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- PDB-7qxb: Cryo-EM map of human telomerase-DNA-TPP1-POT1 complex (sharpened map) -

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Basic information

Entry
Database: PDB / ID: 7qxb
TitleCryo-EM map of human telomerase-DNA-TPP1-POT1 complex (sharpened map)
Components
  • Adrenocortical dysplasia homolog (Mouse), isoform CRA_a
  • Histone H2A
  • Histone H2B
  • Protection of telomeres protein 1
  • Telomerase reverse transcriptase
  • Telomeric DNA
  • human telomerase RNATelomerase RNA component
KeywordsRNA BINDING PROTEIN / Reverse transcriptase / ribonucleoprotein / telomerase / telomere / DNA BINDING PROTEIN
Function / homology
Function and homology information


positive regulation of DNA strand elongation / regulation of DNA helicase activity / G-rich single-stranded DNA binding / telomere assembly / 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 DNA strand elongation / regulation of DNA helicase activity / G-rich single-stranded DNA binding / telomere assembly / 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 / regulation of double-strand break repair via nonhomologous end joining / 8-hydroxy-2'-deoxyguanosine DNA binding / telomeric D-loop binding / telomerase activity / positive regulation of helicase activity / telomerase inhibitor activity / telomerase RNA reverse transcriptase activity / DEAD/H-box RNA helicase binding / RNA-templated DNA biosynthetic process / establishment of protein localization to telomere / positive regulation of DNA helicase activity / telomeric D-loop disassembly / shelterin complex / Telomere C-strand synthesis initiation / regulation of telomere maintenance via telomerase / Telomere C-strand (Lagging Strand) Synthesis / negative regulation of telomerase activity / nuclear telomere cap complex / siRNA processing / single-stranded telomeric DNA binding / positive regulation of telomere maintenance / Processive synthesis on the C-strand of the telomere / Polymerase switching on the C-strand of the telomere / Removal of the Flap Intermediate from the C-strand / G-rich strand telomeric DNA binding / telomerase RNA binding / telomere capping / telomerase holoenzyme complex / positive regulation of vascular associated smooth muscle cell migration / DNA duplex unwinding / telomeric DNA binding / DNA biosynthetic process / RNA-templated transcription / positive regulation of stem cell proliferation / mitochondrial nucleoid / negative regulation of cellular senescence / negative regulation of telomere maintenance via telomerase / 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 / Packaging Of Telomere Ends / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / positive regulation of telomerase activity / positive regulation of vascular associated smooth muscle cell proliferation / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Inhibition of DNA recombination at telomere / Meiotic synapsis / 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 / DNA Damage/Telomere Stress Induced Senescence / PML body / positive regulation of miRNA transcription / RNA-directed DNA polymerase / structural constituent of chromatin / 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 body / 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
Similarity search - Function
: / Protection of telomeres protein 1 (POT1), C-terminal insertion domain / Shelterin complex subunit TPP1/Est3 / Shelterin complex subunit, TPP1/ACD / Adrenocortical dysplasia protein / : / Telomerase reverse transcriptase, C-terminal extension / Protection of telomeres protein 1, ssDNA-binding domain / ssDNA-binding domain of telomere protection protein / Protection of telomeres protein 1 ...: / Protection of telomeres protein 1 (POT1), C-terminal insertion domain / Shelterin complex subunit TPP1/Est3 / Shelterin complex subunit, TPP1/ACD / Adrenocortical dysplasia protein / : / Telomerase reverse transcriptase, C-terminal extension / Protection of telomeres protein 1, ssDNA-binding domain / ssDNA-binding domain of telomere protection protein / Protection of telomeres protein 1 / Telomerase ribonucleoprotein complex - RNA binding domain / Telomeric single stranded DNA binding POT1/Cdc13 / Telomeric single stranded DNA binding POT1/CDC13 / Telomeric single stranded DNA binding POT1/CDC13 / 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 / Nucleic acid-binding, OB-fold / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
: / DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / Adrenocortical dysplasia homolog (Mouse), isoform CRA_a / Histone H2A / Histone H2B / Telomerase reverse transcriptase / Protection of telomeres protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsSekne, Z. / Ghanim, G.E. / van Roon, A.M.M. / Nguyen, T.H.D.
Funding support United Kingdom, United States, 2items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI)MC_UP_1201/19 United Kingdom
Jane Coffin Childs Postdoctoral Fellowship United States
CitationJournal: Science / Year: 2022
Title: Structural basis of human telomerase recruitment by TPP1-POT1.
Authors: Zala Sekne / George E Ghanim / Anne-Marie M van Roon / Thi Hoang Duong Nguyen /
Abstract: Telomerase maintains genome stability by extending the 3' telomeric repeats at eukaryotic chromosome ends, thereby counterbalancing progressive loss caused by incomplete genome replication. In ...Telomerase maintains genome stability by extending the 3' telomeric repeats at eukaryotic chromosome ends, thereby counterbalancing progressive loss caused by incomplete genome replication. In mammals, telomerase recruitment to telomeres is mediated by TPP1, which assembles as a heterodimer with POT1. We report structures of DNA-bound telomerase in complex with TPP1 and with TPP1-POT1 at 3.2- and 3.9-angstrom resolution, respectively. Our structures define interactions between telomerase and TPP1-POT1 that are crucial for telomerase recruitment to telomeres. The presence of TPP1-POT1 stabilizes the DNA, revealing an unexpected path by which DNA exits the telomerase active site and a DNA anchor site on telomerase that is important for telomerase processivity. Our findings rationalize extensive prior genetic and biochemical findings and provide a framework for future mechanistic work on telomerase regulation.
History
DepositionJan 26, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 2, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 9, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Mar 23, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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  • Deposited structure unit
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  • EMDB-14197
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Telomerase reverse transcriptase
B: human telomerase RNA
L: Histone H2A
M: Histone H2B
N: Telomeric DNA
O: Adrenocortical dysplasia homolog (Mouse), isoform CRA_a
P: Protection of telomeres protein 1


Theoretical massNumber of molelcules
Total (without water)434,9247
Polymers434,9247
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25700 Å2
ΔGint-162 kcal/mol
Surface area99330 Å2

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Components

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Protein , 5 types, 5 molecules ALMOP

#1: Protein Telomerase reverse transcriptase / / HEST2 / Telomerase catalytic subunit / Telomerase-associated protein 2 / TP2


Mass: 127195.812 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TERT, EST2, TCS1, TRT / Plasmid: pcDNA3.1 / Details (production host): pcDNA3.1-ZZSS-TERT / Cell (production host): epithelial / Cell line (production host): HEK293T / Organ (production host): Kidney / Production host: Homo sapiens (human) / References: UniProt: O14746, RNA-directed DNA polymerase
#3: Protein Histone H2A /


Mass: 14140.584 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293T / Organ: Kidney / Tissue: Kidney / References: UniProt: B2R5B3
#4: Protein Histone H2B /


Mass: 18074.932 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293T / Organ: Kidney / Tissue: Kidney / References: UniProt: B4DR52
#6: Protein Adrenocortical dysplasia homolog (Mouse), isoform CRA_a / Adrenocortical dysplasia protein homolog


Mass: 49013.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ACD, hCG_27140 / Plasmid: p-FASTBac Dual / Cell (production host): epithelial / Organ (production host): ovary / Production host: Spodoptera (butterflies/moths) / Tissue (production host): ovary / References: UniProt: A0A590TQL1
#7: Protein Protection of telomeres protein 1 / hPot1 / POT1-like telomere end-binding protein


Mass: 71520.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POT1 / Plasmid: p-FASTBac Dual / Cell (production host): epithelial / Organ (production host): ovary / Production host: Spodoptera (butterflies/moths) / Tissue (production host): ovary / References: UniProt: Q9NUX5

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RNA chain / DNA chain , 2 types, 2 molecules BN

#2: 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) / Plasmid: pcDNA3.1 / Details (production host): pcDNA3.1 U3-hTR-HDV / Cell (production host): epithelial / Cell line (production host): HEK293T / Organ (production host): Kidney / Production host: Homo sapiens (human) / References: GenBank: NR_001566
#5: DNA chain Telomeric DNA


Mass: 9501.092 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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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
IDNameTypeEntity IDParent-IDSource
1Complex of telomeric DNA-bound human telomerase with TPP1-POT1COMPLEXall0MULTIPLE SOURCES
2Telomeric DNACOMPLEX#51RECOMBINANT
3Telomerase reverse transcriptase and telomeric RNACOMPLEX#1-#21RECOMBINANT
4HistonesHistoneCOMPLEX#3-#41NATURAL
5TPP1-POT1COMPLEX#6-#71RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Homo sapiens (human)9606
45Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12synthetic construct (others)32630
23Homo sapiens (human)9606
35Spodoptera (butterflies/moths)7106
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaClSodium chloride1
310 %glycerolC3H8O31
42 mMmagnesium chlorideMgCl21
50.05 %IGEPAL CA6301
61 %trehaloseC12H22O111
71 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: 45871 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
Image recordingAverage exposure time: 3 sec. / Electron dose: 48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 3 / Num. of real images: 50775
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
7UCSF ChimeraXmodel fitting
8UCSF Chimeramodel fitting
9Cootmodel fitting
11REFMAC5.8model refinement
12RELION4initial Euler assignment
13RELION4final Euler assignment
14RELION4classification
15RELION43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 21589656
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 192871 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL
Atomic model buildingPDB-ID: 7BG9

7bg9

RefinementResolution: 3.91→164.59 Å / Cor.coef. Fo:Fc: 0.97 / SU B: 119.536 / SU ML: 1.312 / ESU R: 0.905
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.32457 --
obs0.32457 102581 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: 220.612 Å2
Baniso -1Baniso -2Baniso -3
1--4.41 Å216.83 Å211.02 Å2
2--8.23 Å216.28 Å2
3----3.82 Å2
Refinement stepCycle: 1 / Total: 17193
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.010.01618085
ELECTRON MICROSCOPYr_bond_other_d0.0020.0213383
ELECTRON MICROSCOPYr_angle_refined_deg1.4241.69325855
ELECTRON MICROSCOPYr_angle_other_deg1.475330964
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.97951509
ELECTRON MICROSCOPYr_dihedral_angle_2_deg28.35621.228448
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.172151763
ELECTRON MICROSCOPYr_dihedral_angle_4_deg12.85215126
ELECTRON MICROSCOPYr_chiral_restr0.0860.22902
ELECTRON MICROSCOPYr_gen_planes_refined0.0120.0216288
ELECTRON MICROSCOPYr_gen_planes_other0.0110.024003
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_it15.57923.046075
ELECTRON MICROSCOPYr_mcbond_other15.57623.0396074
ELECTRON MICROSCOPYr_mcangle_it24.1934.5167571
ELECTRON MICROSCOPYr_mcangle_other24.1934.5187572
ELECTRON MICROSCOPYr_scbond_it16.66524.65112010
ELECTRON MICROSCOPYr_scbond_other16.66524.65112010
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other26.07937.14118284
ELECTRON MICROSCOPYr_long_range_B_refined37.43121286
ELECTRON MICROSCOPYr_long_range_B_other37.43121286
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.91→4.011 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.6 7628 -
obs--100 %

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