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- EMDB-40985: Structure of a group II intron ribonucleoprotein in the pre-ligat... -

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Basic information

Entry
Database: EMDB / ID: EMD-40985
TitleStructure of a group II intron ribonucleoprotein in the pre-ligation (pre-2F) state
Map dataOverall Reconstruction of Pre-2F
Sample
  • Complex: Intron RNP complex in pre-2f
    • Protein or peptide: Group II intron reverse transcriptase/maturase
    • RNA: 5'exon
    • RNA: Intron
  • Ligand: CALCIUM IONCalcium
  • Ligand: AMMONIUM IONAmmonium
KeywordsRNP / RNA / Transferase-RNA complex
Function / homology
Function and homology information


RNA-directed DNA polymerase / RNA-directed DNA polymerase activity
Similarity search - Function
Group II intron, maturase-specific / Group II intron reverse transcriptase/maturase / Group II intron, maturase-specific domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Group II intron reverse transcriptase/maturase
Similarity search - Component
Biological species[Eubacterium] rectale (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsXu L / Liu T / Chung K / Pyle AM
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2023
Title: Structural insights into intron catalysis and dynamics during splicing.
Authors: Ling Xu / Tianshuo Liu / Kevin Chung / Anna Marie Pyle /
Abstract: The group II intron ribonucleoprotein is an archetypal splicing system with numerous mechanistic parallels to the spliceosome, including excision of lariat introns. Despite the importance of ...The group II intron ribonucleoprotein is an archetypal splicing system with numerous mechanistic parallels to the spliceosome, including excision of lariat introns. Despite the importance of branching in RNA metabolism, structural understanding of this process has remained elusive. Here we present a comprehensive analysis of three single-particle cryogenic electron microscopy structures captured along the splicing pathway. They reveal the network of molecular interactions that specifies the branchpoint adenosine and positions key functional groups to catalyse lariat formation and coordinate exon ligation. The structures also reveal conformational rearrangements of the branch helix and the mechanism of splice site exchange that facilitate the transition from branching to ligation. These findings shed light on the evolution of splicing and highlight the conservation of structural components, catalytic mechanism and dynamical strategies retained through time in premessenger RNA splicing machines.
History
DepositionJun 6, 2023-
Header (metadata) releaseNov 22, 2023-
Map releaseNov 22, 2023-
UpdateJan 3, 2024-
Current statusJan 3, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40985.png

Downloads & links

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Map

FileDownload / File: emd_40985.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOverall Reconstruction of Pre-2F
Voxel sizeX=Y=Z: 0.844 Å
Density
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-1.1132149 - 1.7189692
Average (Standard dev.)0.0010773883 (±0.0283247)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 324.096 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40985_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_40985_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Locally Refined (Left) Reconstruction of Pre-2F

Fileemd_40985_additional_1.map
AnnotationLocally Refined (Left) Reconstruction of Pre-2F
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Locally Refined (Right) Reconstruction of Pre-2F

Fileemd_40985_additional_2.map
AnnotationLocally Refined (Right) Reconstruction of Pre-2F
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Overall Reconstruction of Pre-2F Half Map A

Fileemd_40985_half_map_1.map
AnnotationOverall Reconstruction of Pre-2F Half Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Overall Reconstruction of Pre-2F Half Map B

Fileemd_40985_half_map_2.map
AnnotationOverall Reconstruction of Pre-2F Half Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Intron RNP complex in pre-2f

EntireName: Intron RNP complex in pre-2f
Components
  • Complex: Intron RNP complex in pre-2f
    • Protein or peptide: Group II intron reverse transcriptase/maturase
    • RNA: 5'exon
    • RNA: Intron
  • Ligand: CALCIUM IONCalcium
  • Ligand: AMMONIUM IONAmmonium

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Supramolecule #1: Intron RNP complex in pre-2f

SupramoleculeName: Intron RNP complex in pre-2f / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: [Eubacterium] rectale (bacteria)
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: Group II intron reverse transcriptase/maturase

MacromoleculeName: Group II intron reverse transcriptase/maturase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase
Source (natural)Organism: [Eubacterium] rectale (bacteria)
Molecular weightTheoretical: 49.083914 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDTSNLMEQI LSSDNLNRAY LQVVRNKGAE GVDGMKYTEL KEHLAKNGET IKGQLRTRKY KPQPARRVEI PKPDGGVRNL GVPTVTDRF IQQAIAQVLT PIYEEQFHDH SYGFRPNRCA QQAILTALNI MNDGNDWIVD IDLEKFFDTV NHDKLMTLIG R TIKDGDVI ...String:
MDTSNLMEQI LSSDNLNRAY LQVVRNKGAE GVDGMKYTEL KEHLAKNGET IKGQLRTRKY KPQPARRVEI PKPDGGVRNL GVPTVTDRF IQQAIAQVLT PIYEEQFHDH SYGFRPNRCA QQAILTALNI MNDGNDWIVD IDLEKFFDTV NHDKLMTLIG R TIKDGDVI SIVRKYLVSG IMIDDEYEDS IVGTPQGGNL SPLLANIMLN ELDKEMEKRG LNFVRYADDC IIMVGSEMSA NR VMRNISR FIEEKLGLKV NMTKSKVDRP SGLKYLGFGF YFDPRAHQFK AKPHAKSVAK FKKRMKELTC RSWGVSNSYK VEK LNQLIR GWINYFKIGS MKTLCKELDS RIRYRLRMCI WKQWKTPQNQ EKNLVKLGID RNTARRVAYT GKRIAYVCNK GAVN VAISN KRLASFGLIS MLDYYIEKCV TC

UniProtKB: Group II intron reverse transcriptase/maturase

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Macromolecule #2: 5'exon

MacromoleculeName: 5'exon / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: [Eubacterium] rectale (bacteria)
Molecular weightTheoretical: 2.097219 KDa
SequenceString:
UUUCUUU

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Macromolecule #3: Intron

MacromoleculeName: Intron / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: [Eubacterium] rectale (bacteria)
Molecular weightTheoretical: 208.395812 KDa
SequenceString: GUUUGCGCGC CAUGGGCGCG CUCUAACGGG UGUAAGUCCC GAACAUGCCC AGGUAGUGGG AAAUGUAUAG CCGAACAGCA AGGGUGUCU ACUGUGAGGU GGAAUCUGAA GGAAGCUGUA AGCGAAUCUC UGGUCCGACG GACAGAAAUC GCAUAUAAGG C UAGGCUUC ...String:
GUUUGCGCGC CAUGGGCGCG CUCUAACGGG UGUAAGUCCC GAACAUGCCC AGGUAGUGGG AAAUGUAUAG CCGAACAGCA AGGGUGUCU ACUGUGAGGU GGAAUCUGAA GGAAGCUGUA AGCGAAUCUC UGGUCCGACG GACAGAAAUC GCAUAUAAGG C UAGGCUUC GAGUGAUAAG CUGGCAAAGA ACAGUGAAGU CUAAUAACUA CCACGUUUGU AGAAGCAGAG UAAAUGCGGC GG AUAUAUG GAGAGAAAGA GCGUGCACCU UAAGCGUGGA GGUCUCACAG AGGUUUCAUU AGCCUAGUAA CAACGAACUG UGA GAAGUC AGCCGAGCCC AUAGUAGUGA AGAAGUCUCU GUAAUGGGGA UGGAGCGAAG GGGCGAACAA UCAAUCAGUU UGAG UAUGU CUCGUAUUGC AGAAAUGACA ACAUCUGCCG UAACCAAUCG GGUAAAAGGU GGUCAAAUCA AGCGAGACGG AAAGG AAAG AACGCAUGGA CACAAGUAAU CUAAUUUCGG UUAGAUUACU ACAUCGAAAA GUGUGUUACU UGUUAAGUUG AUUGAA CCG CCGUAUACGG AACCGUACGU ACGGUGGUGU GAGAGGUCGG AAUUUCUCAA UUAAGAGAAA UUCUUCCUAC UCGAUUG AA U

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 13 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #5: AMMONIUM ION

MacromoleculeName: AMMONIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: NH4
Molecular weightTheoretical: 18.038 Da
Chemical component information

ChemComp-NH4:
AMMONIUM ION / Ammonium

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 1.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7uin

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 234625
FSC plot (resolution estimation)

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