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Yorodumi- EMDB-9293: EM structure of Bacillus subtilis ribonucleotide reductase inhibi... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-9293 | |||||||||
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Title | EM structure of Bacillus subtilis ribonucleotide reductase inhibited double-helical filament of NrdE alpha subunit with dATP | |||||||||
Map data | ASU of Bacillus subtilis ribonucleotide reductase inhibited double-helix filament composed of NrdE alpha subunits with dATP | |||||||||
Sample |
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Keywords | ribonucleotide reductase / allostery / nucleotide metabolism / filament / dATP / ATP / OXIDOREDUCTASE / PROTEIN FIBRIL | |||||||||
Function / homology | Function and homology information ribonucleoside-diphosphate reductase complex / ribonucleoside-diphosphate reductase / ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor / deoxyribonucleotide biosynthetic process / DNA replication / ATP binding Similarity search - Function | |||||||||
Biological species | Bacillus subtilis (bacteria) | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 6.0 Å | |||||||||
Authors | Thomas WC / Bacik JP | |||||||||
Funding support | United States, 2 items
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Citation | Journal: Nat Commun / Year: 2019 Title: Convergent allostery in ribonucleotide reductase. Authors: William C Thomas / F Phil Brooks / Audrey A Burnim / John-Paul Bacik / JoAnne Stubbe / Jason T Kaelber / James Z Chen / Nozomi Ando / Abstract: Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for ...Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for being largely restricted to bacteria, including many pathogens, and for lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report the emergence of a distinct and unexpected mechanism of activity regulation in the sole RNR of the model organism Bacillus subtilis. Using a hypothesis-driven structural approach that combines the strengths of small-angle X-ray scattering (SAXS), crystallography, and cryo-electron microscopy (cryo-EM), we describe the reversible interconversion of six unique structures, including a flexible active tetramer and two inhibited helical filaments. These structures reveal the conformational gymnastics necessary for RNR activity and the molecular basis for its control via an evolutionarily convergent form of allostery. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_9293.map.gz | 14.6 MB | EMDB map data format | |
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Header (meta data) | emd-9293-v30.xml emd-9293.xml | 20.1 KB 20.1 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_9293_fsc.xml | 10.7 KB | Display | FSC data file |
Images | emd_9293.png | 44 KB | ||
Filedesc metadata | emd-9293.cif.gz | 7 KB | ||
Others | emd_9293_half_map_1.map.gz emd_9293_half_map_2.map.gz | 59.5 MB 59.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-9293 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-9293 | HTTPS FTP |
-Related structure data
Related structure data | 6myxMC 9272C 6mt9C 6mv9C 6mveC 6mw3C C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_9293.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | ASU of Bacillus subtilis ribonucleotide reductase inhibited double-helix filament composed of NrdE alpha subunits with dATP | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.505 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Half map: Half-set map #1
File | emd_9293_half_map_1.map | ||||||||||||
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Annotation | Half-set map #1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-set map #2
File | emd_9293_half_map_2.map | ||||||||||||
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Annotation | Half-set map #2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Inhibited filament of ribonucleoside-diphosphate reductase compos...
Entire | Name: Inhibited filament of ribonucleoside-diphosphate reductase composed of NrdE alpha subunit |
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Components |
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-Supramolecule #1: Inhibited filament of ribonucleoside-diphosphate reductase compos...
Supramolecule | Name: Inhibited filament of ribonucleoside-diphosphate reductase composed of NrdE alpha subunit type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 Details: The filament is a double helix. Each helix is composed of NrdE subunits dimerizing at alternating canonical and non-canonical interfaces. |
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Source (natural) | Organism: Bacillus subtilis (bacteria) |
-Macromolecule #1: Ribonucleoside-diphosphate reductase
Macromolecule | Name: Ribonucleoside-diphosphate reductase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: ribonucleoside-diphosphate reductase |
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Source (natural) | Organism: Bacillus subtilis (bacteria) |
Molecular weight | Theoretical: 80.791469 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MSQNQVPKWI QLNNEIMIQK DGKFQFDKDK EAVHSYFVDY INQNTVFFHN LKEKLDYLVE NQYYEEEFLS LYSFEDIKEV FKTAYAKKF RFPSFMSAFK FYNDYALKTN DKKKILERYE DRISIVALFF ANGDTEKAKE YVNLMINQEY QPSTPTFLNA G RKRRGELV ...String: MSQNQVPKWI QLNNEIMIQK DGKFQFDKDK EAVHSYFVDY INQNTVFFHN LKEKLDYLVE NQYYEEEFLS LYSFEDIKEV FKTAYAKKF RFPSFMSAFK FYNDYALKTN DKKKILERYE DRISIVALFF ANGDTEKAKE YVNLMINQEY QPSTPTFLNA G RKRRGELV SCFLLEVNDS LNDISRAIDI SMQLSKLGGG VSLNLSKLRA KGEAIKDVEN ATKGVVGVMK LLDNAFRYAD QM GQRQGSG AAYLNIFHRD INDFLDTKKI SADEDVRVKT LSIGVVIPDK FVELAREDKA AYVFYPHTIY KEYGQHMDEM DMN EMYDKF VDNPRVKKEK INPRKLLEKL AMLRSESGYP YIMFQDNVNK VHANNHISKV KFSNLCSEVL QASQVSSYTD YDEE DEIGL DISCNLGSLN ILNVMEHKSI EKTVKLATDS LTHVSETTDI RNAPAVRRAN KAMKSIGLGA MNLHGYLAQN GIAYE SPEA RDFANTFFMM VNFYSIQRSA EIAKEKGETF DQYEGSTYAT GEYFDKYVST DFSPKYEKIA NLFEGMHIPT TEDWKK LKA FVAEHGMYHS YRLCIAPTGS ISYVQSSTAS VMPIMERIEE RTYGNSKTYY PMPGLASNNW FFYKEAYDMD MFKVVDM IA TIQQHIDQGI SFTLFLKDTM TTRDLNRIDL YAHHRGIKTI YYARTKDTGQ DSCLSCVV UniProtKB: Ribonucleoside-diphosphate reductase |
-Macromolecule #2: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
Macromolecule | Name: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 8 / Formula: DTP |
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Molecular weight | Theoretical: 491.182 Da |
Chemical component information | ChemComp-DTP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | filament |
-Sample preparation
Concentration | 0.81 mg/mL | |||||||||||||||
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Buffer | pH: 7.6 Component:
Details: Glycerol in original buffer was diluted to < 0.25% w/v. | |||||||||||||||
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 300 K / Instrument: FEI VITROBOT MARK IV / Details: 3.5 seconds blotting. | |||||||||||||||
Details | Samples of the dATP-induced NrdE filament were produced by incubating 40 uM holo-NrdE with 100 uM dATP and 1 mM CDP in assay buffer. The mixture was then diluted to 10 uM NrdE in the same nucleotide-containing buffer. |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.0 µm / Calibrated defocus min: 1.2 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.2 µm |
Specialist optics | Spherical aberration corrector: None / Chromatic aberration corrector: None |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4000 pixel / Digitization - Dimensions - Height: 4000 pixel / Digitization - Frames/image: 2-90 / Number grids imaged: 2 / Number real images: 500 / Average exposure time: 20.0 sec. / Average electron dose: 20.0 e/Å2 |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
-Image processing
-Atomic model buiding 1
Initial model |
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Refinement | Space: REAL / Protocol: OTHER / Target criteria: Corellation coefficient | ||||||
Output model | PDB-6myx: |