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- PDB-6mec: Structure of a group II intron retroelement after DNA integration -

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Basic information

Entry
Database: PDB / ID: 6mec
TitleStructure of a group II intron retroelement after DNA integration
Components
  • Maturase reverse transcriptase
  • Sense Target DNA
  • T.el4h RNA
KeywordsRNA/DNA/RNA Binding Protein / group II intron / retroelement / retrotransposition / RNA-DNA-RNA Binding Protein complex
Function / homology
Function and homology information


RNA-directed DNA polymerase activity / nucleic acid binding / endonuclease activity
Reverse transcriptase domain / HNH endonuclease / HNH nuclease / Group II intron, maturase-specific / Reverse transcriptase, N-terminal domain / Group II intron reverse transcriptase/maturase / Reverse transcriptase (RNA-dependent DNA polymerase) / HNH endonuclease / Group II intron, maturase-specific domain / N-terminal domain of reverse transcriptase / Reverse transcriptase (RT) catalytic domain profile.
Maturase reverse transcriptase
Biological speciesThermosynechococcus elongatus (Cyanobacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHaack, D. / Yan, X. / Zhang, C. / Hingey, J. / Lyumkis, D. / Baker, T.S. / Toor, N.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences1R01GM123275 United States
National Institutes of Health/National Institute of General Medical Sciences1R01GM033050 United States
National Institutes of Health/Office of the DirectorDP5 OD021396 United States
National Institutes of Health/National Institute of General Medical SciencesU54GM103368 United States
CitationJournal: Cell / Year: 2019
Title: Cryo-EM Structures of a Group II Intron Reverse Splicing into DNA.
Authors: Daniel B Haack / Xiaodong Yan / Cheng Zhang / Jason Hingey / Dmitry Lyumkis / Timothy S Baker / Navtej Toor /
Abstract: Group II introns are a class of retroelements that invade DNA through a copy-and-paste mechanism known as retrotransposition. Their coordinated activities occur within a complex that includes a ...Group II introns are a class of retroelements that invade DNA through a copy-and-paste mechanism known as retrotransposition. Their coordinated activities occur within a complex that includes a maturase protein, which promotes splicing through an unknown mechanism. The mechanism of splice site exchange within the RNA active site during catalysis also remains unclear. We determined two cryo-EM structures at 3.6-Å resolution of a group II intron reverse splicing into DNA. These structures reveal that the branch-site domain VI helix swings 90°, enabling substrate exchange during DNA integration. The maturase assists catalysis through a transient RNA-protein contact with domain VI that positions the branch-site adenosine for lariat formation during forward splicing. These findings provide the first direct evidence of the role the maturase plays during group II intron catalysis. The domain VI dynamics closely parallel spliceosomal branch-site helix movement and provide strong evidence for a retroelement origin of the spliceosome.
Validation Report
SummaryFull reportAbout validation report
History
DepositionSep 6, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 14, 2019Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: T.el4h RNA
B: Sense Target DNA
C: Maturase reverse transcriptase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)361,21555
Polymers359,9523
Non-polymers1,26352
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: DNA/RNA hybrid T.el4h RNA


Mass: 280854.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermosynechococcus elongatus (Cyanobacteria)
Production host: Escherichia coli (E. coli)
#2: DNA chain Sense Target DNA


Mass: 14032.991 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Thermosynechococcus elongatus (Cyanobacteria)
#3: Protein/peptide Maturase reverse transcriptase


Mass: 65065.121 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermosynechococcus elongatus (strain BP-1) (Cyanobacteria)
Strain: BP-1 / Gene: tll0114 / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / References: UniProt: Q8DMK2
#4: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 51 / Source method: obtained synthetically / Formula: Mg / Magnesium
#5: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na / Sodium

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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: T.el4h group II intron retroelement / Type: COMPLEX / Entity ID: 1, 2, 3 / Source: RECOMBINANT
Source (natural)Organism: Thermosynechococcus elongatus (Cyanobacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
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 FIELDBright-field microscopy
Image recordingElectron dose: 58 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.15.2_3472refinement
PHENIX1.15.2_3472refinement
EM software
IDNameCategory
4GctfCTF correction
7Cootmodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 87612 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingPDB-ID: 4R0D
RefinementStereochemistry target values: CDL v1.2
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.010323427
f_angle_d1.219835784
f_chiral_restr0.05784644
f_plane_restr0.00821417
f_dihedral_angle_d17.46712032

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