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- PDB-7u5c: Cryo-EM structure of human CST bound to DNA polymerase alpha-prim... -

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Basic information

Entry
Database: PDB / ID: 7u5c
TitleCryo-EM structure of human CST bound to DNA polymerase alpha-primase in a recruitment state
Components
  • (CST complex subunit ...) x 3
  • (DNA polymerase alpha ...DNA polymerase) x 2
  • DNA primase large subunitPrimase
  • DNA primase small subunitPrimase
  • canonical telomeric DNA sequence
KeywordsDNA BINDING PROTEIN/DNA / Fill-in / Telomere / Replication / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


CST complex / positive regulation of DNA primase activity / DNA primase AEP / ribonucleotide binding / telomerase inhibitor activity / DNA replication initiation / DNA/RNA hybrid binding / telomere maintenance via telomere lengthening / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 ...CST complex / positive regulation of DNA primase activity / DNA primase AEP / ribonucleotide binding / telomerase inhibitor activity / DNA replication initiation / DNA/RNA hybrid binding / telomere maintenance via telomere lengthening / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / alpha DNA polymerase:primase complex / Polymerase switching / regulation of type I interferon production / Processive synthesis on the lagging strand / DNA primase activity / single-stranded telomeric DNA binding / Removal of the Flap Intermediate / Polymerase switching on the C-strand of the telomere / lagging strand elongation / G-rich strand telomeric DNA binding / DNA replication, synthesis of primer / mitotic DNA replication initiation / telomere capping / bone marrow development / intermediate filament cytoskeleton / hematopoietic stem cell proliferation / DNA strand elongation involved in DNA replication / DNA synthesis involved in DNA repair / telomeric DNA binding / leading strand elongation / G1/S-Specific Transcription / DNA replication origin binding / negative regulation of telomere maintenance via telomerase / replicative senescence / Activation of the pre-replicative complex / DNA replication initiation / spleen development / regulation of G2/M transition of mitotic cell cycle / telomere maintenance / thymus development / positive regulation of DNA replication / Defective pyroptosis / multicellular organism growth / fibrillar center / nuclear matrix / double-strand break repair via nonhomologous end joining / protein import into nucleus / positive regulation of fibroblast proliferation / single-stranded DNA binding / nuclear envelope / 4 iron, 4 sulfur cluster binding / DNA replication / chromosome, telomeric region / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / intracellular membrane-bounded organelle / DNA repair / DNA damage response / chromatin binding / chromatin / nucleolus / protein kinase binding / magnesium ion binding / DNA binding / zinc ion binding / nucleoplasm / membrane / metal ion binding / nucleus / cytosol
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 / Replication factor A protein-like ...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 / Replication factor A protein-like / DNA polymerase alpha, subunit B, N-terminal domain superfamily / DNA polymerase alpha subunit B N-terminal / DNA polymerase alpha, subunit B, N-terminal / DNA polymerase alpha, subunit B / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily / Eukaryotic and archaeal DNA primase, large subunit / DNA Polymerase alpha zinc finger / DNA polymerase alpha subunit p180 N terminal / Zinc finger, DNA-directed DNA polymerase, family B, alpha / DNA polymerase alpha catalytic subunit, catalytic domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / DNA polymerase family B, thumb domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Winged helix DNA-binding domain superfamily / Ribonuclease H-like superfamily / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
IRON/SULFUR CLUSTER / DNA / DNA (> 10) / DNA polymerase alpha catalytic subunit / DNA primase small subunit / DNA primase large subunit / DNA polymerase alpha subunit B / CST complex subunit CTC1 / CST complex subunit TEN1 / CST complex subunit STN1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsCai, S.W. / Zinder, J.C. / Svetlov, V. / Bush, M.W. / Nudler, E. / Walz, T. / de Lange, T.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)5 R35 CA210036 United States
Breast Cancer Research FoundationBCRF-19-036 United States
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Cryo-EM structure of the human CST-Polα/primase complex in a recruitment state.
Authors: Sarah W Cai / John C Zinder / Vladimir Svetlov / Martin W Bush / Evgeny Nudler / Thomas Walz / Titia de Lange /
Abstract: The CST-Polα/primase complex is essential for telomere maintenance and functions to counteract resection at double-strand breaks. We report a 4.6-Å resolution cryo-EM structure of human CST- ...The CST-Polα/primase complex is essential for telomere maintenance and functions to counteract resection at double-strand breaks. We report a 4.6-Å resolution cryo-EM structure of human CST-Polα/primase, captured prior to catalysis in a recruitment state stabilized by chemical cross-linking. Our structure reveals an evolutionarily conserved interaction between the C-terminal domain of the catalytic POLA1 subunit and an N-terminal expansion in metazoan CTC1. Cross-linking mass spectrometry and negative-stain EM analysis provide insight into CST binding by the flexible POLA1 N-terminus. Finally, Coats plus syndrome disease mutations previously characterized to disrupt formation of the CST-Polα/primase complex map to protein-protein interfaces observed in the recruitment state. Together, our results shed light on the architecture and stoichiometry of the metazoan fill-in machinery.
History
DepositionMar 2, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 18, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 1, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Aug 24, 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
Revision 1.3Feb 14, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA primase small subunit
B: DNA primase large subunit
C: DNA polymerase alpha catalytic subunit
D: DNA polymerase alpha subunit B
E: CST complex subunit CTC1
F: CST complex subunit STN1
G: CST complex subunit TEN1
H: canonical telomeric DNA sequence
hetero molecules


Theoretical massNumber of molelcules
Total (without water)502,11513
Polymers501,5028
Non-polymers6135
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, cross-linking, equilibrium centrifugation
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein DNA primase small subunit / Primase / DNA primase 49 kDa subunit / p49


Mass: 49981.012 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRIM1 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P49642, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
#2: Protein DNA primase large subunit / Primase / DNA primase 58 kDa subunit / p58


Mass: 59092.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRIM2, PRIM2A / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P49643, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases

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DNA polymerase alpha ... , 2 types, 2 molecules CD

#3: Protein DNA polymerase alpha catalytic subunit / DNA polymerase alpha catalytic subunit p180


Mass: 129635.125 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLA1, POLA / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P09884, DNA-directed DNA polymerase
#4: Protein DNA polymerase alpha subunit B / DNA polymerase / DNA polymerase alpha 70 kDa subunit


Mass: 66015.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLA2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q14181

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

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


Mass: 135050.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CTC1, C17orf68 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q2NKJ3
#6: 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
#7: 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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DNA chain , 1 types, 1 molecules H

#8: DNA chain canonical telomeric DNA sequence


Mass: 5682.672 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 2 types, 5 molecules

#9: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER / Iron–sulfur cluster


Mass: 351.640 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: Human CST bound to DNA polymerase alpha(POLA1 delta N)-primase in a recruitment state
Type: COMPLEX
Details: Sample was GraFix cross-linked with glutaraldehyde.
Entity ID: #1-#8 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.5 MDa / Experimental value: NO
Buffer solutionpH: 7.5
SpecimenConc.: 0.075 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was cross-linked with glutaraldehyde (GraFix)
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1Gautomatch0.56particle selection
2SerialEMimage acquisition
7UCSF Chimera1.13.1model fitting
8UCSF ChimeraX0.93.0model fitting
9ISOLDE1model fitting
11RELION3initial Euler assignment
12RELION3final Euler assignment
14RELION33D reconstruction
15PHENIX1.17.1model refinement
CTF correctionType: NONE
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 131850 / Symmetry type: POINT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
15EXR

5exr

15EXR1PDBexperimental model
26W6W

6w6w

16W6W2PDBexperimental model

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