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- PDB-6aui: Human ribonucleotide reductase large subunit (alpha) with dATP and CDP -

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Basic information

Entry
Database: PDB / ID: 6aui
TitleHuman ribonucleotide reductase large subunit (alpha) with dATP and CDP
ComponentsRibonucleoside-diphosphate reductase large subunitRibonucleotide reductase
KeywordsOXIDOREDUCTASE / Ribonucleotide Reductase Electron transfer Radical chemistry Thiyl radical
Function / homology
Function and homology information


ribonucleoside-diphosphate reductase activity / pyrimidine nucleobase metabolic process / cell proliferation in forebrain / positive regulation of G0 to G1 transition / mitochondrial DNA replication / ribonucleoside diphosphate metabolic process / 2'-deoxyribonucleotide biosynthetic process / ribonucleoside-diphosphate reductase complex / ribonucleoside-diphosphate reductase / ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor ...ribonucleoside-diphosphate reductase activity / pyrimidine nucleobase metabolic process / cell proliferation in forebrain / positive regulation of G0 to G1 transition / mitochondrial DNA replication / ribonucleoside diphosphate metabolic process / 2'-deoxyribonucleotide biosynthetic process / ribonucleoside-diphosphate reductase complex / ribonucleoside-diphosphate reductase / ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor / Interconversion of nucleotide di- and triphosphates / deoxyribonucleotide biosynthetic process / protein heterotetramerization / DNA synthesis involved in DNA repair / response to ionizing radiation / positive regulation of G1/S transition of mitotic cell cycle / positive regulation of G2/M transition of mitotic cell cycle / cell projection / male gonad development / disordered domain specific binding / retina development in camera-type eye / nuclear envelope / DNA repair / neuronal cell body / mitochondrion / ATP binding / identical protein binding / cytosol
Similarity search - Function
Ribonucleotide reductase, class I , alpha subunit / Ribonucleotide reductase large subunit signature. / Ribonucleoside-diphosphate reductase large subunit / ATP-cone domain / ATP cone domain / ATP-cone domain profile. / 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
CYTIDINE-5'-DIPHOSPHATE / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / Ribonucleoside-diphosphate reductase large subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsBrignole, E.J. / Drennan, C.L. / Asturias, F.J. / Tsai, K.L. / Penczek, P.A.
Funding support United States, 3items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM29595 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM67167 United States
CitationJournal: Elife / Year: 2018
Title: 3.3-Å resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound.
Authors: Edward J Brignole / Kuang-Lei Tsai / Johnathan Chittuluru / Haoran Li / Yimon Aye / Pawel A Penczek / JoAnne Stubbe / Catherine L Drennan / Francisco Asturias /
Abstract: Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, a reaction essential for DNA replication and repair. Human RNR requires two subunits for activity, the α subunit ...Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, a reaction essential for DNA replication and repair. Human RNR requires two subunits for activity, the α subunit contains the active site, and the β subunit houses the radical cofactor. Here, we present a 3.3-Å resolution structure by cryo-electron microscopy (EM) of a dATP-inhibited state of human RNR. This structure, which was determined in the presence of substrate CDP and allosteric regulators ATP and dATP, has three α units arranged in an α ring. At near-atomic resolution, these data provide insight into the molecular basis for CDP recognition by allosteric specificity effectors dATP/ATP. Additionally, we present lower-resolution EM structures of human α in the presence of both the anticancer drug clofarabine triphosphate and β. Together, these structures support a model for RNR inhibition in which β is excluded from binding in a radical transfer competent position when α exists as a stable hexamer.
History
DepositionSep 1, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 18, 2018Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.2Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.4Mar 13, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / 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

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Ribonucleoside-diphosphate reductase large subunit
B: Ribonucleoside-diphosphate reductase large subunit
C: Ribonucleoside-diphosphate reductase large subunit
D: Ribonucleoside-diphosphate reductase large subunit
E: Ribonucleoside-diphosphate reductase large subunit
F: Ribonucleoside-diphosphate reductase large subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)562,70736
Polymers554,1026
Non-polymers8,60530
Water64936
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area33640 Å2
ΔGint-252 kcal/mol
Surface area164080 Å2

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Components

#1: Protein
Ribonucleoside-diphosphate reductase large subunit / Ribonucleotide reductase / Ribonucleoside-diphosphate reductase subunit M1 / Ribonucleotide reductase large subunit


Mass: 92350.391 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RRM1, RR1 / Plasmid: pET-28a
Details (production host): Gene inserted between NdeI and NotI sites
Production host: Escherichia coli (E. coli) / Strain (production host): BL21
References: UniProt: P23921, ribonucleoside-diphosphate reductase
#2: Chemical
ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / Deoxyadenosine triphosphate


Mass: 491.182 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H16N5O12P3
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg
#4: Chemical
ChemComp-CDP / CYTIDINE-5'-DIPHOSPHATE / Cytidine diphosphate


Mass: 403.176 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C9H15N3O11P2
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 36 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human ribonucleotide reductase large subnunit (alpha) / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 / Plasmid: pET-28a
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES1
215 mMMagnesium ChlorideMgCl21
31 mMEDTAEthylenediaminetetraacetic acid1
450 mMPotassium ChlorideKCl1
SpecimenConc.: 1.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 14 microM alpha and 0.05 mM dATP, 3 mM ATP, 1 mM CDP in 50 mM HEPES, pH 7.6, 15 mM MgCl2, 1 mM EDTA, 5 mM DTT, and 50 mM KCl
Specimen supportDetails: glow discharged at 20 mA in an EMITech K100X Grid was first cleaned in a Solarus 950 (Gatan) at 25 W for 10 s in 75/25 Ar/O2 gas mixture
Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Protochips C-Flat
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 75 % / Chamber temperature: 277 K
Details: manual blot with a strip of Whatman paper until drop stops wicking determined visually

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Nominal defocus max: 2800 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 7.6 sec. / Electron dose: 44 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2144
Image scansMovie frames/image: 38 / Used frames/image: 5-38

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Processing

SoftwareName: PHENIX / Version: dev_2650: / Classification: refinement
EM software
IDNameCategory
1FindEMparticle selection
2Leginonimage acquisition
4SPARXCTF correction
7UCSF Chimeramodel fitting
10SPARXfinal Euler assignment
12SPARX3D reconstruction
13PHENIXmodel refinement
14Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43885 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00737140
ELECTRON MICROSCOPYf_angle_d0.92850382
ELECTRON MICROSCOPYf_dihedral_angle_d9.01322266
ELECTRON MICROSCOPYf_chiral_restr0.0555526
ELECTRON MICROSCOPYf_plane_restr0.0076336

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