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- EMDB-5824: Negative-stain electron microscopy reconstruction of Tetrahymena ... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-5824 | |||||||||
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Title | Negative-stain electron microscopy reconstruction of Tetrahymena telomerase (f-p50, affinity labeled by one copy of anti-FLAG Fab) | |||||||||
![]() | 3D reconstruction of Tetrahymena telomerase (f-p50, affinity labeled by one copy of anti-FLAG Fab) using Random Conical Tilt (RCT) method | |||||||||
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![]() | telomerase / telomere / Tetrahymena | |||||||||
Biological species | ![]() ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 30.0 Å | |||||||||
![]() | Jiang J / Miracco EJ / Hong K / Eckert B / Chan H / Cash DD / Min B / Zhou ZH / Collins K / Feigon J | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 3.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 10.5 KB 10.5 KB | Display Display | ![]() |
Images | ![]() | 55.1 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 78.5 KB | Display | ![]() |
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Full document | ![]() | 77.6 KB | Display | |
Data in XML | ![]() | 493 B | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 5804C ![]() 5807C ![]() 5808C ![]() 5809C ![]() 5810C ![]() 5811C ![]() 5812C ![]() 5813C ![]() 5814C ![]() 5815C ![]() 5816C ![]() 5817C ![]() 5818C ![]() 5819C ![]() 5820C ![]() 5821C ![]() 5822C ![]() 5823C C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 3D reconstruction of Tetrahymena telomerase (f-p50, affinity labeled by one copy of anti-FLAG Fab) using Random Conical Tilt (RCT) method | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.41 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Telomerase holoenzyme (f-p50, labeled by anti-FLAG Fab) from Tetr...
Entire | Name: Telomerase holoenzyme (f-p50, labeled by anti-FLAG Fab) from Tetrahymena thermophila |
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Components |
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-Supramolecule #1000: Telomerase holoenzyme (f-p50, labeled by anti-FLAG Fab) from Tetr...
Supramolecule | Name: Telomerase holoenzyme (f-p50, labeled by anti-FLAG Fab) from Tetrahymena thermophila type: sample / ID: 1000 / Oligomeric state: Hetero-octamer / Number unique components: 2 |
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Molecular weight | Theoretical: 500 KDa |
-Macromolecule #1: Telomerase holoenzyme
Macromolecule | Name: Telomerase holoenzyme / type: protein_or_peptide / ID: 1 / Name.synonym: Telomerase / Number of copies: 1 / Oligomeric state: Hetero-octamer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 500 KDa |
-Macromolecule #2: Fab from monoclonal anit-FLAG M2 antibody
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 50 KDa |
-Experimental details
-Structure determination
Method | negative staining |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 8 / Details: 20 mM HEPES, 50 mM NaCl, 1 mM MgCl2, 1 mM TCEP |
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Staining | Type: NEGATIVE / Details: 0.8% uranyl formate |
Grid | Details: 200 mesh grid with thin carbon support |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
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Electron microscopy
Microscope | FEI TECNAI F20 |
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Temperature | Average: 298 K |
Date | May 11, 2012 |
Image recording | Category: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Number real images: 802 / Average electron dose: 40 e/Å2 / Bits/pixel: 16 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Calibrated 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 stage | Specimen 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
Details | The RCT 3D reconstruction was done using SPIDER. |
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CTF correction | Details: Each particle |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: OTHER / Software - Name: SPIDER / Number images used: 478 |