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- PDB-8aw3: Cryo-EM structure of the Tb ADAT2/3 deaminase in complex with tRNA -

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Basic information

Entry
Database: PDB / ID: 8aw3
TitleCryo-EM structure of the Tb ADAT2/3 deaminase in complex with tRNA
Components
  • (Deaminase, putativeDeamination) x 2
  • RNA (75-MER)
KeywordsRNA BINDING PROTEIN / ADAT / inosine / tRNA modification / deaminase / cryo-EM structure / Trypanosoma brucei
Function / homology
Function and homology information


tRNA wobble adenosine to inosine editing / tRNA-specific adenosine-34 deaminase activity / Hydrolases; Acting on carbon-nitrogen bonds, other than peptide bonds; In cyclic amidines / hydrolase activity / zinc ion binding / nucleoplasm / cytoplasm
Similarity search - Function
Cytidine and deoxycytidylate deaminase zinc-binding region / Cytidine and deoxycytidylate deaminase domain / Cytidine and deoxycytidylate deaminases domain profile. / Cytidine deaminase-like
Similarity search - Domain/homology
: / RNA / RNA (> 10) / Deaminase, putative / Deaminase, putative
Similarity search - Component
Biological speciesTrypanosoma brucei brucei (eukaryote)
Trypanosoma brucei (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsDolce, L.G. / Tengo, L. / Weis, F. / Kowalinski, E.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-20-CE11-0016 France
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis for sequence-independent substrate selection by eukaryotic wobble base tRNA deaminase ADAT2/3.
Authors: Luciano G Dolce / Aubree A Zimmer / Laura Tengo / Félix Weis / Mary Anne T Rubio / Juan D Alfonzo / Eva Kowalinski /
Abstract: The essential deamination of adenosine A to inosine at the wobble base is the individual tRNA modification with the greatest effects on mRNA decoding, empowering a single tRNA to translate three ...The essential deamination of adenosine A to inosine at the wobble base is the individual tRNA modification with the greatest effects on mRNA decoding, empowering a single tRNA to translate three different codons. To date, many aspects of how eukaryotic deaminases specifically select their multiple substrates remain unclear. Here, using cryo-EM, we present the structure of a eukaryotic ADAT2/3 deaminase bound to a full-length tRNA, revealing that the enzyme distorts the anticodon loop, but in contrast to the bacterial enzymes, selects its substrate via sequence-independent contacts of eukaryote-acquired flexible or intrinsically unfolded motifs distal from the conserved catalytic core. A gating mechanism for substrate entry to the active site is identified. Our multi-step tRNA recognition model yields insights into how RNA editing by A deamination evolved, shaped the genetic code, and directly impacts the eukaryotic proteome.
History
DepositionAug 29, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 29, 2023Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: RNA (75-MER)
2: Deaminase, putative
3: Deaminase, putative
hetero molecules


Theoretical massNumber of molelcules
Total (without water)88,4115
Polymers88,2803
Non-polymers1312
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5860 Å2
ΔGint-75 kcal/mol
Surface area33330 Å2
MethodPISA

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Components

#1: RNA chain RNA (75-MER)


Mass: 24132.311 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Trypanosoma brucei (eukaryote) / References: GenBank: 62359426
#2: Protein Deaminase, putative / Deamination


Mass: 23547.535 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma brucei brucei (eukaryote) / Strain: 927/4 GUTat10.1 / Gene: Tb08.29H22.100, Tb927.8.4180 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q57W17, Hydrolases; Acting on carbon-nitrogen bonds, other than peptide bonds; In cyclic amidines
#3: Protein Deaminase, putative / Deamination


Mass: 40600.254 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma brucei brucei (eukaryote) / Strain: 927/4 GUTat10.1 / Gene: Tb11.01.6930 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q381Q7
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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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: ADAT2/3 in complex with tRNA / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.09 MDa / Experimental value: NO
Source (natural)Organism: Trypanosoma brucei brucei (eukaryote)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
SpecimenConc.: 0.07 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1800 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 55 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 105718 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035410
ELECTRON MICROSCOPYf_angle_d0.5917704
ELECTRON MICROSCOPYf_dihedral_angle_d12.4121349
ELECTRON MICROSCOPYf_chiral_restr0.035934
ELECTRON MICROSCOPYf_plane_restr0.005706

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