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride
1.0 % (w/v)C3H8O3glycerol
7.55 mMMgCl2magnesium chloride

Details: 50 mM HEPES, 150 mM NaCl, 1% v/v glycerol, 7.55 mM MgCl2
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.039 kPa
Details: glow discharged on a PELCO easiGlow system for 45 s with 15 mA current
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 4 seconds before plunging.
Details4 uM ribonucleotide reductase from Synechococcus phage S-CBP4 with 200 uM TTP, 200 uM GDP

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Electron microscopy

MicroscopeTFS TALOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 79000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
SoftwareName: SerialEM (ver. 3.8)
DetailsData was collected on a Thermo Fisher Talos Arcica Cryo-TEM with a Gatan K3 camera and BioQuantum energy filter.
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 856 / Average exposure time: 2.164 sec. / Average electron dose: 50.0 e/Å2

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Image processing

Particle selectionNumber 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.
CTF correctionSoftware - Name: cryoSPARC (ver. 3.3.1)
Software - details: cryoSPARC patchCTF was used to estimate CTF parameters.
Startup modelType of model: OTHER
Details: obtained from ab initial reconstruction in cryoSPARC
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.3.1) / Software - details: from cryoSPARC ab initio reconstruction / Details: from ab into result
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1) / Software - details: from cryoSPARC non-uniform refinement / Details: using cryoSPARC non-uniform refinement
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 107885
FSC plot (resolution estimation)

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Atomic model buiding 1

SoftwareName: PHENIX (ver. 1.20.1)
Details: PHENIX was used to dock the alpha fold model into the EM map.
DetailsThe 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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 74.37
Output model

PDB-7urg:
cryo-EM structure of ribonucleotide reductase from Synechococcus phage S-CBP4 bound with TTP

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