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- EMDB-12174: The catalytic core lobe of human telomerase in complex with a tel... -

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Basic information

Entry
Database: EMDB / ID: EMD-12174
TitleThe catalytic core lobe of human telomerase in complex with a telomeric DNA substrate
Map dataPost-processed map
Sample
  • Complex: Human telomerase catalytic core in complex with a telomeric DNA substrate
    • Complex: Telomerase reverse transcriptase
      • Protein or peptide: Telomerase reverse transcriptase,Telomerase reverse transcriptase
      • RNA: RNA (256-MER)
    • Complex: Histone
      • Protein or peptide: Histone H2B
      • Protein or peptide: Histone H2A
    • Complex: DNA
      • DNA: DNA (5'-D(P*TP*TP*AP*GP*GP*G)-3')
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 / siRNA transcription / positive regulation of protein localization to nucleolus / 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 / siRNA transcription / positive regulation of protein localization to nucleolus / telomerase activity / telomerase RNA reverse transcriptase activity / 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 / telomeric DNA binding / DNA biosynthetic process / RNA-templated transcription / positive regulation of stem cell proliferation / mitochondrial nucleoid / negative regulation of cellular senescence / 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 / response to cadmium ion / negative regulation of endothelial cell apoptotic process / positive regulation of vascular associated smooth muscle cell proliferation / 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 / positive regulation of miRNA transcription / structural constituent of chromatin / RNA-directed DNA polymerase / positive regulation of angiogenesis / RNA-directed DNA polymerase activity / nucleosome / positive regulation of protein binding / cellular response to hypoxia / protein-folding chaperone binding / negative regulation of neuron apoptotic process / tRNA binding / chromosome, telomeric region / nuclear speck / protein heterodimerization activity / RNA-dependent RNA polymerase activity / negative regulation of gene expression / nucleolus / protein homodimerization activity / DNA binding / RNA binding / nucleoplasm / identical protein binding / metal ion binding / nucleus / plasma membrane / cytosol
Similarity search - Function
: / Telomerase reverse transcriptase, C-terminal extension / Telomerase ribonucleoprotein complex - RNA binding domain / Telomerase reverse transcriptase / Telomerase ribonucleoprotein complex - RNA-binding domain / Telomerase ribonucleoprotein complex - RNA binding domain / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site ...: / Telomerase reverse transcriptase, C-terminal extension / Telomerase ribonucleoprotein complex - RNA binding domain / Telomerase reverse transcriptase / Telomerase ribonucleoprotein complex - RNA-binding domain / Telomerase ribonucleoprotein complex - RNA binding domain / 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 / 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 / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Histone H2A / Histone H2B / Telomerase reverse transcriptase
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others) / Human (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 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: 0.014
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bg9
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7bg9
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12174.png

Downloads & links

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Map

FileDownload / File: emd_12174.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed map
Voxel sizeX=Y=Z: 1.11 Å
Density
Contour LevelBy AUTHOR: 0.014 / Movie #1: 0.014
Minimum - Maximum-0.052251868 - 0.08463936
Average (Standard dev.)0.00036842295 (±0.0027956716)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 266.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.111.111.11
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z266.400266.400266.400
α/β/γ90.00090.00090.000
start NX/NY/NZ278280246
NX/NY/NZ474891
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0520.0850.000

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Supplemental data

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Half map: #2

Fileemd_12174_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_12174_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 : Human telomerase catalytic core in complex with a telomeric DNA s...

EntireName: Human telomerase catalytic core in complex with a telomeric DNA substrate
Components
  • Complex: Human telomerase catalytic core in complex with a telomeric DNA substrate
    • Complex: Telomerase reverse transcriptase
      • Protein or peptide: Telomerase reverse transcriptase,Telomerase reverse transcriptase
      • RNA: RNA (256-MER)
    • Complex: Histone
      • Protein or peptide: Histone H2B
      • Protein or peptide: Histone H2A
    • Complex: DNA
      • DNA: DNA (5'-D(P*TP*TP*AP*GP*GP*G)-3')

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Supramolecule #1: Human telomerase catalytic core in complex with a telomeric DNA s...

SupramoleculeName: Human telomerase catalytic core in complex with a telomeric DNA substrate
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5

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Supramolecule #2: Telomerase reverse transcriptase

SupramoleculeName: Telomerase reverse transcriptase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #5
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #3: Histone

SupramoleculeName: Histone / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: DNA

SupramoleculeName: DNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: Telomerase reverse transcriptase,Telomerase reverse transcriptase

MacromoleculeName: Telomerase reverse transcriptase,Telomerase reverse transcriptase
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human) / Organ: Kidney
Molecular weightTheoretical: 149.158578 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKTAALAQHD EAVDNKFNKE QQNAFYEILH LPNLNEEQRN AFIQSLKDDP SQSANLLAEA KKLNDAQAPK VDNKFNKEQQ NAFYEILHL PNLNEEQRNA FIQSLKDDPS QSANLLAEAK KLNGAQAPKV DANSAGKSTD YDIPTTASEN LYFQGHKLGT F QGPWSHPQ ...String:
MKTAALAQHD EAVDNKFNKE QQNAFYEILH LPNLNEEQRN AFIQSLKDDP SQSANLLAEA KKLNDAQAPK VDNKFNKEQQ NAFYEILHL PNLNEEQRNA FIQSLKDDPS QSANLLAEAK KLNGAQAPKV DANSAGKSTD YDIPTTASEN LYFQGHKLGT F QGPWSHPQ FEKGSAGSAA GSGAGWSHPQ FEKSRPTTAS GTMPRAPRCR AVRSLLRSHY REVLPLATFV RRLGPQGWRL VQ RGDPAAF RALVAQCLVC VPWDARPPPA APSFRQVSCL KELVARVLQR LCERGAKNVL AFGFALLDGA RGGPPEAFTT SVR SYLPNT VTDALRGSGA WGLLLRRVGD DVLVHLLARC ALFVLVAPSC AYQVCGPPLY QLGAATQARP PPHASGPRRR LGCE RAWNH SVREAGVPLG LPAPGARRRG GSASRSLPLP KRPRRGAAPE PERTPVGQGS WAHPGRTRGP SDRGFCVVSP ARPAE EATS LEGALSGTRH SHPSVGRQHH AGPPSTSRPP RPWDTPCPPV YAETKHFLYS SGDKEQLRPS FLLSSLRPSL TGARRL VET IFLGSRPWMP GTPRRLPRLP QRYWQMRPLF LELLGNHAQC PYGVLLKTHC PLRAAVTPAA GVCAREKPQG SVAAPEE ED TDPRRLVQLL RQHSSPWQVY GFVRACLRRL VPPGLWGSRH NERRFLRNTK KFISLGKHAK LSLQELTWKM SVRDCAWL R RSPGVGCVPA AEHRLREEIL AKFLHWLMSV YVVELLRSFF YVTETTFQKN RLFFYRKSVW SKLQSIGIRQ HLKRVQLRE LSEAEVRQHR EARPALLTSR LRFIPKPDGL RPIVNMDYVV GARTFRREKR AERLTSRVKA LFSVLNYERA RRPGLLGASV LGLDDIHRA WRTFVLRVRA QDPPPELYFV KVDVTGAYDT IPQDRLTEVI ASIIKPQNTY CVRRYAVVQK AAHGHVRKAF K SHVSTLTD LQPYMRQFVA HLQETSPLRD AVVIEQSSSL NEASSGLFDV FLRFMCHHAV RIRGKSYVQC QGIPQGSILS TL LCSLCYG DMENKLFAGI RRDGLLLRLV DDFLLVTPHL THAKTFLRTL VRGVPEYGCV VNLRKTVVNF PVEDEALGGT AFV QMPAHG LFPWCGLLLD TRTLEVQSDY SSYARTSIRA SLTFNRGFKA GRNMRRKLFG VLRLKCHSLF LDLQVNSLQT VCTN IYKIL LLQAYRFHAC VLQLPFHQQV WKNPTFFLRV ISDTASLCYS ILKAKNAGMS LGAKGAAGPL PSEAVQWLCH QAFLL KLTR HRVTYVPLLG SLRTAQTQLS RKLPGTTLTA LEAAANPALP SDFKTILD

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Macromolecule #3: Histone H2B

MacromoleculeName: Histone H2B / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human (human) / Organ: Kidney
Molecular weightTheoretical: 18.074932 KDa
SequenceString:
MPDPAKSAPA PKKGSKKAVT KVQKKDGKKR KRSRKESYSV YVYKVLKQVH PDTGISSKAM GIMNSFVNDI FERIAGEASR LAHYNKRST ITSREIQTAV RLLLPGELAK HAVSEGTKAV TKYTSSNPRN LSPTKPGGSE DRQPPPSQLS AIPPFCLVLR A GIAGQV

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Macromolecule #4: Histone H2A

MacromoleculeName: Histone H2A / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human (human) / Organ: Kidney
Molecular weightTheoretical: 14.140584 KDa
SequenceString:
MSGRGKQGGK ARAKAKTRSS RAGLQFPVGR VRRLLRKGNY AERVGAGAPV YLAAVLEYLT AEILELAGNA ARDNKKTRII PRHLQLAIR NDEELNKLLG KVTIAQGGVL PNIQAVLLPK KTESHHKAKG K

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Macromolecule #2: DNA (5'-D(P*TP*TP*AP*GP*GP*G)-3')

MacromoleculeName: DNA (5'-D(P*TP*TP*AP*GP*GP*G)-3') / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.514567 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DA)(DG)(DG)(DG)

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Macromolecule #5: RNA (256-MER)

MacromoleculeName: RNA (256-MER) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Homo sapiens (human) / Organ: Kidney
Molecular weightTheoretical: 145.477797 KDa
SequenceString: GGGUUGCGGA GGGUGGGCCU GGGAGGGGUG GUGGCCAUUU UUUGUCUAAC CCUAACUGAG AAGGGCGUAG GCGCCGUGCU UUUGCUCCC CGCGCGCUGU UUUUCUCGCU GACUUUCAGC GGGCGGAAAA GCCUCGGCCU GCCGCCUUCC ACCGUUCAUU C UAGAGCAA ...String:
GGGUUGCGGA GGGUGGGCCU GGGAGGGGUG GUGGCCAUUU UUUGUCUAAC CCUAACUGAG AAGGGCGUAG GCGCCGUGCU UUUGCUCCC CGCGCGCUGU UUUUCUCGCU GACUUUCAGC GGGCGGAAAA GCCUCGGCCU GCCGCCUUCC ACCGUUCAUU C UAGAGCAA ACAAAAAAUG UCAGCUGCUG GCCCGUUCGC CCCUCCCGGG GACCUGCGGC GGGUCGCCUG CCCAGCCCCC GA ACCCCGC CUGGAGGCCG CGGUCGGCCC GGGGCUUCUC CGGAGGCACC CACUGCCACC GCGAAGAGUU GGGCUCUGUC AGC CGCGGG UCUCUCGGGG GCGAGGGCGA GGUUCAGGCC UUUCAGGCCG CAGGAAGAGG AACGGAGCGA GUCCCCGCGC GCGG CGCGA UUCCCUGAGC UGUGGGACGU GCACCCAGGA CUCGGCUCAC ACAUGC

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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 / Details: Blot for 4-5 seconds before plunging.

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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
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
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: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 168538
DetailsAll images were processed using RELION 3.1.
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 80
Output model

PDB-7bg9:
The catalytic core lobe of human telomerase in complex with a telomeric DNA substrate

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