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- PDB-6w4x: Holocomplex of E. coli class Ia ribonucleotide reductase with GDP... -

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Basic information

Entry
Database: PDB / ID: 6w4x
TitleHolocomplex of E. coli class Ia ribonucleotide reductase with GDP and TTP
Components(Ribonucleoside-diphosphate reductase 1 subunit ...Ribonucleotide reductase) x 2
KeywordsOXIDOREDUCTASE / complex
Function / homology
Function and homology information


ribonucleoside diphosphate metabolic process / 2'-deoxyribonucleotide biosynthetic process / nucleobase-containing small molecule interconversion / ribonucleoside-diphosphate reductase complex / ribonucleoside-diphosphate reductase / ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor / deoxyribonucleotide biosynthetic process / protein folding chaperone / DNA replication / iron ion binding ...ribonucleoside diphosphate metabolic process / 2'-deoxyribonucleotide biosynthetic process / nucleobase-containing small molecule interconversion / ribonucleoside-diphosphate reductase complex / ribonucleoside-diphosphate reductase / ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor / deoxyribonucleotide biosynthetic process / protein folding chaperone / DNA replication / iron ion binding / ATP binding / identical protein binding / cytosol / cytoplasm
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, 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 / Anaerobic Ribonucleotide-triphosphate Reductase Large Chain / Anaerobic Ribonucleotide-triphosphate Reductase Large Chain - #20 / Ribonucleotide reductase small subunit, acitve site / Ribonucleotide reductase small subunit signature. / Ribonucleotide reductase small subunit / Ribonucleotide reductase small subunit family / Ribonucleotide reductase, small chain / Ribonucleotide Reductase, subunit A / Ribonucleotide Reductase, subunit A / Ribonucleotide reductase-like / Ferritin-like superfamily / Alpha-Beta Barrel / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
MU-OXO-DIIRON / GUANOSINE-5'-DIPHOSPHATE / THYMIDINE-5'-TRIPHOSPHATE / Ribonucleoside-diphosphate reductase 1 subunit alpha / Ribonucleoside-diphosphate reductase 1 subunit beta
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsKang, G. / Taguchi, A. / Stubbe, J. / Drennan, C.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Science / Year: 2020
Title: Structure of a trapped radical transfer pathway within a ribonucleotide reductase holocomplex.
Authors: Gyunghoon Kang / Alexander T Taguchi / JoAnne Stubbe / Catherine L Drennan /
Abstract: Ribonucleotide reductases (RNRs) are a diverse family of enzymes that are alone capable of generating 2'-deoxynucleotides de novo and are thus critical in DNA biosynthesis and repair. The nucleotide ...Ribonucleotide reductases (RNRs) are a diverse family of enzymes that are alone capable of generating 2'-deoxynucleotides de novo and are thus critical in DNA biosynthesis and repair. The nucleotide reduction reaction in all RNRs requires the generation of a transient active site thiyl radical, and in class I RNRs, this process involves a long-range radical transfer between two subunits, α and β. Because of the transient subunit association, an atomic resolution structure of an active α2β2 RNR complex has been elusive. We used a doubly substituted β2, E52Q/(2,3,5)-trifluorotyrosine122-β2, to trap wild-type α2 in a long-lived α2β2 complex. We report the structure of this complex by means of cryo-electron microscopy to 3.6-angstrom resolution, allowing for structural visualization of a 32-angstrom-long radical transfer pathway that affords RNR activity.
History
DepositionMar 11, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 8, 2020Provider: repository / Type: Initial release
Revision 1.1May 6, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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  • Deposited structure unit
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  • EMDB-21540
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Ribonucleoside-diphosphate reductase 1 subunit alpha
B: Ribonucleoside-diphosphate reductase 1 subunit alpha
C: Ribonucleoside-diphosphate reductase 1 subunit beta
D: Ribonucleoside-diphosphate reductase 1 subunit beta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)260,68811
Polymers258,9764
Non-polymers1,7127
Water724
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization, Enzyme is only active in the tetramic form described here and biochemical assays support activity.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area20770 Å2
ΔGint-115 kcal/mol
Surface area75390 Å2

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Components

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Ribonucleoside-diphosphate reductase 1 subunit ... , 2 types, 4 molecules ABCD

#1: Protein Ribonucleoside-diphosphate reductase 1 subunit alpha / Ribonucleotide reductase / Protein B1 / Ribonucleoside-diphosphate reductase 1 R1 subunit / Ribonucleotide reductase 1


Mass: 85877.086 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: nrdA, dnaF, b2234, JW2228 / Production host: Escherichia coli (E. coli)
References: UniProt: P00452, ribonucleoside-diphosphate reductase
#2: Protein Ribonucleoside-diphosphate reductase 1 subunit beta / Ribonucleotide reductase / Protein B2 / Protein R2 / Ribonucleotide reductase 1


Mass: 43611.039 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: nrdB, ftsB, b2235, JW2229 / Production host: Escherichia coli (E. coli)
References: UniProt: P69924, ribonucleoside-diphosphate reductase

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Non-polymers , 5 types, 11 molecules

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


Mass: 482.168 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N2O14P3
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#6: Chemical ChemComp-FEO / MU-OXO-DIIRON


Mass: 127.689 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe2O
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: Holocomplex of E. coli class Ia ribonucleotide reductase with GDP and TTP
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 283 K / Details: blot for 4.5 seconds before plunging

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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
Image recordingAverage exposure time: 6 sec. / Electron dose: 47.1 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 6238
Image scansMovie frames/image: 30

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
1Topazparticle selection
2SerialEMimage acquisition
4RELION3CTF correction
7Coot0.8.9.2model fitting
9PHENIX1.15.2model refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80386 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 5CNV

5cnv

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00318061
ELECTRON MICROSCOPYf_angle_d0.61224502
ELECTRON MICROSCOPYf_dihedral_angle_d7.0410852
ELECTRON MICROSCOPYf_chiral_restr0.0432689
ELECTRON MICROSCOPYf_plane_restr0.0043180

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