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- PDB-7bg9: The catalytic core lobe of human telomerase in complex with a tel... -

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Basic information

Entry
Database: PDB / ID: 7bg9
TitleThe catalytic core lobe of human telomerase in complex with a telomeric DNA substrate
Components
  • DNA (5'-D(P*TP*TP*AP*GP*GP*G)-3')
  • Histone H2A
  • Histone H2B
  • RNA (256-MER)
  • Telomerase reverse transcriptase,Telomerase reverse transcriptase
KeywordsRNA BINDING PROTEIN / Reverse transcriptase / ribonucleoprotein / complex / DNA
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
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / Histone H2A / Histone H2B / Telomerase reverse transcriptase
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsNguyen, T.H.D. / Ghanim, G.E. / Fountain, A.J. / van Roon, A.M.M. / Rangan, R. / Das, R. / Collins, K.
Funding support United Kingdom, United States, 2items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 28, 2021Provider: repository / Type: Initial release
Revision 1.1May 5, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_id_CSD / _citation.pdbx_database_id_PubMed ..._citation.journal_id_CSD / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jun 2, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: Telomerase reverse transcriptase,Telomerase reverse transcriptase
N: DNA (5'-D(P*TP*TP*AP*GP*GP*G)-3')
M: Histone H2B
L: Histone H2A
B: RNA (256-MER)


Theoretical massNumber of molelcules
Total (without water)332,3665
Polymers332,3665
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area19800 Å2
ΔGint-161 kcal/mol
Surface area99610 Å2
MethodPISA

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Components

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


Mass: 149158.578 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line: 293T / Gene: TERT, EST2, TCS1, TRT / Organ: Kidney / Plasmid: pcDNA3.1 / Cell line (production host): 293T / Organ (production host): Kidney / Production host: Homo sapiens (human) / References: UniProt: O14746, RNA-directed DNA polymerase
#2: DNA chain DNA (5'-D(P*TP*TP*AP*GP*GP*G)-3')


Mass: 5514.567 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: 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: 293T / Organ: Kidney / Plasmid details: endogeneous / References: UniProt: B4DR52
#4: 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: 293T / Organ: Kidney / Plasmid details: endogeneous / References: UniProt: B2R5B3
#5: RNA chain RNA (256-MER)


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: pcDNA3.1
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)

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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
1Human telomerase catalytic core in complex with a telomeric DNA substrateCOMPLEXall0MULTIPLE SOURCES
2Telomerase reverse transcriptaseCOMPLEX#1, #51RECOMBINANT
3HistoneCOMPLEX#3-#41NATURAL
4DNACOMPLEX#21RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
24synthetic construct (others)32630
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaClSodium chloride1
32 mMmagnesium chlorideMgCl21
40.05 %Igepal CA630(C2H4O)nC14H22O1
51 %TrehaloseC12H22O111
61 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 / Details: Blot for 4-5 seconds before plunging

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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 / Nominal defocus max: 3500 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (min): 78 K
Image recordingAverage exposure time: 1 sec. / Electron dose: 47 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 43639
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
IDNameVersionCategoryDetails
1RELION3.1particle selectionAutomatic picking using 2D references
2SerialEMimage acquisition
4CTFFINDCTF correction
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13REFMACmodel refinement
Image processingDetails: All images were processed using RELION 3.1.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 15760434
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 168538 / Symmetry type: POINT
Atomic model buildingB value: 80 / Protocol: AB INITIO MODEL / Space: RECIPROCAL
RefinementResolution: 3.8→138.75 Å / Cor.coef. Fo:Fc: 0.864 / SU B: 30.041 / SU ML: 0.393 / ESU R: 0.822
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.34525 --
obs0.34525 93181 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: 219.916 Å2
Baniso -1Baniso -2Baniso -3
1--5.3 Å2-0.16 Å2-0.98 Å2
2--0.64 Å2-7.31 Å2
3---4.66 Å2
Refinement stepCycle: 1 / Total: 14233
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0130.01615063
ELECTRON MICROSCOPYr_bond_other_d0.0030.0211190
ELECTRON MICROSCOPYr_angle_refined_deg1.5741.66221642
ELECTRON MICROSCOPYr_angle_other_deg1.236326051
ELECTRON MICROSCOPYr_dihedral_angle_1_deg8.16651078
ELECTRON MICROSCOPYr_dihedral_angle_2_deg29.17421.08389
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.308151551
ELECTRON MICROSCOPYr_dihedral_angle_4_deg14.39915110
ELECTRON MICROSCOPYr_chiral_restr0.0980.22396
ELECTRON MICROSCOPYr_gen_planes_refined0.0080.02112793
ELECTRON MICROSCOPYr_gen_planes_other0.0020.023390
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_it22.84819.7334333
ELECTRON MICROSCOPYr_mcbond_other22.83819.7354332
ELECTRON MICROSCOPYr_mcangle_it36.39729.525404
ELECTRON MICROSCOPYr_mcangle_other36.39529.525405
ELECTRON MICROSCOPYr_scbond_it24.05724.48410730
ELECTRON MICROSCOPYr_scbond_other24.05424.48210729
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other38.65736.56116239
ELECTRON MICROSCOPYr_long_range_B_refined59.34658154
ELECTRON MICROSCOPYr_long_range_B_other59.34658155
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.8→3.899 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.639 6901 -
obs--100 %

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