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- EMDB-16592: Cryo-EM structure of the RESC1-RESC2 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-16592
TitleCryo-EM structure of the RESC1-RESC2 complex
Map data
Sample
  • Complex: RESC1-RESC2 complex
    • Protein or peptide: Guide_RNA_associated_protein_-_putative
    • Protein or peptide: Mitochondrial guide RNA binding complex subunit 2
KeywordsRESC / RNA editing / cryo-EM structure / Trypanosoma brucei / RNA BINDING PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsDolce LG / Weis F / 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-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16592.png

Downloads & links

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Map

FileDownload / File: emd_16592.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.81 Å/pix.
x 300 pix.
= 243. Å		0.81 Å/pix.
x 300 pix.
= 243. Å		0.81 Å/pix.
x 300 pix.
= 243. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.81 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.015303235 - 0.04658992
Average (Standard dev.)0.000020117335 (±0.0011337915)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 243.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16592_msk_1.map
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Additional map: deepEMhancer post process

Fileemd_16592_additional_1.map
AnnotationdeepEMhancer post process
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Additional map: relion post process

Fileemd_16592_additional_2.map
Annotationrelion post process
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Half map: #2

Fileemd_16592_half_map_1.map
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Half map: #1

Fileemd_16592_half_map_2.map
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Sample components

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Entire : RESC1-RESC2 complex

EntireName: RESC1-RESC2 complex
Components
  • Complex: RESC1-RESC2 complex
    • Protein or peptide: Guide_RNA_associated_protein_-_putative
    • Protein or peptide: Mitochondrial guide RNA binding complex subunit 2

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Supramolecule #1: RESC1-RESC2 complex

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

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Macromolecule #1: Guide_RNA_associated_protein_-_putative

MacromoleculeName: Guide_RNA_associated_protein_-_putative / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Trypanosoma brucei brucei (eukaryote)
Molecular weightTheoretical: 52.724535 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MKHHHHHHSA GLEVLFQGPD SMQNQGSVSQ GALNMRDQQA AAAENVTPER VWALWNEGNL FSLSLAQLQG FLSRCGVRTD PAAKKAAVV RQVEEYLHSK DTTVKGGGQG AASPQQHQQH GQQGGYGRWN QASVMQPETL LDLSQAGFYE GAANMVPKAF Q LLVSDTAP ...String:
MKHHHHHHSA GLEVLFQGPD SMQNQGSVSQ GALNMRDQQA AAAENVTPER VWALWNEGNL FSLSLAQLQG FLSRCGVRTD PAAKKAAVV RQVEEYLHSK DTTVKGGGQG AASPQQHQQH GQQGGYGRWN QASVMQPETL LDLSQAGFYE GAANMVPKAF Q LLVSDTAP DVVVSRVNTT AFPGFPSNTE CYTLGASEKD VAIRSRYSKV LQWCCLNMSN LQMDGELYVD FGKLLLKPSV MR KNRRIVS SYTLQQRLQV NHPYTWVPTL PESCLSKIQE QFLQPEGFAP IGKGVQLTYS GTIKRSKDQL HVDLDNKGKV LAV NSAWVN LQTAWCTHAK GPDVRLLLRS RPPIRRQDVE LFASTPIIKL ADDDVADVLP PEHGQLVYLS EDETRLFERV SDRG VTITV REVKRQPLII LRDEEEDPRV EYSLSAHIPA NAAKATDVRA VGLTAFELAG RLAGLVAEDF VREYGCEAKL

UniProtKB: Guide RNA associated protein, GAP2

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Macromolecule #2: Mitochondrial guide RNA binding complex subunit 2

MacromoleculeName: Mitochondrial guide RNA binding complex subunit 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Trypanosoma brucei brucei (eukaryote)
Molecular weightTheoretical: 52.210234 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MQSFSAAAPA ASGDFSHITR NTVWGLWNEG NLFSLSVPEL AFFLQEHCRV ANVDPRAKKS ALVRQVEEIL SAEQQASATV PQEDNPHAI VVTDYDRAED ALEEADEYGD WGAEPGFEDR RELDFMELSP GRMGERYDPL SPRAFQLLHS ETATDVGIAS I DPSKLPGQ ...String:
MQSFSAAAPA ASGDFSHITR NTVWGLWNEG NLFSLSVPEL AFFLQEHCRV ANVDPRAKKS ALVRQVEEIL SAEQQASATV PQEDNPHAI VVTDYDRAED ALEEADEYGD WGAEPGFEDR RELDFMELSP GRMGERYDPL SPRAFQLLHS ETATDVGIAS I DPSKLPGQ SKVKNALAAI HVAPNDANKM RFRMAFEWCL MNIWNMNMPG ELNIGAGKAL YYRSVAKQNR NVMPLWTVQK HL YAQHPYA WFAIASESNV AAMESLAAAL NMSIQQERTT SYKVTIRRMA EFFDCELNGQ LKCTMMNKPW DRFFVSHYIR SKM PDLRYV VRARHPIKKR IADAYLEADI LRSTRDSVQS VLSPELGDVV YCCERVVRKW AKKTATGVTL QLVETKRTPL IITK AGDEG ERLEYEWIVP LPQQAERIDI AALTDELWEY GNKLAAALEE GMEELMVHTM TAVSAY

UniProtKB: Guide RNA associated protein, GAP1

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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
GridModel: Quantifoil R1.2/1.3 / Material: GOLD
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 63.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 447858
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC
Final angle assignmentType: OTHER / Software - Name: RELION
FSC plot (resolution estimation)

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