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- EMDB-16593: Cryo-EM map of RESC1-RESC2 bound to gRNA -

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Basic information

Entry
Database: EMDB / ID: EMD-16593
TitleCryo-EM map of RESC1-RESC2 bound to gRNA
Map data
Sample
  • Complex: RESC1-RESC2-gRNA complex
    • Protein or peptide: RESC1
    • Protein or peptide: RESC2
    • RNA: gRNA gND7(506)
KeywordsRESC / RNA editing / cryo-EM structure / Trypanosoma brucei / RNA BINDING PROTEIN
Biological speciesTrypanosoma brucei brucei (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsDolce LG / Kowalinski E
Funding support France, 1 items
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, 2023-
Header (metadata) releaseMar 29, 2023-
Map releaseMar 29, 2023-
UpdateMay 31, 2023-
Current statusMay 31, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16593.png

Downloads & links

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Map

FileDownload / File: emd_16593.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.009076782 - 0.03053051
Average (Standard dev.)0.000025206835 (±0.00093813153)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 251.99998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16593_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Additional map: Relion post process

Fileemd_16593_additional_1.map
AnnotationRelion post process
Projections & Slices
AxesZYX

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Additional map: deepEMhancer post process

Fileemd_16593_additional_2.map
AnnotationdeepEMhancer post process
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_16593_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_16593_half_map_2.map
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Sample components

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Entire : RESC1-RESC2-gRNA complex

EntireName: RESC1-RESC2-gRNA complex
Components
  • Complex: RESC1-RESC2-gRNA complex
    • Protein or peptide: RESC1
    • Protein or peptide: RESC2
    • RNA: gRNA gND7(506)

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Supramolecule #1: RESC1-RESC2-gRNA complex

SupramoleculeName: RESC1-RESC2-gRNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Trypanosoma brucei brucei (eukaryote)
Molecular weightTheoretical: 118 KDa

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Macromolecule #1: RESC1

MacromoleculeName: RESC1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Trypanosoma brucei brucei (eukaryote)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MKHHHHHHSA GLEVLFQGPD SMQNQGSVSQ GALNMRDQQA AAAENVTPER VWALWNEGNL FSLSLAQLQG FLSRCGVRTD PAAKKAAVVR QVEEYLHSKD TTVKGGGQGA ASPQQHQQHG QQGGYGRWNQ ASVMQPETLL DLSQAGFYEG AANMVPKAFQ LLVSDTAPDV ...String:
MKHHHHHHSA GLEVLFQGPD SMQNQGSVSQ GALNMRDQQA AAAENVTPER VWALWNEGNL FSLSLAQLQG FLSRCGVRTD PAAKKAAVVR QVEEYLHSKD TTVKGGGQGA ASPQQHQQHG QQGGYGRWNQ ASVMQPETLL DLSQAGFYEG AANMVPKAFQ LLVSDTAPDV VVSRVNTTAF PGFPSNTECY TLGASEKDVA IRSRYSKVLQ WCCLNMSNLQ MDGELYVDFG KLLLKPSVMR KNRRIVSSYT LQQRLQVNHP YTWVPTLPES CLSKIQEQFL QPEGFAPIGK GVQLTYSGTI KRSKDQLHVD LDNKGKVLAV NSAWVNLQTA WCTHAKGPDV RLLLRSRPPI RRQDVELFAS TPIIKLADDD VADVLPPEHG QLVYLSEDET RLFERVSDRG VTITVREVKR QPLIILRDEE EDPRVEYSLS AHIPANAAKA TDVRAVGLTA FELAGRLAGL VAEDFVREYG CEAKL

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Macromolecule #2: RESC2

MacromoleculeName: RESC2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Trypanosoma brucei brucei (eukaryote)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MQSFSAAAPA ASGDFSHITR NTVWGLWNEG NLFSLSVPEL AFFLQEHCRV ANVDPRAKKS ALVRQVEEIL SAEQQASATV PQEDNPHAIV VTDYDRAEDA LEEADEYGDW GAEPGFEDRR ELDFMELSPG RMGERYDPLS PRAFQLLHSE TATDVGIASI DPSKLPGQSK ...String:
MQSFSAAAPA ASGDFSHITR NTVWGLWNEG NLFSLSVPEL AFFLQEHCRV ANVDPRAKKS ALVRQVEEIL SAEQQASATV PQEDNPHAIV VTDYDRAEDA LEEADEYGDW GAEPGFEDRR ELDFMELSPG RMGERYDPLS PRAFQLLHSE TATDVGIASI DPSKLPGQSK VKNALAAIHV APNDANKMRF RMAFEWCLMN IWNMNMPGEL NIGAGKALYY RSVAKQNRNV MPLWTVQKHL YAQHPYAWFA IASESNVAAM ESLAAALNMS IQQERTTSYK VTIRRMAEFF DCELNGQLKC TMMNKPWDRF FVSHYIRSKM PDLRYVVRAR HPIKKRIADA YLEADILRST RDSVQSVLSP ELGDVVYCCE RVVRKWAKKT ATGVTLQLVE TKRTPLIITK AGDEGERLEY EWIVPLPQQA ERIDIAALTD ELWEYGNKLA AALEEGMEEL MVHTMTAVSA Y

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Macromolecule #3: gRNA gND7(506)

MacromoleculeName: gRNA gND7(506) / type: rna / ID: 3
Source (natural)Organism: Trypanosoma brucei brucei (eukaryote)
SequenceString:
GGGAUAUAAA UACGAUGUAA AUAACCUGUA GUAUAGUUAG UGUAUAUAGU GAAA

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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

Concentration0.07 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K

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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: 3.5 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Average electron dose: 62.9 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC
Final angle assignmentType: OTHER / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 202502
FSC plot (resolution estimation)

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