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- PDB-8cdp: Cryo-EM structure of the RESC1-RESC2 complex -

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Basic information

Entry
Database: PDB / ID: 8cdp
TitleCryo-EM structure of the RESC1-RESC2 complex
Components
  • Guide_RNA_associated_protein_-_putative
  • Mitochondrial guide RNA binding complex subunit 2
KeywordsRNA BINDING PROTEIN / RESC / RNA editing / cryo-EM structure / Trypanosoma brucei
Function / homologymitochondrial mRNA processing / mitochondrial mRNA editing complex / mitochondrial RNA processing / kinetoplast / mRNA modification / mRNA binding / mitochondrion / Guide RNA associated protein, GAP2 / Guide RNA associated protein, GAP1
Function and homology information
Biological speciesTrypanosoma brucei brucei (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsDolce, L.G. / Weis, F. / Kowalinski, E.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-20-CE11-0016 France
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Structural basis for guide RNA selection by the RESC1-RESC2 complex.
Authors: Luciano G Dolce / Yevheniia Nesterenko / Leon Walther / Félix Weis / Eva Kowalinski /
Abstract: Kinetoplastid parasites, such as trypanosomes or leishmania, rely on RNA-templated RNA editing to mature mitochondrial cryptic pre-mRNAs into functional protein-coding transcripts. Processive pan- ...Kinetoplastid parasites, such as trypanosomes or leishmania, rely on RNA-templated RNA editing to mature mitochondrial cryptic pre-mRNAs into functional protein-coding transcripts. Processive pan-editing of multiple editing blocks within a single transcript is dependent on the 20-subunit RNA editing substrate binding complex (RESC) that serves as a platform to orchestrate the interactions between pre-mRNA, guide RNAs (gRNAs), the catalytic RNA editing complex (RECC), and a set of RNA helicases. Due to the lack of molecular structures and biochemical studies with purified components, neither the spacio-temporal interplay of these factors nor the selection mechanism for the different RNA components is understood. Here we report the cryo-EM structure of Trypanosoma brucei RESC1-RESC2, a central hub module of the RESC complex. The structure reveals that RESC1 and RESC2 form an obligatory domain-swapped dimer. Although the tertiary structures of both subunits closely resemble each other, only RESC2 selectively binds 5'-triphosphate-nucleosides, a defining characteristic of gRNAs. We therefore propose RESC2 as the protective 5'-end binding site for gRNAs within the RESC complex. Overall, our structure provides a starting point for the study of the assembly and function of larger RNA-bound kinetoplast RNA editing modules and might aid in the design of anti-parasite drugs.
History
DepositionJan 31, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 29, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 12, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2May 31, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Guide_RNA_associated_protein_-_putative
B: Mitochondrial guide RNA binding complex subunit 2


Theoretical massNumber of molelcules
Total (without water)104,9352
Polymers104,9352
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering, SEC-MALS
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Guide_RNA_associated_protein_-_putative


Mass: 52724.535 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma brucei brucei (eukaryote) / Gene: Tb07.22O10.680, Tb927.7.2570 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q57XL7
#2: Protein Mitochondrial guide RNA binding complex subunit 2


Mass: 52210.234 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma brucei brucei (eukaryote) / Gene: 28H13.250, Tb927.2.3800 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q586X1

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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: RESC1-RESC2 complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
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: Quantifoil R1.2/1.3
VitrificationCryogen 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 63 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameCategory
1Warpparticle selection
4RELIONCTF correction
10cryoSPARCinitial Euler assignment
11RELIONfinal Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 447858 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035305
ELECTRON MICROSCOPYf_angle_d0.577189
ELECTRON MICROSCOPYf_dihedral_angle_d4.926717
ELECTRON MICROSCOPYf_chiral_restr0.042804
ELECTRON MICROSCOPYf_plane_restr0.004928

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