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- PDB-6mt9: X-ray crystal structure of Bacillus subtilis ribonucleotide reduc... -

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Basic information

Entry
Database: PDB / ID: 6mt9
TitleX-ray crystal structure of Bacillus subtilis ribonucleotide reductase NrdE alpha subunit with TTP, ATP, and ADP
ComponentsRibonucleoside-diphosphate reductaseRibonucleotide reductase
KeywordsOXIDOREDUCTASE / ribonucleotide reductase / allostery / nucleotide metabolism / 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 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 / Anaerobic Ribonucleotide-triphosphate Reductase Large Chain / Anaerobic Ribonucleotide-triphosphate Reductase Large Chain - #20 / Alpha-Beta Barrel / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / THYMIDINE-5'-TRIPHOSPHATE / Ribonucleoside-diphosphate reductase / Ribonucleoside-diphosphate reductase subunit alpha
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.5 Å
AuthorsThomas, W.C. / Brooks, F.P. / Bacik, J.P. / 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 19, 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 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Oct 11, 2023Group: 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 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr2_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ribonucleoside-diphosphate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)82,2325
Polymers80,7911
Non-polymers1,4414
Water3,495194
1
A: Ribonucleoside-diphosphate reductase
hetero molecules

A: Ribonucleoside-diphosphate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)164,46510
Polymers161,5832
Non-polymers2,8828
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation8_555-y,-x,-z+1/21
Buried area6870 Å2
ΔGint-30 kcal/mol
Surface area49470 Å2
MethodPISA
Unit cell
Length a, b, c (Å)126.337, 126.337, 125.436
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Ribonucleoside-diphosphate reductase / Ribonucleotide reductase


Mass: 80791.469 Da / Num. of mol.: 1
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

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Non-polymers , 5 types, 198 molecules

#2: Chemical ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE / Thymidine triphosphate


Mass: 482.168 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O14P3
#3: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#4: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 194 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.1 Å3/Da / Density % sol: 60.29 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.6
Details: The crystal was grown by vapor diffusion from 4.5 mg/ml holo-NrdE in 50 mM HEPES (pH 7.6), 50 mM NaCl, 5 mM MgCl2, 2 mM TCEP, and 1% glycerol, supplemented with 5 mM ATP, 0.5 mM TTP, and 1 ...Details: The crystal was grown by vapor diffusion from 4.5 mg/ml holo-NrdE in 50 mM HEPES (pH 7.6), 50 mM NaCl, 5 mM MgCl2, 2 mM TCEP, and 1% glycerol, supplemented with 5 mM ATP, 0.5 mM TTP, and 1 mM GDP. The protein solution was incubated for 10 minutes with freshly added nucleotides prior to being mixed in a 1:1 hanging drop with a precipitating solution of 6% PEG 3350, 1% w/v tryptone, and 50 mM HEPES at pH 7. Crystals were cryoprotected by soaking for 5-10 seconds in well solution mixed with 8% w/v sucrose, 2% w/v glucose, 8% v/v glycerol, 8% v/v ethylene glycol and supplemented with nucleotides, TCEP, and MgCl2 adjusted to the same concentrations used in the original protein solutions.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: CHESS / Beamline: F1 / Wavelength: 0.9775 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: May 5, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9775 Å / Relative weight: 1
ReflectionResolution: 2.5→16 Å / Num. obs: 35371 / % possible obs: 99.2 % / Redundancy: 17 % / CC1/2: 0.998 / Rmerge(I) obs: 0.175 / Rpim(I) all: 0.041 / Net I/σ(I): 14.7
Reflection shellResolution: 2.5→2.6 Å / Redundancy: 17.6 % / Rmerge(I) obs: 1.445 / Mean I/σ(I) obs: 2.2 / Num. unique obs: 3958 / CC1/2: 0.734 / Rpim(I) all: 0.333 / % possible all: 99.9

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Processing

Software
NameVersionClassification
PHENIX(1.13_2998: ???)refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6CGM

6cgm


Resolution: 2.5→15.998 Å / SU ML: 0.26 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 21.99
RfactorNum. reflection% reflection
Rfree0.2165 2778 7.87 %
Rwork0.1762 --
obs0.1795 35313 99.31 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.5→15.998 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5433 0 88 194 5715
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0065650
X-RAY DIFFRACTIONf_angle_d0.9337655
X-RAY DIFFRACTIONf_dihedral_angle_d20.7652078
X-RAY DIFFRACTIONf_chiral_restr0.048829
X-RAY DIFFRACTIONf_plane_restr0.005971
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.5-2.5430.30861350.24181619X-RAY DIFFRACTION100
2.543-2.5890.29451400.23241600X-RAY DIFFRACTION100
2.589-2.63860.30911340.21911609X-RAY DIFFRACTION100
2.6386-2.69220.2691400.22651606X-RAY DIFFRACTION100
2.6922-2.75050.26561340.2151609X-RAY DIFFRACTION100
2.7505-2.81410.24571340.19891616X-RAY DIFFRACTION100
2.8141-2.88410.24341340.20161619X-RAY DIFFRACTION100
2.8841-2.96160.26651410.19671601X-RAY DIFFRACTION100
2.9616-3.04820.22971390.21551617X-RAY DIFFRACTION100
3.0482-3.14580.2471350.20661630X-RAY DIFFRACTION100
3.1458-3.25740.23391400.2011622X-RAY DIFFRACTION100
3.2574-3.38660.25121390.19171612X-RAY DIFFRACTION100
3.3866-3.53910.22711390.19091627X-RAY DIFFRACTION100
3.5391-3.72350.21891410.17571627X-RAY DIFFRACTION99
3.7235-3.95350.21991400.16291645X-RAY DIFFRACTION100
3.9535-4.25340.20151380.15321626X-RAY DIFFRACTION99
4.2534-4.67180.1881420.13911643X-RAY DIFFRACTION99
4.6718-5.32580.17431380.14781658X-RAY DIFFRACTION99
5.3258-6.62960.1921430.1731655X-RAY DIFFRACTION98
6.6296-15.99870.16561520.14791694X-RAY DIFFRACTION96

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