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- EMDB-7006: Human ribonucleotide reductase large subunit (alpha) with dATP and CDP -

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Basic information

Entry
Database: EMDB / ID: EMD-7006
TitleHuman ribonucleotide reductase large subunit (alpha) with dATP and CDP
Map dataHuman ribonucleotide reductase large subunit (alpha) with dATP and CDP
Sample
  • Complex: Human ribonucleotide reductase large subnunit (alpha)
    • Protein or peptide: Ribonucleoside-diphosphate reductase large subunitRibonucleotide reductase
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: CYTIDINE-5'-DIPHOSPHATE
  • Ligand: water
KeywordsRibonucleotide Reductase Electron transfer Radical chemistry Thiyl radical / OXIDOREDUCTASE
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
Ribonucleoside-diphosphate reductase large subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsBrignole EJ / Drennan CL
Funding support United States, 3 items
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, 2017-
Header (metadata) releaseOct 18, 2017-
Map releaseApr 18, 2018-
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: 0.0275
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0275
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6aui
  • Surface level: 0.0275
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7006.png

Downloads & links

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Map

FileDownload / File: emd_7006.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman ribonucleotide reductase large subunit (alpha) with dATP and CDP
Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.0275 / Movie #1: 0.0275
Minimum - Maximum-0.06736154 - 0.20922565
Average (Standard dev.)0.0005886731 (±0.0061916388)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 314.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z314.400314.400314.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-128-128-128
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0670.2090.001

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Supplemental data

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Sample components

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Entire : Human ribonucleotide reductase large subnunit (alpha)

EntireName: Human ribonucleotide reductase large subnunit (alpha)
Components
  • Complex: Human ribonucleotide reductase large subnunit (alpha)
    • Protein or peptide: Ribonucleoside-diphosphate reductase large subunitRibonucleotide reductase
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: CYTIDINE-5'-DIPHOSPHATE
  • Ligand: water

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Supramolecule #1: Human ribonucleotide reductase large subnunit (alpha)

SupramoleculeName: Human ribonucleotide reductase large subnunit (alpha) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Ribonucleoside-diphosphate reductase large subunit

MacromoleculeName: Ribonucleoside-diphosphate reductase large subunit / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: ribonucleoside-diphosphate reductase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 92.350391 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MHVIKRDGRQ ERVMFDKITS RIQKLCYGLN MDFVDPAQIT MKVIQGLYSG VTTVELDTLA AETAATLTT KHPDYAILAA RIAVSNLHKE TKKVFSDVME DLYNYINPHN GKHSPMVAKS TLDIVLANKD RLNSAIIYDR D FSYNYFGF ...String:
MGSSHHHHHH SSGLVPRGSH MHVIKRDGRQ ERVMFDKITS RIQKLCYGLN MDFVDPAQIT MKVIQGLYSG VTTVELDTLA AETAATLTT KHPDYAILAA RIAVSNLHKE TKKVFSDVME DLYNYINPHN GKHSPMVAKS TLDIVLANKD RLNSAIIYDR D FSYNYFGF KTLERSYLLK INGKVAERPQ HMLMRVSVGI HKEDIDAAIE TYNLLSERWF THASPTLFNA GTNRPQLSSC FL LSMKDDS IEGIYDTLKQ CALISKSAGG IGVAVSCIRA TGSYIAGTNG NSNGLVPMLR VYNNTARYVD QGGNKRPGAF AIY LEPWHL DIFEFLDLKK NTGKEEQRAR DLFFALWIPD LFMKRVETNQ DWSLMCPNEC PGLDEVWGEE FEKLYASYEK QGRV RKVVK AQQLWYAIIE SQTETGTPYM LYKDSCNRKS NQQNLGTIKC SNLCTEIVEY TSKDEVAVCN LASLALNMYV TSEHT YDFK KLAEVTKVVV RNLNKIIDIN YYPVPEACLS NKRHRPIGIG VQGLADAFIL MRYPFESAEA QLLNKQIFET IYYGAL EAS CDLAKEQGPY ETYEGSPVSK GILQYDMWNV TPTDLWDWKV LKEKIAKYGI RNSLLIAPMP TASTAQILGN NESIEPY TS NIYTRRVLSG EFQIVNPHLL KDLTERGLWH EEMKNQIIAC NGSIQSIPEI PDDLKQLYKT VWEISQKTVL KMAAERGA F IDQSQSLNIH IAEPNYGKLT SMHFYGWKQG LKTGMYYLRT RPAANPIQFT LNKEKLKDKE KVSKEEEEKE RNTAAMVCS LENRDECLMC GS

UniProtKB: Ribonucleoside-diphosphate reductase large subunit

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

MacromoleculeName: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 12 / Formula: DTP
Molecular weightTheoretical: 491.182 Da
Chemical component information

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

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 12 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: CYTIDINE-5'-DIPHOSPHATE

MacromoleculeName: CYTIDINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 6 / Formula: CDP
Molecular weightTheoretical: 403.176 Da
Chemical component information

ChemComp-CDP:
CYTIDINE-5'-DIPHOSPHATE / Cytidine diphosphate

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 36 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.3 mg/mL
BufferpH: 7.6
Component:
ConcentrationNameFormula
50.0 mMHEPES
15.0 mMMagnesium ChlorideMgCl2
1.0 mMEDTAEthylenediaminetetraacetic acid
50.0 mMPotassium ChlorideKCl
GridModel: Protochips C-Flat / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
Details: 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
VitrificationCryogen name: ETHANE / Chamber humidity: 75 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER
Details: manual blot with a strip of Whatman paper until drop stops wicking determined visually.
Details14 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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 22500
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 - Frames/image: 5-38 / Number real images: 2144 / Average exposure time: 7.6 sec. / Average electron dose: 44.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: Calculated ab initio
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: PROJECTION MATCHING / Software - Name: SPARX
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPARX / Number images used: 43885

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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