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- EMDB-12176: Low resolution reconstruction of whole human telomerase class 5 -

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Basic information

Entry
Database: EMDB / ID: EMD-12176
TitleLow resolution reconstruction of whole human telomerase class 5
Map data
Sample
  • Complex: Whole human telomerase reconstruction class 5
Function / homology
Function and homology information


telomere formation via telomerase / box H/ACA scaRNP complex / box H/ACA telomerase RNP complex / telomerase RNA localization to Cajal body / protein localization to Cajal body / Cajal body organization / snoRNA guided rRNA pseudouridine synthesis / box H/ACA snoRNP complex / rRNA pseudouridine synthesis / box H/ACA sno(s)RNA 3'-end processing ...telomere formation via telomerase / box H/ACA scaRNP complex / box H/ACA telomerase RNP complex / telomerase RNA localization to Cajal body / protein localization to Cajal body / Cajal body organization / snoRNA guided rRNA pseudouridine synthesis / box H/ACA snoRNP complex / rRNA pseudouridine synthesis / box H/ACA sno(s)RNA 3'-end processing / snRNA pseudouridine synthesis / Isomerases; Intramolecular transferases; Transferring other groups / positive regulation of hair cycle / template-free RNA nucleotidyltransferase / positive regulation of transdifferentiation / TERT-RMRP complex / DNA strand elongation / mRNA pseudouridine synthesis / RNA-directed RNA polymerase complex / telomerase catalytic core complex / enzyme-directed rRNA pseudouridine synthesis / siRNA transcription / pseudouridine synthesis / telomerase RNA stabilization / positive regulation of protein localization to nucleolus / telomerase activity / box H/ACA snoRNA binding / regulation of telomerase RNA localization to Cajal body / pseudouridine synthase activity / telomerase RNA reverse transcriptase activity / RNA-templated DNA biosynthetic process / establishment of protein localization to telomere / scaRNA localization to Cajal body / positive regulation of protein localization to Cajal body / positive regulation of establishment of protein localization to telomere / positive regulation of telomerase RNA localization to Cajal body / nuclear telomere cap complex / siRNA processing / sno(s)RNA-containing ribonucleoprotein complex / telomerase RNA binding / telomerase holoenzyme complex / positive regulation of vascular associated smooth muscle cell migration / rRNA modification in the nucleus and cytosol / positive regulation of double-strand break repair / telomeric DNA binding / positive regulation of double-strand break repair via homologous recombination / DNA biosynthetic process / RNA-templated transcription / positive regulation of stem cell proliferation / positive regulation of double-strand break repair via nonhomologous end joining / mitochondrial nucleoid / Association of TriC/CCT with target proteins during biosynthesis / negative regulation of cellular senescence / RNA folding / Telomere Extension By Telomerase / positive regulation of Wnt signaling pathway / telomere maintenance via telomerase / replicative senescence / negative regulation of extrinsic apoptotic signaling pathway in absence of ligand / positive regulation of G1/S transition of mitotic cell cycle / Cajal body / RNA processing / response to cadmium ion / negative regulation of endothelial cell apoptotic process / maturation of LSU-rRNA / positive regulation of telomerase activity / positive regulation of vascular associated smooth muscle cell proliferation / positive regulation of DNA repair / positive regulation of telomere maintenance via telomerase / telomere maintenance / mitochondrion organization / positive regulation of nitric-oxide synthase activity / positive regulation of glucose import / Formation of the beta-catenin:TCF transactivating complex / regulation of protein stability / transcription coactivator binding / PML body / fibrillar center / rRNA processing / positive regulation of miRNA transcription / structural constituent of chromatin / RNA-directed DNA polymerase / positive regulation of angiogenesis / RNA-directed DNA polymerase activity / nucleosome / site of double-strand break / positive regulation of protein binding / cytosolic large ribosomal subunit / histone binding / cellular response to hypoxia / protein-folding chaperone binding / negative regulation of neuron apoptotic process / tRNA binding / chromosome, telomeric region / nuclear body / nuclear speck / protein heterodimerization activity / RNA-dependent RNA polymerase activity / negative regulation of gene expression / DNA repair
Similarity search - Function
H/ACA ribonucleoprotein complex, subunit Gar1/Naf1 / H/ACA RNP complex subunit Gar1/Naf1, Cbf5-binding domain / Gar1/Naf1 RNA binding region / tRNA pseudouridine synthase B family / Dyskerin-like / DKCLD (NUC011) domain / DKCLD (NUC011) domain / : / Telomerase reverse transcriptase, C-terminal extension / H/ACA ribonucleoprotein complex, subunit Nop10 ...H/ACA ribonucleoprotein complex, subunit Gar1/Naf1 / H/ACA RNP complex subunit Gar1/Naf1, Cbf5-binding domain / Gar1/Naf1 RNA binding region / tRNA pseudouridine synthase B family / Dyskerin-like / DKCLD (NUC011) domain / DKCLD (NUC011) domain / : / Telomerase reverse transcriptase, C-terminal extension / H/ACA ribonucleoprotein complex, subunit Nop10 / H/ACA ribonucleoprotein complex, subunit Nop10 superfamily / Nucleolar RNA-binding protein, Nop10p family / Pseudouridine synthase II, N-terminal / tRNA pseudouridylate synthase B, C-terminal / TruB family pseudouridylate synthase (N terminal domain) / tRNA pseudouridylate synthase B C-terminal domain / Uncharacterised domain CHP00451 / PUA domain / Telomerase ribonucleoprotein complex - RNA binding domain / Telomerase reverse transcriptase / Telomerase ribonucleoprotein complex - RNA-binding domain / Telomerase ribonucleoprotein complex - RNA binding domain / Pseudouridine synthase, catalytic domain superfamily / Putative RNA-binding Domain in PseudoUridine synthase and Archaeosine transglycosylase / PUA domain / PUA domain profile. / PUA domain superfamily / H/ACA ribonucleoprotein complex, subunit Nhp2-like / PUA-like superfamily / Ribosomal protein L7Ae/L8/Nhp2 family / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A / Histone 2A / Ribosomal protein L7Ae/L30e/S12e/Gadd45 / Ribosomal protein L7Ae/L30e/S12e/Gadd45 family / 50S ribosomal protein L30e-like / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / Histone-fold / Translation protein, beta-barrel domain superfamily / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Histone H2A / Histone H2B / Telomerase reverse transcriptase / H/ACA ribonucleoprotein complex subunit DKC1 / Telomerase Cajal body protein 1 / H/ACA ribonucleoprotein complex subunit 3 / H/ACA ribonucleoprotein complex subunit 2 / H/ACA ribonucleoprotein complex subunit 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsNguyen THD / Ghanim GE / Fountain AJ / van Roon AMM / Rangan R / Das R / Collins K
Funding support United Kingdom, United States, 2 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_1201/19 United Kingdom
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM054198 United States
CitationJournal: Nature / Year: 2021
Title: Structure of human telomerase holoenzyme with bound telomeric DNA.
Authors: George E Ghanim / Adam J Fountain / Anne-Marie M van Roon / Ramya Rangan / Rhiju Das / Kathleen Collins / Thi Hoang Duong Nguyen /
Abstract: Telomerase adds telomeric repeats at chromosome ends to compensate for the telomere loss that is caused by incomplete genome end replication. In humans, telomerase is upregulated during embryogenesis ...Telomerase adds telomeric repeats at chromosome ends to compensate for the telomere loss that is caused by incomplete genome end replication. In humans, telomerase is upregulated during embryogenesis and in cancers, and mutations that compromise the function of telomerase result in disease. A previous structure of human telomerase at a resolution of 8 Å revealed a vertebrate-specific composition and architecture, comprising a catalytic core that is flexibly tethered to an H and ACA (hereafter, H/ACA) box ribonucleoprotein (RNP) lobe by telomerase RNA. High-resolution structural information is necessary to develop treatments that can effectively modulate telomerase activity as a therapeutic approach against cancers and disease. Here we used cryo-electron microscopy to determine the structure of human telomerase holoenzyme bound to telomeric DNA at sub-4 Å resolution, which reveals crucial DNA- and RNA-binding interfaces in the active site of telomerase as well as the locations of mutations that alter telomerase activity. We identified a histone H2A-H2B dimer within the holoenzyme that was bound to an essential telomerase RNA motif, which suggests a role for histones in the folding and function of telomerase RNA. Furthermore, this structure of a eukaryotic H/ACA RNP reveals the molecular recognition of conserved RNA and protein motifs, as well as interactions that are crucial for understanding the molecular pathology of many mutations that cause disease. Our findings provide the structural details of the assembly and active site of human telomerase, which paves the way for the development of therapeutic agents that target this enzyme.
History
DepositionJan 6, 2021-
Header (metadata) releaseApr 28, 2021-
Map releaseApr 28, 2021-
UpdateJun 2, 2021-
Current statusJun 2, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12176.png

Downloads & links

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Map

FileDownload / File: emd_12176.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.11 Å
Density
Contour LevelBy AUTHOR: 0.008 / Movie #1: 0.008
Minimum - Maximum-0.023158103 - 0.05311676
Average (Standard dev.)0.00017281379 (±0.0013025529)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 444.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.111.111.11
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z444.000444.000444.000
α/β/γ90.00090.00090.000
start NX/NY/NZ7297100
NX/NY/NZ181127145
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0230.0530.000

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Supplemental data

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Half map: #2

Fileemd_12176_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_12176_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Whole human telomerase reconstruction class 5

EntireName: Whole human telomerase reconstruction class 5
Components
  • Complex: Whole human telomerase reconstruction class 5

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Supramolecule #1: Whole human telomerase reconstruction class 5

SupramoleculeName: Whole human telomerase reconstruction class 5 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Details: Low resolution reconstruction of human telomerase containing both catalytic core and H/ACA lobe linked by flexible RNA linkers.
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: 293T / Recombinant plasmid: pcDNA3.1

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride
2.0 mMMgCl2magnesium chloride
0.05 %(C2H4O)nC14H22OIgepal CA630
1.0 %C12H22O11Trehalose
1.0 mMC4H10O2S2DTT
GridModel: C-flat / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 5.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 78.0 K
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 2 / Number real images: 43639 / Average exposure time: 1.0 sec. / Average electron dose: 47.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 15760434
CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: EMDB MAP
EMDB ID:

7521

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 463013
DetailsAll images were processed using RELION 3.1.
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT

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