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- EMDB-50173: Trypanosoma brucei nuclear cap-binding complex (CBC) bound to cap0 -

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Basic information

Entry
Database: EMDB / ID: EMD-50173
TitleTrypanosoma brucei nuclear cap-binding complex (CBC) bound to cap0
Map data
Sample
  • Complex: Trypanosoma brucei nuclear cap binding complex (CBC) bound to m7GMP
    • Protein or peptide: Nuclear cap binding complex subunit CBP110
    • Protein or peptide: Nuclear cap-binding protein subunit 2
    • Protein or peptide: Nuclear cap binding complex subunit CBP30
  • Ligand: 7N-METHYL-8-HYDROGUANOSINE-5'-MONOPHOSPHATE
Keywordsnuclear cap binding complex / CBC / cap0 / RNA BINDING PROTEIN
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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsBernhard H / Dolce LG / Kowalinski E
Funding support France, 1 items
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, 2024-
Header (metadata) releaseDec 25, 2024-
Map releaseDec 25, 2024-
UpdateJan 29, 2025-
Current statusJan 29, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50173.png

Downloads & links

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Map

FileDownload / File: emd_50173.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.81 Å/pix.
x 350 pix.
= 284.445 Å		0.81 Å/pix.
x 350 pix.
= 284.445 Å		0.81 Å/pix.
x 350 pix.
= 284.445 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8127 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.027627308 - 0.067383744
Average (Standard dev.)-0.000005646636 (±0.0013209092)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 284.44498 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: DeepEMhancer sharpened cryo-EM map

Fileemd_50173_additional_1.map
AnnotationDeepEMhancer sharpened cryo-EM map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_50173_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_50173_half_map_2.map
Projections & Slices
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Sample components

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Entire : Trypanosoma brucei nuclear cap binding complex (CBC) bound to m7GMP

EntireName: Trypanosoma brucei nuclear cap binding complex (CBC) bound to m7GMP
Components
  • Complex: Trypanosoma brucei nuclear cap binding complex (CBC) bound to m7GMP
    • Protein or peptide: Nuclear cap binding complex subunit CBP110
    • Protein or peptide: Nuclear cap-binding protein subunit 2
    • Protein or peptide: Nuclear cap binding complex subunit CBP30
  • Ligand: 7N-METHYL-8-HYDROGUANOSINE-5'-MONOPHOSPHATE

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Supramolecule #1: Trypanosoma brucei nuclear cap binding complex (CBC) bound to m7GMP

SupramoleculeName: Trypanosoma brucei nuclear cap binding complex (CBC) bound to m7GMP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Trypanosoma brucei brucei (eukaryote) / Location in cell: Nucleus

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Macromolecule #1: Nuclear cap binding complex subunit CBP110

MacromoleculeName: Nuclear cap binding complex subunit CBP110 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Trypanosoma brucei brucei (eukaryote)
Molecular weightTheoretical: 112.342773 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: HHHHHHHHPP SGADPMYAMP GNSGDLQCDD GKKRHIPPLS LLLSGYGSSE GRELDTSYTP KQWLWFLYVT SWARPFFTYA SSGGASEAT EGSSDNRSGS AYWGRLSFDE LLKLSGSPIP PAMVSLWMGL CMPTDDVVQE LRIMTEGKAE GRQRIYPFLP R VAESTFGR ...String:
HHHHHHHHPP SGADPMYAMP GNSGDLQCDD GKKRHIPPLS LLLSGYGSSE GRELDTSYTP KQWLWFLYVT SWARPFFTYA SSGGASEAT EGSSDNRSGS AYWGRLSFDE LLKLSGSPIP PAMVSLWMGL CMPTDDVVQE LRIMTEGKAE GRQRIYPFLP R VAESTFGR ALRSLAVRQH ISSWMPSHAG DVCAALDVLV RDTLEVIQNS ADVRKTARVR CEERDASGEE ALEGAFRSML SA STTAPKV VEATTYRPLF DVKASIPEGG TAQVLIRVAN AARLFAALSV EAFGRVKSEC AVLLLAHINQ RDAPEHVDAR AYG VVTGVV EYAMAYRYCR DDGTGRCPLT CAALLLHRLV ELQGIVEKDT AAGDRCMGMA VSASRFANMT VACIQELLFC VVAG DTVRW HREHQPDGVS VCPTAARTLT LHETDCLLQV FIPALLQQVG FEWPWSESLR HAKMLDRART QHIVMEDGVR LDSRS VFEE LLVSVARRTY GLRLRAILPQ SFDVIAENIF VSIHDVPNEG KSSGGNPEKV ADNSSSRFAL PLYYRTAGEV LLEYFD RCG PSGITAEETE RVLRRATDVQ PMVVQLQALG GDTNGGITDC VSYCGELSRT VYFSAREKER LLQRYRCEVL LASLVVY TQ LRTVSVVQQL TRQLAPLFEQ LLLPLAHERT LSRCPVIASR KGDGVDDNGT PLVDLTPEFK MLVDEIHYEF YPLEWVPE A VDAHIRQECG VREKEGAPYH EGGNGPPCFA QYSLFAAIAH QFGLVLEGNP RGFRGGDGSS SEVRTKAYRF FTLMLLNNL GDAVSSSCEL DGAAALAATR RVKDCQDGSE NCKQRQAAVR QRGGASFHSV VSACDVVVTM TQCLLPAHLS SHPRMATTGS MSNEWMRRV GEWTRSAYSK YTAYQQQVHG LDLPVPLISL YNSLTFDSVP LARETIRAVR SRLLEKMSVV TASPPGDVET A GKQLLEQH LSSLTVTLTA VGLLPLCVRG GSHVPCATQL LWASPFFSHE LLHCGRYEVS V

UniProtKB: Nuclear cap binding complex subunit CBP110

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Macromolecule #2: Nuclear cap-binding protein subunit 2

MacromoleculeName: Nuclear cap-binding protein subunit 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Trypanosoma brucei brucei (eukaryote)
Molecular weightTheoretical: 21.292068 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MAEYLIDLTP RMAYVDRHEL LRSLLTEKEF IERRQEQLNK STTVYVGNLS FYTTEDQIWE HFSRCGHIRD LVMGLSEVTR TPCGFCFVV FESQDGAMSA VIDLHGTLLD DRVITVSWDV GCDHTRRWGR GAHGGQVVDG VRQNLDSARG GLGVLRREEL G VGAAVAED QLVHYTWIPP RRVEKRGRS

UniProtKB: Nuclear cap-binding protein subunit 2

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Macromolecule #3: Nuclear cap binding complex subunit CBP30

MacromoleculeName: Nuclear cap binding complex subunit CBP30 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Trypanosoma brucei brucei (eukaryote)
Molecular weightTheoretical: 30.235732 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MRRGSGFVHL NTPAAISYSP GQQVSLSDLR QKRRREECVV LPPIMTVWRS AFSQYTKMWG LTKFAGDIEA EREGEGPILP PIREETVVK STTHSFKGKS TTVAAAVATP PGEKGIEIVS HHKLCGKQRF VYPDHDGVLS SGIVPKVVIS KEEEEEMEAN K KYYISPQE ...String:
MRRGSGFVHL NTPAAISYSP GQQVSLSDLR QKRRREECVV LPPIMTVWRS AFSQYTKMWG LTKFAGDIEA EREGEGPILP PIREETVVK STTHSFKGKS TTVAAAVATP PGEKGIEIVS HHKLCGKQRF VYPDHDGVLS SGIVPKVVIS KEEEEEMEAN K KYYISPQE MTEEERNAFE ELDAMWKSYA RGRSEQGARH RAFAGTVTPG DAMTEVEEET GNNRGNARRR LESHTPRQHP QE SAAEGNS VAHNTEEALD DALRLIDELL EFN

UniProtKB: Nuclear cap binding complex subunit CBP30

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Macromolecule #4: 7N-METHYL-8-HYDROGUANOSINE-5'-MONOPHOSPHATE

MacromoleculeName: 7N-METHYL-8-HYDROGUANOSINE-5'-MONOPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: 7MG
Molecular weightTheoretical: 379.263 Da
Chemical component information

ChemComp-7MG:
7N-METHYL-8-HYDROGUANOSINE-5'-MONOPHOSPHATE

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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

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 51.56 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN
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: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 251312
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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