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- PDB-9f3f: Trypanosoma brucei nuclear cap-binding complex (CBC) bound to cap0 -

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Basic information

Entry
Database: PDB / ID: 9f3f
TitleTrypanosoma brucei nuclear cap-binding complex (CBC) bound to cap0
Components
  • Nuclear cap binding complex subunit CBP110
  • Nuclear cap binding complex subunit CBP30
  • Nuclear cap-binding protein subunit 2
KeywordsRNA BINDING PROTEIN / nuclear cap binding complex / CBC / cap0
Function / homology
Function and homology information


snRNA export from nucleus / nuclear cap binding complex / RNA cap binding / mRNA cis splicing, via spliceosome / mRNA splicing, via spliceosome / nucleolus / nucleoplasm / nucleus
Similarity search - Function
Nuclear cap-binding complex subunit CBP110 / Nuclear cap-binding complex subunit CBP30 / Nuclear cap binding complex subunit CBP30 / Nuclear cap binding complex subunit CBP110 / Nuclear cap-binding protein subunit 2 / NCBP2, RNA recognition motif / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain ...Nuclear cap-binding complex subunit CBP110 / Nuclear cap-binding complex subunit CBP30 / Nuclear cap binding complex subunit CBP30 / Nuclear cap binding complex subunit CBP110 / Nuclear cap-binding protein subunit 2 / NCBP2, RNA recognition motif / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / RNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily
Similarity search - Domain/homology
7N-METHYL-8-HYDROGUANOSINE-5'-MONOPHOSPHATE / Nuclear cap binding complex subunit CBP30 / Nuclear cap binding complex subunit CBP110 / Nuclear cap-binding protein subunit 2
Similarity search - Component
Biological speciesTrypanosoma brucei brucei (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsBernhard, H. / Dolce, L.G. / Kowalinski, E.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-20-CE11-0016 France
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis of Spliced Leader RNA recognition by the Trypanosoma brucei cap-binding complex.
Authors: Harald Bernhard / Hana Petržílková / Barbora Popelářová / Kamil Ziemkiewicz / Karolina Bartosik / Marcin Warmiński / Laura Tengo / Henri Gröger / Luciano G Dolce / Cameron D ...Authors: Harald Bernhard / Hana Petržílková / Barbora Popelářová / Kamil Ziemkiewicz / Karolina Bartosik / Marcin Warmiński / Laura Tengo / Henri Gröger / Luciano G Dolce / Cameron D Mackereth / Ronald Micura / Jacek Jemielity / Eva Kowalinski /
Abstract: Kinetoplastids are a clade of eukaryotic protozoans that include human parasitic pathogens like trypanosomes and Leishmania species. In these organisms, protein-coding genes are transcribed as ...Kinetoplastids are a clade of eukaryotic protozoans that include human parasitic pathogens like trypanosomes and Leishmania species. In these organisms, protein-coding genes are transcribed as polycistronic pre-mRNAs, which need to be processed by the coupled action of trans-splicing and polyadenylation to yield monogenic mature mRNAs. During trans-splicing, a universal RNA sequence, the spliced leader RNA (SL RNA) mini-exon, is added to the 5'-end of each mRNA. The 5'-end of this mini-exon carries a hypermethylated cap structure and is bound by a trypanosomatid-specific cap-binding complex (CBC). The function of three of the kinetoplastid CBC subunits is unknown, but an essential role in cap-binding and trans-splicing has been suggested. Here, we report cryo-EM structures that reveal the molecular architecture of the Trypanosoma brucei CBC (TbCBC) complex. We find that TbCBC interacts with two distinct features of the SL RNA. The TbCBP20 subunit interacts with the mG cap while TbCBP66 recognizes double-stranded portions of the SL RNA. Our findings pave the way for future research on mRNA maturation in kinetoplastids. Moreover, the observed structural similarities and differences between TbCBC and the mammalian cap-binding complex will be crucial for considering the potential of TbCBC as a target for anti-trypanosomatid drug development.
History
DepositionApr 25, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 25, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 29, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: Nuclear cap binding complex subunit CBP110
2: Nuclear cap-binding protein subunit 2
3: Nuclear cap binding complex subunit CBP30
hetero molecules


Theoretical massNumber of molelcules
Total (without water)164,2504
Polymers163,8713
Non-polymers3791
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Nuclear cap binding complex subunit CBP110


Mass: 112342.773 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma brucei brucei (eukaryote) / Gene: Tb10.70.4560 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q38BU6
#2: Protein Nuclear cap-binding protein subunit 2 / 20 kDa nuclear cap-binding protein


Mass: 21292.068 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma brucei brucei (eukaryote) / Gene: Tb06.28P18.1190, Tb927.6.1970 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q585L4
#3: Protein Nuclear cap binding complex subunit CBP30


Mass: 30235.732 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma brucei brucei (eukaryote) / Gene: Tb10.61.1300 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q387Z0
#4: Chemical ChemComp-7MG / 7N-METHYL-8-HYDROGUANOSINE-5'-MONOPHOSPHATE


Type: RNA linking / Mass: 379.263 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H18N5O8P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: Trypanosoma brucei nuclear cap binding complex (CBC) bound to m7GMP
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Trypanosoma brucei brucei (eukaryote) / Cellular location: Nucleus
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
SpecimenEmbedding 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

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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: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 51.56 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 251312 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0177669
ELECTRON MICROSCOPYf_angle_d0.81610420
ELECTRON MICROSCOPYf_dihedral_angle_d6.7041047
ELECTRON MICROSCOPYf_chiral_restr0.0431186
ELECTRON MICROSCOPYf_plane_restr0.0061325

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