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- EMDB-2310: Three-dimensional structure of active, full-length human telomera... -

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Basic information

Entry
Database: EMDB / ID: EMD-2310
TitleThree-dimensional structure of active, full-length human telomerase dimer, determined by single-particle electron microscopy in negative stain
Map data3D map of human telomerase dimer bound to G-overhang oligonucleotide (TTAGGG)2
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 / telomerase catalytic core complex / positive regulation of protein localization to nucleolus / siRNA transcription / telomerase activity ...positive regulation of hair cycle / template-free RNA nucleotidyltransferase / positive regulation of transdifferentiation / TERT-RMRP complex / DNA strand elongation / RNA-directed RNA polymerase complex / telomerase catalytic core complex / positive regulation of protein localization to nucleolus / siRNA transcription / telomerase activity / Regulation of MITF-M-dependent genes involved in DNA damage repair and senescence / : / RNA-templated DNA biosynthetic process / establishment of protein localization to telomere / nuclear telomere cap complex / siRNA processing / telomerase RNA binding / telomerase holoenzyme complex / positive regulation of vascular associated smooth muscle cell migration / DNA biosynthetic process / telomeric DNA binding / RNA-templated transcription / positive regulation of stem cell proliferation / mitochondrial nucleoid / negative regulation of cellular senescence / positive regulation of Wnt signaling pathway / telomere maintenance via telomerase / Telomere Extension By Telomerase / replicative senescence / negative regulation of extrinsic apoptotic signaling pathway in absence of ligand / positive regulation of G1/S transition of mitotic cell cycle / 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 / mitochondrion organization / positive regulation of D-glucose import / Formation of the beta-catenin:TCF transactivating complex / regulation of protein stability / PML body / transcription coactivator binding / positive regulation of miRNA transcription / RNA-directed DNA polymerase / positive regulation of angiogenesis / RNA-directed DNA polymerase activity / positive regulation of protein binding / protein-folding chaperone binding / cellular response to hypoxia / negative regulation of neuron apoptotic process / tRNA binding / chromosome, telomeric region / nuclear speck / negative regulation of gene expression / RNA-dependent RNA polymerase activity / nucleolus / protein homodimerization activity / DNA binding / RNA binding / nucleoplasm / identical protein binding / nucleus / metal ion binding / 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 (RT) catalytic domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Telomerase reverse transcriptase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 30.0 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 5, 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: 0.0974
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0974
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2310.png

Downloads & links

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Map

FileDownload / File: emd_2310.map.gz / Format: CCP4 / Size: 144.5 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D map of human telomerase dimer bound to G-overhang oligonucleotide (TTAGGG)2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.6 Å/pix.
x 28 pix.
= 184.8 Å		6.6 Å/pix.
x 28 pix.
= 184.8 Å		6.6 Å/pix.
x 48 pix.
= 316.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 6.6 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0974 / Movie #1: 0.0974
Minimum - Maximum-0.20594661 - 0.41761535
Average (Standard dev.)-0.01318102 (±0.08440322)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin261725
Dimensions284828
Spacing284828
CellA: 316.8 Å / B: 184.8 Å / C: 184.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.66.66.6
M x/y/z482828
origin x/y/z0.0000.0000.000
length x/y/z316.800184.800184.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS172625
NC/NR/NS482828
D min/max/mean-0.2060.418-0.013

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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: 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 ...Details: 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).
Oligomeric state: Dimer / Number unique components: 1
Molecular weightExperimental: 670 KDa / Theoretical: 626 KDa / Method: Sedimentation

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Macromolecule #1: Telomerase reverse transcriptase

MacromoleculeName: Telomerase reverse transcriptase / type: protein_or_peptide / ID: 1 / Name.synonym: TERT / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: Human Embryonic Kidney cells
Molecular weightExperimental: 127 KDa / Theoretical: 670 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

DetailsElectron tomography was used to calculate a low-resolution reference model. Distinct conformations were further classified by three-dimensional maximum-likelihood analysis in Fourier space (MLF3D).Density map calculated from a sub-population of 3,659 particles.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: OTHER / Software - Name: EMAN2, XMIPP
Details: 3D structure was refined using the low-resolution subtomogram average as a reference model for 3D single particle refinement with EMAN2. Distinct conformations were further classified by ...Details: 3D structure was refined using the low-resolution subtomogram average as a reference model for 3D single particle refinement with EMAN2. Distinct conformations were further classified by three-dimensional maximum-likelihood analysis in Fourier space (MLF3D). Density map calculated from a sub-population of 3,659 particles. The absolute hand and the overall correctness of the dimer structure were assessed using a modified version of tilt-pair validation method.
Number images used: 20127
Final two d classificationNumber classes: 100

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