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- PDB-6mw3: EM structure of Bacillus subtilis ribonucleotide reductase inhibi... -

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Basic information

Entry
Database: PDB / ID: 6mw3
TitleEM structure of Bacillus subtilis ribonucleotide reductase inhibited filament composed of NrdE alpha subunit and NrdF beta subunit with dATP
Components
  • Ribonucleoside-diphosphate reductase NrdF beta subunit
  • Ribonucleoside-diphosphate reductaseRibonucleotide reductase
KeywordsOXIDOREDUCTASE / 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: 4.65 Å
AuthorsThomas, W.C. / Bacik, J.P. / Kaelber, J.T. / 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
DepositionOct 29, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 19, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 26, 2019Group: Data collection / Database references / Structure summary
Category: audit_author / citation / citation_author
Item: _audit_author.identifier_ORCID / _audit_author.name ..._audit_author.identifier_ORCID / _audit_author.name / _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 1.2Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _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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Assembly

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


Theoretical massNumber of molelcules
Total (without water)164,9458
Polymers162,9814
Non-polymers1,9654
Water0
1
C: Ribonucleoside-diphosphate reductase
D: Ribonucleoside-diphosphate reductase
I: Ribonucleoside-diphosphate reductase NrdF beta subunit
J: Ribonucleoside-diphosphate reductase NrdF beta subunit
hetero molecules
x 6


Theoretical massNumber of molelcules
Total (without water)989,67248
Polymers977,88424
Non-polymers11,78824
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
helical symmetry operation5
2


  • Idetical with deposited unit
  • helical asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: yes / N subunits divisor: 1 / Num. of operations: 6 / Rise per n subunits: 73.8 Å / Rotation per n subunits: 88.6 °)

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Components

#1: Protein Ribonucleoside-diphosphate reductase / Ribonucleotide reductase


Mass: 80791.469 Da / Num. of mol.: 2
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 (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: A0A162Q3J9, UniProt: P50620*PLUS, ribonucleoside-diphosphate reductase
#2: Protein/peptide Ribonucleoside-diphosphate reductase NrdF beta subunit


Mass: 698.854 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: C-terminus modeled as a polyalanine chain / Source: (gene. exp.) Bacillus subtilis (bacteria) / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
#3: Chemical
ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / Deoxyadenosine triphosphate


Mass: 491.182 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O12P3
Sequence detailsB. subtilis Ribonucleoside-diphosphate reductase beta subunit (NrdF) is modeled as an 8-residue ...B. subtilis Ribonucleoside-diphosphate reductase beta subunit (NrdF) is modeled as an 8-residue polyA chain. The complete sequence is provided below: MGSSHHHHHHSSGLVPRGSHMVTKIYDAANWSKHEDDFTQMFYNQNVKQFWLPEEIALNGDLLTWKYL GKNEQDTYMKVLAGLTLLDTEQGNTGMPIVAEHVDGHQRKAVLNFMAMMENAVHAKSYSNIFMTLAPT ETINEVFEWVKQNKYLQKKAQMIVGLYKAIQKDDEISLFKAMVASVYLESFLFYSGFYYPLYFYGQGK LMQSGEIINLILRDEAIHGVYVGLLAQEIYNKQTEEKKAELREFAIDLLNQLYENELEYTEDLYDQVG LSHDVKKFIRYNANKALMNLGFDPYFEEEDINPIVLNGLNTKTKSHDFFSMKGNGYKKATVEPLKDDD FYFEDEKEQI

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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 subunits and NrdF beta subunit tails
Type: COMPLEX
Details: Beta subunit core density only visible at low threshold. Beta subunit tail is bound with strong density to alpha subunit and modeled as a poly-A peptide in the model.
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7.6
Details: Glycerol in original storage buffer was diluted to < 0.25% w/v.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaClSodium chloride1
315 mMmagnesium chlorideMgCl21
41 mMTCEPC9H15O6P1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Cryo-EM samples of the NrdEF filament were prepared by mixing 20 uM C382S holo-NrdE with 20 or 40 uM Mn-reconstituted NrdF in assay buffer with 100 uM dATP and 1 mM CDP, prior to dilution ...Details: Cryo-EM samples of the NrdEF filament were prepared by mixing 20 uM C382S holo-NrdE with 20 or 40 uM Mn-reconstituted NrdF in assay buffer with 100 uM dATP and 1 mM CDP, prior to dilution with nucleotide-containing buffer to a concentration of 5 uM protein. A subset of the grids were pre-coated with a support film of continuous, amorphous carbon by flotation of cleaved mica. For these grids, the sample was diluted to a final protein concentration of 2 uM.
Specimen supportDetails: unspecified
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 %

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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 FIELDBright-field microscopy / Nominal magnification: 130000 X / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2843
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4cryoSPARC2.0.27CTF correction
7PHENIXmodel fitting
10cryoSPARC2.0.27final Euler assignment
11cryoSPARC2.0.27classification
12cryoSPARC2.0.273D reconstruction
13PHENIXmodel refinement
14Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 88.6 ° / Axial rise/subunit: 73.8 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 281591
3D reconstructionResolution: 4.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 126224 / Algorithm: FOURIER SPACE / Symmetry type: HELICAL
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6CGN

6cgn


Accession code: 6CGN / Source name: PDB / Type: experimental model

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