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- EMDB-2311: Three-dimensional structure of active, full-length human telomera... -

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Entry
Database: EMDB / ID: EMD-2311
TitleThree-dimensional structure of active, full-length human telomerase. Independently refined open monomer structure, determined by single-particle electron microscopy in negative stain
Map dataThree-dimensional structure of active, full-length human telomerase. Independently refined open monomer structure, determined by single-particle electron microscopy in negative stain
Sample
  • Sample: Three-dimensional structure of active, full-length human telomerase dimer, determined by single-particle electron microscopy in negative stain
  • Protein or peptide: Telomerase reverse transcriptase
KeywordsTelomerase reverse transcriptase / human / telomere length extension
Function / homology
Function and homology information


positive regulation of hair cycle / template-free RNA nucleotidyltransferase / positive regulation of transdifferentiation / TERT-RMRP complex / DNA strand elongation / RNA-directed RNA polymerase complex / positive regulation of protein localization to nucleolus / siRNA transcription / telomerase catalytic core complex / RNA-templated DNA biosynthetic process ...positive regulation of hair cycle / template-free RNA nucleotidyltransferase / positive regulation of transdifferentiation / TERT-RMRP complex / DNA strand elongation / RNA-directed RNA polymerase complex / positive regulation of protein localization to nucleolus / siRNA transcription / telomerase catalytic core complex / RNA-templated DNA biosynthetic process / establishment of protein localization to telomere / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / nuclear telomere cap complex / siRNA processing / telomere maintenance via recombination / positive regulation of vascular associated smooth muscle cell migration / telomerase holoenzyme complex / telomerase RNA binding / RNA-templated transcription / DNA biosynthetic process / telomeric DNA binding / positive regulation of stem cell proliferation / mitochondrial nucleoid / negative regulation of cellular senescence / Telomere Extension By Telomerase / replicative senescence / positive regulation of Wnt signaling pathway / negative regulation of extrinsic apoptotic signaling pathway in absence of ligand / positive regulation of G1/S transition of mitotic cell cycle / telomere maintenance via telomerase / RNA-directed DNA polymerase activity / negative regulation of endothelial cell apoptotic process / response to cadmium ion / positive regulation of vascular associated smooth muscle cell proliferation / telomere maintenance / positive regulation of nitric-oxide synthase activity / positive regulation of D-glucose import / mitochondrion organization / Formation of the beta-catenin:TCF transactivating complex / transcription coactivator binding / regulation of protein stability / PML body / positive regulation of miRNA transcription / RNA-directed DNA polymerase / telomerase activity / protein import into nucleus / positive regulation of angiogenesis / positive regulation of protein binding / protein-folding chaperone binding / heart development / cellular response to hypoxia / negative regulation of neuron apoptotic process / tRNA binding / chromosome, telomeric region / nuclear speck / negative regulation of gene expression / RNA-directed RNA polymerase activity / nucleolus / protein homodimerization activity / DNA binding / RNA binding / nucleoplasm / metal ion binding / identical protein binding / nucleus / plasma membrane / cytosol
Similarity search - Function
: / Telomerase reverse transcriptase, C-terminal extension / Telomerase ribonucleoprotein complex - RNA binding domain / Telomerase reverse transcriptase / Telomerase reverse transcriptase / Telomerase ribonucleoprotein complex - RNA-binding domain / Telomerase ribonucleoprotein complex - RNA binding domain / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Telomerase reverse transcriptase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 Å
AuthorsSauerwald A / Sandin S / Cristofari G / Scheres SHW / Lingner J / Rhodes D
CitationJournal: Nat Struct Mol Biol / Year: 2013
Title: Structure of active dimeric human telomerase.
Authors: Anselm Sauerwald / Sara Sandin / Gaël Cristofari / Sjors H W Scheres / Joachim Lingner / Daniela Rhodes /
Abstract: Telomerase contains a large RNA subunit, TER, and a protein catalytic subunit, TERT. Whether telomerase functions as a monomer or dimer has been a matter of debate. Here we report biochemical and ...Telomerase contains a large RNA subunit, TER, and a protein catalytic subunit, TERT. Whether telomerase functions as a monomer or dimer has been a matter of debate. Here we report biochemical and labeling data that show that in vivo-assembled human telomerase contains two TERT subunits and binds two telomeric DNA substrates. Notably, catalytic activity requires both TERT active sites to be functional, which demonstrates that human telomerase functions as a dimer. We also present the three-dimensional structure of the active full-length human telomerase dimer, determined by single-particle EM in negative stain. Telomerase has a bilobal architecture with the two monomers linked by a flexible interface. The monomer reconstruction at 23-Å resolution and fitting of the atomic structure of the TERT subunit from beetle Tribolium castaneum into the EM density reveals the spatial relationship between RNA and protein subunits, providing insights into telomerase architecture.
History
DepositionFeb 7, 2013-
Header (metadata) releaseMar 13, 2013-
Map releaseMar 20, 2013-
UpdateApr 17, 2013-
Current statusApr 17, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0767
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0767
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2311.png

Downloads & links

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Map

FileDownload / File: emd_2311.map.gz / Format: CCP4 / Size: 53.7 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThree-dimensional structure of active, full-length human telomerase. Independently refined open monomer structure, determined by single-particle electron microscopy in negative stain
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.6 Å/pix.
x 24 pix.
= 158.4 Å		6.6 Å/pix.
x 24 pix.
= 158.4 Å		6.6 Å/pix.
x 24 pix.
= 158.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 6.6 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0767 / Movie #1: 0.0767
Minimum - Maximum-0.21785364 - 0.27877218
Average (Standard dev.)-0.01974571 (±0.06051278)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin553
Dimensions242424
Spacing242424
CellA=B=C: 158.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.66.66.6
M x/y/z242424
origin x/y/z0.0000.0000.000
length x/y/z158.400158.400158.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS553
NC/NR/NS242424
D min/max/mean-0.2180.279-0.020

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Supplemental data

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Sample components

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Entire : Three-dimensional structure of active, full-length human telomera...

EntireName: Three-dimensional structure of active, full-length human telomerase dimer, determined by single-particle electron microscopy in negative stain
Components
  • Sample: Three-dimensional structure of active, full-length human telomerase dimer, determined by single-particle electron microscopy in negative stain
  • Protein or peptide: Telomerase reverse transcriptase

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Supramolecule #1000: Three-dimensional structure of active, full-length human telomera...

SupramoleculeName: Three-dimensional structure of active, full-length human telomerase dimer, determined by single-particle electron microscopy in negative stain
type: sample / ID: 1000
Details: Independently refined open monomer structure. The composition analysed by mass spectroscopy.Purified telomerase contains the hTERT subunits and two accessory proteins, Nop10 and dyskerin. ...Details: Independently refined open monomer structure. The composition analysed by mass spectroscopy.Purified telomerase contains the hTERT subunits and two accessory proteins, Nop10 and dyskerin. Telomerase complexes have a molecular weight consistent with that of a dimer consisting of two hTERT (127 kDa) and two hTER (153 kDa) subunits, as well as the two accessory proteins Nop10 (7.7 kDa) and dyskerin (58 kDA).
Number unique components: 1
Molecular weightMethod: Sedimentation

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Macromolecule #1: Telomerase reverse transcriptase

MacromoleculeName: Telomerase reverse transcriptase / type: protein_or_peptide / ID: 1 / Name.synonym: TERT / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: Human Embryonic Kidney cells
Molecular weightExperimental: 127 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK239T / Recombinant plasmid: pCDNA6
SequenceUniProtKB: Telomerase reverse transcriptase / GO: telomere maintenance / InterPro: Telomerase reverse transcriptase

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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

Concentration0.01 mg/mL
BufferpH: 7.6 / Details: 20 mM Tris, 150 mM KCl, 1 mM MgCl2
StainingType: NEGATIVE
Details: Continuous carbon-coated grids were freshly prepared and glow-discharged before use. 13 microl of telomerase sample (8-10 nM) were deposited on the grid for 15-30 minutes, blotted with ...Details: Continuous carbon-coated grids were freshly prepared and glow-discharged before use. 13 microl of telomerase sample (8-10 nM) were deposited on the grid for 15-30 minutes, blotted with filter paper and negatively stained with 2 drops of 1-2% (w/v) uranyl acetate solution.
GridDetails: 200 mesh carbon coated with thin carbon, glow discharged
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Alignment procedureLegacy - Astigmatism: Corrected at 100,000 times magnification
DetailsThe films were developed in Kodak developer at full strength for 12 min.
DateJan 1, 2011
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 482 / Average electron dose: 10 e/Å2 / Details: The micrographs were compressed x4 / Od range: 1.4
Electron beamAcceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsCalibrated magnification: 42550 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.0015 µm / Nominal defocus min: 0.001 µm / Nominal magnification: 42000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC

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Image processing

Details4690 sub-images were selected of the open monomer from 2D class averages of telomerase dimer side-views, used for independent monomer refinement.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: EMAN2, XMIPP / Number images used: 4690

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Atomic model buiding 1

Initial modelPDB ID:

3kyl


Chain - Chain ID: A
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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