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- PDB-8sok: Cryo-EM structure of human CST bound to POT1(ESDL)/TPP1 in the pr... -

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Basic information

Entry
Database: PDB / ID: 8sok
TitleCryo-EM structure of human CST bound to POT1(ESDL)/TPP1 in the presence of telomeric ssDNA
Components
  • (CST complex subunit ...) x 3
  • DNA (5'-D(*TP*TP*AP*GP*GP*GP*TP*TP*AP*G)-3')
  • Protection of telomeres protein 1
  • TPP1
KeywordsDNA BINDING PROTEIN / telomere / shelterin / cst / complex
Function / homology
Function and homology information


positive regulation of single-stranded telomeric DNA binding / positive regulation of DNA strand elongation / regulation of DNA helicase activity / positive regulation of DNA helicase activity / G-rich single-stranded DNA binding / telomere assembly / CST complex / segmentation / urogenital system development / 8-hydroxy-2'-deoxyguanosine DNA binding ...positive regulation of single-stranded telomeric DNA binding / positive regulation of DNA strand elongation / regulation of DNA helicase activity / positive regulation of DNA helicase activity / G-rich single-stranded DNA binding / telomere assembly / CST complex / segmentation / urogenital system development / 8-hydroxy-2'-deoxyguanosine DNA binding / telomeric D-loop binding / regulation of double-strand break repair via nonhomologous end joining / positive regulation of helicase activity / protection from non-homologous end joining at telomere / telomerase inhibitor activity / DEAD/H-box RNA helicase binding / establishment of protein localization to telomere / telomere maintenance via telomere lengthening / 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 / positive regulation of telomere maintenance / nuclear telomere cap complex / single-stranded telomeric DNA binding / 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 / telomere capping / telomerase holoenzyme complex / embryonic limb morphogenesis / bone marrow development / DNA duplex unwinding / intermediate filament cytoskeleton / hematopoietic stem cell proliferation / protein localization to chromosome, telomeric region / telomeric DNA binding / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MDA-5 activity / negative regulation of telomere maintenance via telomerase / Telomere Extension By Telomerase / carbohydrate transmembrane transporter activity / maltose binding / telomere maintenance via telomerase / replicative senescence / maltose transport / maltodextrin transmembrane transport / DNA polymerase binding / Packaging Of Telomere Ends / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / positive regulation of telomerase activity / spleen development / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / positive regulation of telomere maintenance via telomerase / Inhibition of DNA recombination at telomere / Meiotic synapsis / regulation of G2/M transition of mitotic cell cycle / picornain 2A / symbiont-mediated suppression of host mRNA export from nucleus / telomere maintenance / symbiont genome entry into host cell via pore formation in plasma membrane / bioluminescence / picornain 3C / thymus development / T=pseudo3 icosahedral viral capsid / positive regulation of DNA replication / generation of precursor metabolites and energy / skeletal system development / host cell cytoplasmic vesicle membrane / intracellular protein transport / cytoplasmic vesicle membrane / endocytosis involved in viral entry into host cell / multicellular organism growth / DNA Damage/Telomere Stress Induced Senescence / fibrillar center / positive regulation of fibroblast proliferation / nucleoside-triphosphate phosphatase / protein complex oligomerization / monoatomic ion channel activity / single-stranded DNA binding / symbiont-mediated suppression of host gene expression / outer membrane-bounded periplasmic space / chromosome, telomeric region / RNA helicase activity / nuclear body / induction by virus of host autophagy / RNA-directed RNA polymerase / viral RNA genome replication / cysteine-type endopeptidase activity / intracellular membrane-bounded organelle / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / DNA-templated transcription / DNA damage response / host cell nucleus
Similarity search - Function
CST complex subunit Ten1, animal and plant type / CST complex subunit CTC1 / CST complex subunit CTC1-like / CST, telomere maintenance, complex subunit CTC1 / Telomere-capping, CST complex subunit / CST complex subunit Stn1 / Stn1, C-terminal / CST complex subunit Stn1, wHTH1 motif superfamily / CST, complex subunit STN1, C terminal / : ...CST complex subunit Ten1, animal and plant type / CST complex subunit CTC1 / CST complex subunit CTC1-like / CST, telomere maintenance, complex subunit CTC1 / Telomere-capping, CST complex subunit / CST complex subunit Stn1 / Stn1, C-terminal / CST complex subunit Stn1, wHTH1 motif superfamily / CST, complex subunit STN1, C terminal / : / Protection of telomeres protein 1 (POT1), C-terminal insertion domain / Shelterin complex subunit TPP1/Est3 / Shelterin complex subunit, TPP1/ACD / Adrenocortical dysplasia protein / Protection of telomeres protein 1, ssDNA-binding domain / ssDNA-binding domain of telomere protection protein / Replication factor A protein-like / Protection of telomeres protein 1 / Telomeric single stranded DNA binding POT1/Cdc13 / Telomeric single stranded DNA binding POT1/CDC13 / Telomeric single stranded DNA binding POT1/CDC13 / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Green fluorescent protein, GFP / Bacterial extracellular solute-binding protein / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Poliovirus 3A protein-like / Poliovirus 3A protein like / Picornavirus 2B protein / Poliovirus core protein 3a, soluble domain / Picornavirus 2B protein / Peptidase C3, picornavirus core protein 2A / Picornavirus core protein 2A / Picornavirus coat protein VP4 / Picornavirus coat protein (VP4) / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) / Picornavirus capsid / picornavirus capsid protein / Helicase, superfamily 3, single-stranded RNA virus / Superfamily 3 helicase of positive ssRNA viruses domain profile. / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA helicase / Picornavirus/Calicivirus coat protein / Viral coat protein subunit / RNA-directed RNA polymerase, C-terminal domain / Viral RNA-dependent RNA polymerase / Winged helix DNA-binding domain superfamily / Reverse transcriptase/Diguanylate cyclase domain / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / Winged helix-like DNA-binding domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / Genome polyprotein / Maltose/maltodextrin-binding periplasmic protein / CST complex subunit CTC1 / CST complex subunit TEN1 / Adrenocortical dysplasia protein homolog / CST complex subunit STN1 / Protection of telomeres protein 1
Similarity search - Component
Biological speciesEscherichia coli O157:H7 (bacteria)
Homo sapiens (human)
Human enterovirus 71
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsCai, S.W.
Funding support United States, 2items
OrganizationGrant numberCountry
Other privateBCRF-22-036
National Institutes of Health/National Cancer Institute (NIH/NCI)R35 CA210036 United States
CitationJournal: bioRxiv / Year: 2023
Title: POT1 recruits and regulates CST-Polα/Primase at human telomeres.
Authors: Sarah W Cai / Hiroyuki Takai / Thomas Walz / Titia de Lange /
Abstract: Telomere maintenance requires extension of the G-rich telomeric repeat strand by telomerase and fill-in synthesis of the C-rich strand by Polα/Primase. Telomeric Polα/Primase is bound to Ctc1-Stn1- ...Telomere maintenance requires extension of the G-rich telomeric repeat strand by telomerase and fill-in synthesis of the C-rich strand by Polα/Primase. Telomeric Polα/Primase is bound to Ctc1-Stn1-Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/Primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Phosphorylation of POT1 is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/Primase in an inactive auto-inhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/Primase into an active state that completes telomere replication through fill-in synthesis.
History
DepositionApr 28, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 7, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 15, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CST complex subunit CTC1
B: CST complex subunit STN1
C: CST complex subunit TEN1
D: Protection of telomeres protein 1
E: TPP1
F: DNA (5'-D(*TP*TP*AP*GP*GP*GP*TP*TP*AP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)371,1628
Polymers371,0316
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: equilibrium centrifugation
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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CST complex subunit ... , 3 types, 3 molecules ABC

#1: Protein CST complex subunit CTC1 / Conserved telomere maintenance component 1 / HBV DNAPTP1-transactivated protein B


Mass: 178300.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O157:H7 (bacteria), (gene. exp.) Homo sapiens (human)
Gene: malE, Z5632, ECs5017, CTC1, C17orf68 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P0AEY0, UniProt: Q2NKJ3
#2: Protein CST complex subunit STN1 / Oligonucleotide/oligosaccharide-binding fold-containing protein 1 / Suppressor of cdc thirteen homolog


Mass: 42172.949 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: STN1, OBFC1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9H668
#3: Protein CST complex subunit TEN1 / Protein telomeric pathways with STN1 homolog / Telomere length regulation protein TEN1 homolog


Mass: 13872.013 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TEN1, C17orf106 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q86WV5

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Protein , 2 types, 2 molecules DE

#4: Protein Protection of telomeres protein 1 / hPot1 / POT1-like telomere end-binding protein


Mass: 72938.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POT1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9NUX5
#5: Protein TPP1 / POT1 and TIN2-interacting protein


Mass: 58064.863 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human enterovirus 71, (gene. exp.) Homo sapiens (human)
Gene: ACD, PIP1, PTOP, TINT1, TPP1 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: B6F2F5, UniProt: Q96AP0, picornain 2A, nucleoside-triphosphate phosphatase, picornain 3C, RNA-directed RNA polymerase

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DNA chain / Non-polymers , 2 types, 3 molecules F

#6: DNA chain DNA (5'-D(*TP*TP*AP*GP*GP*GP*TP*TP*AP*G)-3')


Mass: 5682.672 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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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

ComponentName: ssDNA-bound human CST-POT1(ESDL)/TPP1 complex / Type: COMPLEX / Details: CST-POT1(ESDL)/TPP1 complex in the presence of DNA / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 0.37 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
SpecimenConc.: 0.02 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50.3 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 76359 / Symmetry type: POINT
RefinementCross valid method: NONE

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