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- PDB-8ouf: The H/ACA RNP lobe of human telomerase with the dyskerin thumb lo... -

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Basic information

Entry
Database: PDB / ID: 8ouf
TitleThe H/ACA RNP lobe of human telomerase with the dyskerin thumb loop in an open conformation
Components
  • (H/ACA ribonucleoprotein complex subunit ...) x 4
  • Human telomerase RNATelomerase RNA component
  • Telomerase Cajal body protein 1
KeywordsRNA BINDING PROTEIN / telomerase / H/ACA / pseudouridylation / ribonucleoprotein
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 / mRNA pseudouridine synthesis / enzyme-directed rRNA pseudouridine synthesis / pseudouridine synthesis / telomerase RNA stabilization / telomerase activity / box H/ACA snoRNA binding / regulation of telomerase RNA localization to Cajal body / pseudouridine synthase activity / 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 / sno(s)RNA-containing ribonucleoprotein complex / telomerase RNA binding / telomerase holoenzyme complex / rRNA modification in the nucleus and cytosol / positive regulation of double-strand break repair / positive regulation of double-strand break repair via homologous recombination / positive regulation of double-strand break repair via nonhomologous end joining / Association of TriC/CCT with target proteins during biosynthesis / RNA folding / Telomere Extension By Telomerase / telomere maintenance via telomerase / Cajal body / RNA processing / maturation of LSU-rRNA / positive regulation of telomerase activity / positive regulation of DNA repair / positive regulation of telomere maintenance via telomerase / fibrillar center / rRNA processing / site of double-strand break / cytosolic large ribosomal subunit / histone binding / protein-folding chaperone binding / chromosome, telomeric region / nuclear body / DNA repair / ubiquitin protein ligase binding / protein-containing complex binding / nucleolus / RNA binding / nucleoplasm / identical protein binding / nucleus / cytosol / cytoplasm
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 / H/ACA ribonucleoprotein complex, subunit Nop10 / H/ACA ribonucleoprotein complex, subunit Nop10 superfamily / Nucleolar RNA-binding protein, Nop10p family ...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 / 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 / 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 / Ribosomal protein L7Ae/L30e/S12e/Gadd45 / Ribosomal protein L7Ae/L30e/S12e/Gadd45 family / 50S ribosomal protein L30e-like / 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
Similarity search - Domain/homology
: / RNA / RNA (> 10) / RNA (> 100) / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsGhanim, G.E. / Sekne, Z. / van Roon, A.M.M. / Balch, S. / Nguyen, T.H.D.
Funding support United Kingdom, United States, European Union, 3items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI)MC_UP_1201/19 United Kingdom
Jane Coffin Childs (JCC) Fund United States
European Molecular Biology Organization (EMBO)European Union
CitationJournal: Nat Commun / Year: 2024
Title: 2.7 Å cryo-EM structure of human telomerase H/ACA ribonucleoprotein.
Authors: George E Ghanim / Zala Sekne / Sebastian Balch / Anne-Marie M van Roon / Thi Hoang Duong Nguyen /
Abstract: Telomerase is a ribonucleoprotein (RNP) enzyme that extends telomeric repeats at eukaryotic chromosome ends to counterbalance telomere loss caused by incomplete genome replication. Human telomerase ...Telomerase is a ribonucleoprotein (RNP) enzyme that extends telomeric repeats at eukaryotic chromosome ends to counterbalance telomere loss caused by incomplete genome replication. Human telomerase is comprised of two distinct functional lobes tethered by telomerase RNA (hTR): a catalytic core, responsible for DNA extension; and a Hinge and ACA (H/ACA) box RNP, responsible for telomerase biogenesis. H/ACA RNPs also have a general role in pseudouridylation of spliceosomal and ribosomal RNAs, which is critical for the biogenesis of the spliceosome and ribosome. Much of our structural understanding of eukaryotic H/ACA RNPs comes from structures of the human telomerase H/ACA RNP. Here we report a 2.7 Å cryo-electron microscopy structure of the telomerase H/ACA RNP. The significant improvement in resolution over previous 3.3 Å to 8.2 Å structures allows us to uncover new molecular interactions within the H/ACA RNP. Many disease mutations are mapped to these interaction sites. The structure also reveals unprecedented insights into a region critical for pseudouridylation in canonical H/ACA RNPs. Together, our work advances understanding of telomerase-related disease mutations and the mechanism of pseudouridylation by eukaryotic H/ACA RNPs.
History
DepositionApr 22, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 7, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Human telomerase RNA
C: H/ACA ribonucleoprotein complex subunit DKC1
D: H/ACA ribonucleoprotein complex subunit 1
E: H/ACA ribonucleoprotein complex subunit 2
F: H/ACA ribonucleoprotein complex subunit 3
G: H/ACA ribonucleoprotein complex subunit DKC1
H: H/ACA ribonucleoprotein complex subunit 1
I: H/ACA ribonucleoprotein complex subunit 2
J: H/ACA ribonucleoprotein complex subunit 3
K: Telomerase Cajal body protein 1


Theoretical massNumber of molelcules
Total (without water)415,06110
Polymers415,06110
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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H/ACA ribonucleoprotein complex subunit ... , 4 types, 8 molecules CGDHEIFJ

#2: Protein H/ACA ribonucleoprotein complex subunit DKC1 / CBF5 homolog / Dyskerin / Nopp140-associated protein of 57 kDa / Nucleolar protein NAP57 / ...CBF5 homolog / Dyskerin / Nopp140-associated protein of 57 kDa / Nucleolar protein NAP57 / Nucleolar protein family A member 4 / snoRNP protein DKC1


Mass: 57779.211 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: 293T / Organ: Kidney / Plasmid details: endogenous / References: UniProt: O60832
#3: Protein H/ACA ribonucleoprotein complex subunit 1 / Nucleolar protein family A member 1 / snoRNP protein GAR1


Mass: 22387.963 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: 293T / Organ: Kidney / Plasmid details: endogenous / References: UniProt: Q9NY12
#4: Protein H/ACA ribonucleoprotein complex subunit 2 / Nucleolar protein family A member 2 / snoRNP protein NHP2


Mass: 17226.070 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: 293T / Organ: Kidney / Plasmid details: endogenous / References: UniProt: Q9NX24
#5: Protein H/ACA ribonucleoprotein complex subunit 3 / Nucleolar protein 10 / Nucleolar protein family A member 3 / snoRNP protein NOP10


Mass: 7719.989 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: 293T / Organ: Kidney / Plasmid details: endogenous / References: UniProt: Q9NPE3

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RNA chain / Protein , 2 types, 2 molecules BK

#1: RNA chain Human telomerase RNA / Telomerase RNA component


Mass: 145477.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line: 293T / Organ: Kidney / Plasmid: pcDNA 3.1
Details (production host): pcDNA 3.1 inserted with U3 promoter-hTR gene-hepatitis virus D ribozyme
Cell line (production host): 293T / Organ (production host): Kidney / Production host: Homo sapiens (human) / References: GenBank: 1932797
#6: Protein Telomerase Cajal body protein 1 / WD repeat-containing protein 79 / WD40 repeat-containing protein antisense to TP53 gene / WRAP53beta


Mass: 59357.070 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: 293T / Organ: Kidney / Plasmid details: endogenous / References: UniProt: Q9BUR4

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Human telomerase H/ACA RNP lobeCOMPLEXThe hTR H/ACA motif, which adopts a double-hairpin structure hinged by the H-box and flanked by the ACA box, scaffolds the assembly of two copies each of dyskerin, NHP2, NOP10 and GAR1, one on each hairpin. TCAB1 binds the CAB box.all0MULTIPLE SOURCES
2Telomerase H/ACA RNP lobeCOMPLEX#2-#61NATURAL
3Human telomerase RNATelomerase RNA componentCOMPLEX#11RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.415 MDaNO
22
33
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
11Homo sapiens (human)9606
22Homo sapiens (human)9606
33Homo sapiens (human)9606293TpcDNA3.1 U3-hTR-HDV
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120.0 mMHEPESC8H18N2O4S1
2150.0 mMSodium chlorideNaClSodium chloride1
32.0 mMMagnesium chlorideMgCl21
40.05 %Igepal CA630(C2H4O)nC14H22O1
51.0 %TrehaloseC12H22O111
61.0 mMDTTC4H10O2S21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Calibrated magnification: 45872 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (min): 78 K
Image recordingAverage exposure time: 3 sec. / Electron dose: 48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 41053
Details: Images were collected in movie-mode and fractionated into 48 movie frames
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: REFMAC / Version: 5.8.0256 / Classification: refinement
EM software
IDNameVersionCategory
1RELION4particle selection
2EPU2.13.0.3175RELimage acquisition
4CTFFIND4CTF correction
7Coot0.9.8.1model fitting
8ISOLDE1.5model fitting
9UCSF ChimeraX1.5model fitting
11RELION4initial Euler assignment
12RELION4final Euler assignment
13RELION4classification
14RELION43D reconstruction
15REFMAC5.8model refinement
16PHENIX1.20-4459model refinement
Image processingDetails: All images were processed using RELION4.0 and CryoSPARC 4.1.2
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 18744992
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 145627 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingPDB-ID: 7BGB

7bgb


Accession code: 7BGB / Source name: PDB / Type: experimental model
RefinementResolution: 3.1→3.1 Å / Cor.coef. Fo:Fc: 0.901 / SU B: 16.611 / SU ML: 0.267 / ESU R: 0.422
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.29752 --
obs0.29752 134803 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 119.935 Å2
Baniso -1Baniso -2Baniso -3
1--5.09 Å2-0.18 Å20.58 Å2
2--6.64 Å2-5.75 Å2
3----1.54 Å2
Refinement stepCycle: 1 / Total: 15237
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0050.01815747
ELECTRON MICROSCOPYr_bond_other_d0.0020.0213833
ELECTRON MICROSCOPYr_angle_refined_deg1.0921.87121707
ELECTRON MICROSCOPYr_angle_other_deg0.937332208
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.51451661
ELECTRON MICROSCOPYr_dihedral_angle_2_deg36.86323.344601
ELECTRON MICROSCOPYr_dihedral_angle_3_deg12.529152471
ELECTRON MICROSCOPYr_dihedral_angle_4_deg18.67315106
ELECTRON MICROSCOPYr_chiral_restr0.0610.22386
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.02115972
ELECTRON MICROSCOPYr_gen_planes_other0.0020.023243
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it4.51111.7746683
ELECTRON MICROSCOPYr_mcbond_other4.51111.7736682
ELECTRON MICROSCOPYr_mcangle_it7.49417.6478331
ELECTRON MICROSCOPYr_mcangle_other7.49317.6498332
ELECTRON MICROSCOPYr_scbond_it4.33512.6689064
ELECTRON MICROSCOPYr_scbond_other4.33512.6689064
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other7.3518.83513376
ELECTRON MICROSCOPYr_long_range_B_refined14.36161441
ELECTRON MICROSCOPYr_long_range_B_other14.3661442
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.12→3.201 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.54 9923 -
obs--100 %

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