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- EMDB-5822: Negative-stain electron microscopy reconstruction of Tetrahymena ... -

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Basic information

Entry
Database: EMDB / ID: EMD-5822
TitleNegative-stain electron microscopy reconstruction of Tetrahymena telomerase (Teb1-f, affinity labeled by anti-FLAG Fab)
Map data3D reconstruction of Tetrahymena telomerase (Teb1-f, affinity labeled by anti-FLAG Fab) using Random Conical Tilt (RCT) method
Sample
  • Sample: Telomerase holoenzyme (Teb1-f, labeled by anti-FLAG Fab) from Tetrahymena thermophila
  • Protein or peptide: Telomerase holoenzyme
  • Protein or peptide: Fab from monoclonal anit-FLAG M2 antibody
Keywordstelomerase / telomere / Tetrahymena
Biological speciesTetrahymena thermophila (eukaryote) / Mus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 30.0 Å
AuthorsJiang J / Miracco EJ / Hong K / Eckert B / Chan H / Cash DD / Min B / Zhou ZH / Collins K / Feigon J
CitationJournal: Nature / Year: 2013
Title: The architecture of Tetrahymena telomerase holoenzyme.
Authors: Jiansen Jiang / Edward J Miracco / Kyungah Hong / Barbara Eckert / Henry Chan / Darian D Cash / Bosun Min / Z Hong Zhou / Kathleen Collins / Juli Feigon /
Abstract: Telomerase adds telomeric repeats to chromosome ends using an internal RNA template and a specialized telomerase reverse transcriptase (TERT), thereby maintaining genome integrity. Little is known ...Telomerase adds telomeric repeats to chromosome ends using an internal RNA template and a specialized telomerase reverse transcriptase (TERT), thereby maintaining genome integrity. Little is known about the physical relationships among protein and RNA subunits within a biologically functional holoenzyme. Here we describe the architecture of Tetrahymena thermophila telomerase holoenzyme determined by electron microscopy. Six of the seven proteins and the TERT-binding regions of telomerase RNA (TER) have been localized by affinity labelling. Fitting with high-resolution structures reveals the organization of TERT, TER and p65 in the ribonucleoprotein (RNP) catalytic core. p50 has an unanticipated role as a hub between the RNP catalytic core, p75-p19-p45 subcomplex, and the DNA-binding Teb1. A complete in vitro holoenzyme reconstitution assigns function to these interactions in processive telomeric repeat synthesis. These studies provide the first view of the extensive network of subunit associations necessary for telomerase holoenzyme assembly and physiological function.
History
DepositionDec 2, 2013-
Header (metadata) releaseJan 15, 2014-
Map releaseJan 15, 2014-
UpdateJan 15, 2014-
Current statusJan 15, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0123
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0123
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5822.png

Downloads & links

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Map

FileDownload / File: emd_5822.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of Tetrahymena telomerase (Teb1-f, affinity labeled by anti-FLAG Fab) using Random Conical Tilt (RCT) method
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.41 Å/pix.
x 96 pix.
= 423.36 Å		4.41 Å/pix.
x 96 pix.
= 423.36 Å		4.41 Å/pix.
x 96 pix.
= 423.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.41 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0123 / Movie #1: 0.0123
Minimum - Maximum-0.0827905 - 0.08638466
Average (Standard dev.)-0.00027157 (±0.00551023)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-48-48-48
Dimensions969696
Spacing969696
CellA=B=C: 423.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.414.414.41
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z423.360423.360423.360
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-48-48-48
NC/NR/NS969696
D min/max/mean-0.0830.086-0.000

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Supplemental data

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Sample components

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Entire : Telomerase holoenzyme (Teb1-f, labeled by anti-FLAG Fab) from Tet...

EntireName: Telomerase holoenzyme (Teb1-f, labeled by anti-FLAG Fab) from Tetrahymena thermophila
Components
  • Sample: Telomerase holoenzyme (Teb1-f, labeled by anti-FLAG Fab) from Tetrahymena thermophila
  • Protein or peptide: Telomerase holoenzyme
  • Protein or peptide: Fab from monoclonal anit-FLAG M2 antibody

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Supramolecule #1000: Telomerase holoenzyme (Teb1-f, labeled by anti-FLAG Fab) from Tet...

SupramoleculeName: Telomerase holoenzyme (Teb1-f, labeled by anti-FLAG Fab) from Tetrahymena thermophila
type: sample / ID: 1000 / Oligomeric state: Hetero-octamer / Number unique components: 2
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: Telomerase holoenzyme

MacromoleculeName: Telomerase holoenzyme / type: protein_or_peptide / ID: 1 / Name.synonym: Telomerase / Number of copies: 1 / Oligomeric state: Hetero-octamer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Tetrahymena thermophila (eukaryote) / synonym: Tetrahymena
Molecular weightTheoretical: 500 KDa

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Macromolecule #2: Fab from monoclonal anit-FLAG M2 antibody

MacromoleculeName: Fab from monoclonal anit-FLAG M2 antibody / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Mus musculus (house mouse) / synonym: Mouse
Molecular weightTheoretical: 50 KDa

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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: 8 / Details: 20 mM HEPES, 50 mM NaCl, 1 mM MgCl2, 1 mM TCEP
StainingType: NEGATIVE / Details: 0.8% uranyl formate
GridDetails: 200 mesh grid with thin carbon support
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 298 K
DateMay 18, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Number real images: 339 / Average electron dose: 40 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 68027 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.7 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 70000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle min: 65 / Tilt angle max: 65
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe RCT 3D reconstruction was done using SPIDER.
CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: OTHER / Software - Name: SPIDER / Number images used: 654

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