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- PDB-8x9o: Cte-U537ins-Branching,Ca,inactive -

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Basic information

Entry
Database: PDB / ID: 8x9o
TitleCte-U537ins-Branching,Ca,inactive
ComponentsRNA (811-mer)
KeywordsRNA / SPLICING / CP group ii intron / Cte / back-splicing / circular RNA
Function / homology: / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesComamonas testosteroni KF-1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsLing, X.B. / Ma, J.B.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)NSFC 31971130 China
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Structures of a natural circularly permuted group II intron reveal mechanisms of branching and backsplicing.
Authors: Xiaobin Ling / Yuqi Yao / Jinbiao Ma /
Abstract: Circularly permuted (CP) group II introns, identified in various bacteria phyla, swap domains D5 and D6 near the 5' end and have reversed splice sites (SSs), leading to backsplicing and circular RNA ...Circularly permuted (CP) group II introns, identified in various bacteria phyla, swap domains D5 and D6 near the 5' end and have reversed splice sites (SSs), leading to backsplicing and circular RNA formation. In this study, we present multiple high-resolution cryo-electron microscopy structures of a natural CP group II intron from Comamonas testosteroni KF-1 (Cte 1), elucidating the molecular mechanisms of branching and backsplicing. During branching, the 5' SS is positioned by an auxiliary sequence (AUX)-enhanced interaction between the exon-binding site and intron-binding site (IBS) and stacks on the branch-site adenosine within D6, allowing the attacking 2'-OH group to coordinate with a metal ion in the active center. In backsplicing, the 3' SS is aligned with the branching step, leaving IBS in the active center, stabilized by base pairing with the AUX, which enables the free 3'-end hydroxyl group to directly attack the scissile phosphate of 3' SS. Furthermore, a groove in Cte 1 may stabilize the circular RNA. These findings highlight a conserved catalytic mechanism for canonical group II introns, albeit facilitated by the versatile AUX, opening avenues for designing potent ribozymes producing circular RNAs.
History
DepositionNov 30, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNA (811-mer)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)264,64434
Polymers263,3401
Non-polymers1,30433
Water1448
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: RNA chain RNA (811-mer)


Mass: 263339.688 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Comamonas testosteroni KF-1 (bacteria)
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 19 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: H2O
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: Cte-U537ins-Branching / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Comamonas testosteroni KF-1 (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 1.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 126130 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00718098
ELECTRON MICROSCOPYf_angle_d0.93428241
ELECTRON MICROSCOPYf_dihedral_angle_d17.7729003
ELECTRON MICROSCOPYf_chiral_restr0.0473755
ELECTRON MICROSCOPYf_plane_restr0.006751

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