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- PDB-6myx: EM structure of Bacillus subtilis ribonucleotide reductase inhibi... -

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Basic information

Entry
Database: PDB / ID: 6myx
TitleEM structure of Bacillus subtilis ribonucleotide reductase inhibited double-helical filament of NrdE alpha subunit with dATP
ComponentsRibonucleoside-diphosphate reductase
KeywordsOXIDOREDUCTASE / PROTEIN FIBRIL / ribonucleotide reductase / allostery / nucleotide metabolism / filament / dATP / ATP
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
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / Ribonucleoside-diphosphate reductase / Ribonucleoside-diphosphate reductase subunit alpha
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 6 Å
AuthorsThomas, W.C. / Bacik, J.P. / Chen, J.Z. / Ando, N.
Funding support United States, 2items
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, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 19, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 26, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 13, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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  • Biological unit as representative helical assembly
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Structure viewerMolecule:
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Assembly

Deposited unit
C: Ribonucleoside-diphosphate reductase
D: Ribonucleoside-diphosphate reductase
I: Ribonucleoside-diphosphate reductase
J: Ribonucleoside-diphosphate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)327,09512
Polymers323,1664
Non-polymers3,9298
Water00
1
C: Ribonucleoside-diphosphate reductase
D: Ribonucleoside-diphosphate reductase
I: Ribonucleoside-diphosphate reductase
J: Ribonucleoside-diphosphate reductase
hetero molecules

C: Ribonucleoside-diphosphate reductase
D: Ribonucleoside-diphosphate reductase
I: Ribonucleoside-diphosphate reductase
J: Ribonucleoside-diphosphate reductase
hetero molecules

C: Ribonucleoside-diphosphate reductase
D: Ribonucleoside-diphosphate reductase
I: Ribonucleoside-diphosphate reductase
J: Ribonucleoside-diphosphate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)981,28636
Polymers969,49812
Non-polymers11,78824
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
helical symmetry operation2
2


  • Idetical with deposited unit
  • helical asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 3 / Rise per n subunits: 74.24 Å / Rotation per n subunits: 81.28 °)

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Components

#1: Protein
Ribonucleoside-diphosphate reductase


Mass: 80791.469 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: nrdE_1, B4417_3413, NCTC3610_03984 / Plasmid: pE-SUMO / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A162Q3J9, UniProt: P50620*PLUS, ribonucleoside-diphosphate reductase
#2: Chemical
ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE


Mass: 491.182 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H16N5O12P3

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Inhibited filament of ribonucleoside-diphosphate reductase composed of NrdE alpha subunit
Type: COMPLEX
Details: The filament is a double helix. Each helix is composed of NrdE subunits dimerizing at alternating canonical and non-canonical interfaces.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.6
Details: Glycerol in original buffer was diluted to < 0.25% w/v.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
315 mMmagnesium chlorideMgCl21
41 mMTCEPC9H15O6P1
SpecimenConc.: 0.81 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 ...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.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 300 K / Details: 3.5 seconds blotting

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm / Calibrated defocus min: 1200 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 20 sec. / Electron dose: 20 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 500
EM imaging opticsChromatic aberration corrector: None / Spherical aberration corrector: None
Image scansSampling size: 5 µm / Width: 4000 / Height: 4000 / Movie frames/image: 100 / Used frames/image: 2-90

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM4image acquisition
7PHENIXmodel fitting
9PHENIXmodel refinement
10Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 81.28 ° / Axial rise/subunit: 74.24 Å / Axial symmetry: C1
3D reconstructionResolution: 6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4 / Symmetry type: HELICAL
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: Corellation coefficient
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16CGL

6cgl

16CGL1PDBexperimental model
26MT9

6mt9

16MT92PDBexperimental model

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