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- EMDB-5581: Negatively Stained TEM structure of trypanosomatid core editing c... -

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Basic information

Entry
Database: EMDB / ID: EMD-5581
TitleNegatively Stained TEM structure of trypanosomatid core editing complex (L-complex)
Map datareconstruction of negatively stained L-complex
Sample
  • Sample: L-Complex from L. major
  • Protein or peptide: Core editing complex
KeywordsRNA editing
Biological speciesLeishmania major (eukaryote)
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 Å
AuthorsLi F / Ge P / Hui WH / Atanasov I / Rogersa K / Guo Q / Osatoa D / Falickd AM / Zhou ZH / Simpson L
CitationJournal: Proc Natl Acad Sci U S A / Year: 2009
Title: Structure of the core editing complex (L-complex) involved in uridine insertion/deletion RNA editing in trypanosomatid mitochondria.
Authors: Feng Li / Peng Ge / Wong H Hui / Ivo Atanasov / Kestrel Rogers / Qiang Guo / Daren Osato / Arnold M Falick / Z Hong Zhou / Larry Simpson /
Abstract: Uridine insertion/deletion RNA editing is a unique form of posttranscriptional RNA processing that occurs in mitochondria of kinetoplastid protists. We have carried out 3D structural analyses of the ...Uridine insertion/deletion RNA editing is a unique form of posttranscriptional RNA processing that occurs in mitochondria of kinetoplastid protists. We have carried out 3D structural analyses of the core editing complex or "L (ligase)-complex" from Leishmania tarentolae mitochondria isolated by the tandem affinity purification procedure (TAP). The purified material, sedimented at 20-25S, migrated in a blue native gel at 1 MDa and exhibited both precleaved and full-cycle gRNA-mediated U-insertion and U-deletion in vitro activities. The purified L-complex was analyzed by electron tomography to determine the extent of heterogeneity. Three-dimensional structural comparisons of individual particles in the tomograms revealed that a majority of the complexes have a similar shape of a slender triangle. An independent single-particle reconstruction, using a featureless Gaussian ball as the initial model, converged to a similar triangular structure. Another single-particle reconstruction, using the averaged tomography structure as the initial model, yielded a similar structure. The REL1 ligase was localized on the model to the base of the apex by decoration with REL1-specific IgG. This structure should prove useful for a detailed analysis of the editing reaction.
History
DepositionJan 31, 2013-
Header (metadata) releaseFeb 13, 2013-
Map releaseFeb 13, 2013-
UpdateFeb 13, 2013-
Current statusFeb 13, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 5.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5581_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5581.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of negatively stained L-complex
Voxel sizeX=Y=Z: 2.2 Å
Density
Contour LevelBy AUTHOR: 5.82 / Movie #1: 5.5
Minimum - Maximum-2.79889107 - 12.01914024
Average (Standard dev.)0.0 (±0.90487319)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-44-44-44
Dimensions192192192
Spacing192192192
CellA=B=C: 422.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.22.22.2
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z422.400422.400422.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-5029166
NX/NY/NZ106122134
MAP C/R/S123
start NC/NR/NS-44-44-44
NC/NR/NS192192192
D min/max/mean-2.79912.0190.000

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Supplemental data

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Sample components

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Entire : L-Complex from L. major

EntireName: L-Complex from L. major
Components
  • Sample: L-Complex from L. major
  • Protein or peptide: Core editing complex

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Supramolecule #1000: L-Complex from L. major

SupramoleculeName: L-Complex from L. major / type: sample / ID: 1000 / Oligomeric state: A single complex / Number unique components: 1
Molecular weightExperimental: 1.0 MDa / Method: blue native gel

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Macromolecule #1: Core editing complex

MacromoleculeName: Core editing complex / type: protein_or_peptide / ID: 1 / Name.synonym: L-Complex / Number of copies: 1 / Oligomeric state: complex of many subunits / Recombinant expression: Yes
Source (natural)Organism: Leishmania major (eukaryote) / synonym: Leishmania / Organelle: Mitochondria
Molecular weightExperimental: 1.0 MDa
Recombinant expressionOrganism: Leishmania tarentolae (eukaryote) / Recombinant plasmid: pMRP1-TAP

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6 / Details: 20 mM Tris, pH 7.6, 60 mM KCl, 10 mM MgCl2
StainingType: NEGATIVE / Details: 2% uranyl acetate for 2 min
GridDetails: continuous carbon grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 68200 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 70000
Sample stageSpecimen holder: Single tilt room temperature holder / Specimen holder model: SIDE ENTRY, EUCENTRIC
TemperatureAverage: 300 K
DateAug 1, 2008
Image recordingCategory: CCD / Film or detector model: GENERIC CCD / Digitization - Sampling interval: 15 µm / Number real images: 26 / Average electron dose: 400 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: phase flip
Final two d classificationNumber classes: 879
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 12500

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