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- PDB-8fli: Cryo-EM structure of a group II intron immediately before branching -

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Basic information

Entry
Database: PDB / ID: 8fli
TitleCryo-EM structure of a group II intron immediately before branching
Components
  • Group II Intron
  • Maturase reverse transcriptase
KeywordsSPLICING/RNA / group II intron / splicing / branching / maturase / SPLICING-RNA complex
Function / homology
Function and homology information


RNA-directed DNA polymerase activity / endonuclease activity / nucleic acid binding / zinc ion binding
Similarity search - Function
Reverse transcriptase, N-terminal domain / N-terminal domain of reverse transcriptase / Group II intron, maturase-specific / Group II intron reverse transcriptase/maturase / Group II intron, maturase-specific domain / HNH endonuclease / HNH endonuclease / HNH nucleases / : / HNH nuclease ...Reverse transcriptase, N-terminal domain / N-terminal domain of reverse transcriptase / Group II intron, maturase-specific / Group II intron reverse transcriptase/maturase / Group II intron, maturase-specific domain / HNH endonuclease / HNH endonuclease / HNH nucleases / : / HNH nuclease / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / Maturase reverse transcriptase
Similarity search - Component
Biological speciesThermosynechococcus vestitus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsHaack, D.B. / Rudolfs, B.G. / Zhang, C. / Lyumkis, D. / Toor, N.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structural basis of branching during RNA splicing.
Authors: Daniel B Haack / Boris Rudolfs / Cheng Zhang / Dmitry Lyumkis / Navtej Toor /
Abstract: Branching is a critical step in RNA splicing that is essential for 5' splice site selection. Recent spliceosome structures have led to competing models for the recognition of the invariant adenosine ...Branching is a critical step in RNA splicing that is essential for 5' splice site selection. Recent spliceosome structures have led to competing models for the recognition of the invariant adenosine at the branch point. However, there are no structures of any splicing complex with the adenosine nucleophile docked in the active site and positioned to attack the 5' splice site. Thus we lack a mechanistic understanding of adenosine selection and splice site recognition during RNA splicing. Here we present a cryo-electron microscopy structure of a group II intron that reveals that active site dynamics are coupled to the formation of a base triple within the branch-site helix that positions the 2'-OH of the adenosine for nucleophilic attack on the 5' scissile phosphate. This structure, complemented with biochemistry and comparative analyses to splicing complexes, supports a base triple model of adenosine recognition for branching within group II introns and the evolutionarily related spliceosome.
History
DepositionDec 21, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 20, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Feb 7, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Group II Intron
B: Maturase reverse transcriptase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)354,3965
Polymers354,3232
Non-polymers733
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain Group II Intron


Mass: 289257.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermosynechococcus vestitus (bacteria)
Production host: Escherichia coli (E. coli)
#2: Protein Maturase reverse transcriptase


Mass: 65065.121 Da / Num. of mol.: 1 / Mutation: G275D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermosynechococcus vestitus (bacteria)
Gene: tll0114 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8DMK2
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Group IIB intron in complex with its maturase protein / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Thermosynechococcus vestitus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
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: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 31.6 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 38881 / Symmetry type: POINT
RefinementCross valid method: NONE

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