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- EMDB-9293: EM structure of Bacillus subtilis ribonucleotide reductase inhibi... -

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Basic information

Entry
Database: EMDB / ID: EMD-9293
TitleEM structure of Bacillus subtilis ribonucleotide reductase inhibited double-helical filament of NrdE alpha subunit with dATP
Map dataASU of Bacillus subtilis ribonucleotide reductase inhibited double-helix filament composed of NrdE alpha subunits with dATP
Sample
  • Complex: Inhibited filament of ribonucleoside-diphosphate reductase composed of NrdE alpha subunit
    • Protein or peptide: Ribonucleoside-diphosphate reductaseRibonucleotide reductase
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
Keywordsribonucleotide 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
Ribonucleotide reductase N-terminal / Ribonucleotide reductase, class 1b, subunit NrdE / Ribonucleotide reductase N-terminal / Ribonucleotide reductase, class I , alpha subunit / Ribonucleotide reductase large subunit signature. / Ribonucleoside-diphosphate reductase large subunit / Ribonucleotide reductase R1 subunit, N-terminal / Ribonucleotide reductase large subunit, N-terminal / Ribonucleotide reductase, all-alpha domain / Ribonucleotide reductase large subunit, C-terminal / Ribonucleotide reductase, barrel domain
Similarity search - Domain/homology
Ribonucleoside-diphosphate reductase / Ribonucleoside-diphosphate reductase subunit alpha
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 6.0 Å
AuthorsThomas WC / Bacik JP
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM124847 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM100008 United States
CitationJournal: 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
DepositionNov 2, 2018-
Header (metadata) releaseNov 14, 2018-
Map releaseJun 19, 2019-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.45
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 4.45
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6myx
  • Surface level: 4.45
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6myx
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9293.png

Downloads & links

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Map

FileDownload / File: emd_9293.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationASU of Bacillus subtilis ribonucleotide reductase inhibited double-helix filament composed of NrdE alpha subunits with dATP
Voxel sizeX=Y=Z: 1.505 Å
Density
Contour LevelBy AUTHOR: 4.45 / Movie #1: 4.45
Minimum - Maximum-4.277737 - 9.984742000000001
Average (Standard dev.)0.30920467 (±1.5092593)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 240.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.5051.5051.505
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z240.800240.800240.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-163-114-126
NX/NY/NZ210124170
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-4.2789.9850.309

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Supplemental data

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Half map: Half-set map #1

Fileemd_9293_half_map_1.map
AnnotationHalf-set map #1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-set map #2

Fileemd_9293_half_map_2.map
AnnotationHalf-set map #2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Inhibited filament of ribonucleoside-diphosphate reductase compos...

EntireName: Inhibited filament of ribonucleoside-diphosphate reductase composed of NrdE alpha subunit
Components
  • Complex: Inhibited filament of ribonucleoside-diphosphate reductase composed of NrdE alpha subunit
    • Protein or peptide: Ribonucleoside-diphosphate reductaseRibonucleotide reductase
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

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Supramolecule #1: Inhibited filament of ribonucleoside-diphosphate reductase compos...

SupramoleculeName: 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.
Source (natural)Organism: Bacillus subtilis (bacteria)

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Macromolecule #1: Ribonucleoside-diphosphate reductase

MacromoleculeName: Ribonucleoside-diphosphate reductase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: ribonucleoside-diphosphate reductase
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 80.791469 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: 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

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Macromolecule #2: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 8 / Formula: DTP
Molecular weightTheoretical: 491.182 Da
Chemical component information

ChemComp-DTP:
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / Deoxyadenosine triphosphate

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.81 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride
15.0 mMMgCl2magnesium chloride
1.0 mMC9H15O6PTCEP

Details: Glycerol in original buffer was diluted to < 0.25% w/v.
GridModel: 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.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 300 K / Instrument: FEI VITROBOT MARK IV / Details: 3.5 seconds blotting.
DetailsSamples 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.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 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 opticsSpherical aberration corrector: None / Chromatic aberration corrector: None
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm 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

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Image processing

Startup modelType of model: NONE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 74.24 Å
Applied symmetry - Helical parameters - Δ&Phi: 81.28 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 4
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6cgl

source_name: PDB, initial_model_type: experimental model

6mt9

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: OTHER / Target criteria: Corellation coefficient
Output model

PDB-6myx:
EM structure of Bacillus subtilis ribonucleotide reductase inhibited double-helical filament of NrdE alpha subunit with dATP

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