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- EMDB-21561: The structure of human CST reveals a decameric assembly bound to ... -

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Basic information

Entry
Database: EMDB / ID: EMD-21561
TitleThe structure of human CST reveals a decameric assembly bound to telomeric DNA
Map dataCST decamer sharpened map with D5 symmetry refinement
Sample
  • Complex: Decameric assembly of human CST complex with telomeric single-stranded DNA
    • Protein or peptide: Homo sapiens CST complex subunit CTC1
    • Protein or peptide: Homo sapiens CST complex subunit STN1
    • Protein or peptide: Homo sapiens CST complex subunit TEN1
    • DNA: 3xTEL
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsLim C / Barbour AT / Zaug AJ / Goodrich KJ / McKay AE / Wuttke DS / Cech TR
Funding support United States, 6 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI)Tom Cech HHMI Investigator United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM059414 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM099705 United States
National Science Foundation (NSF, United States)MCB 1716425 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)K99GM131023 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM008759 United States
CitationJournal: Science / Year: 2020
Title: The structure of human CST reveals a decameric assembly bound to telomeric DNA.
Authors: Ci Ji Lim / Alexandra T Barbour / Arthur J Zaug / Karen J Goodrich / Allison E McKay / Deborah S Wuttke / Thomas R Cech /
Abstract: The CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and resolution of stalled replication forks genome-wide. Here, we report the 3.0-angstrom cryo-electron microscopy structure of ...The CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and resolution of stalled replication forks genome-wide. Here, we report the 3.0-angstrom cryo-electron microscopy structure of human CST bound to telomeric single-stranded DNA (ssDNA), which assembles as a decameric supercomplex. The atomic model of the 134-kilodalton CTC1 subunit, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. The carboxyl-terminal domain of STN1 interacts with CTC1 at two separate docking sites, allowing allosteric mediation of CST decamer assembly. Furthermore, ssDNA appears to staple two monomers to nucleate decamer assembly. CTC1 has stronger structural similarity to Replication Protein A than the expected similarity to yeast Cdc13. The decameric structure suggests that CST can organize ssDNA analogously to the nucleosome's organization of double-stranded DNA.
History
DepositionMar 17, 2020-
Header (metadata) releaseJun 3, 2020-
Map releaseJun 3, 2020-
UpdateJun 17, 2020-
Current statusJun 17, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.331
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.331
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21561.png

Downloads & links

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Map

FileDownload / File: emd_21561.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCST decamer sharpened map with D5 symmetry refinement
Voxel sizeX=Y=Z: 1.078 Å
Density
Contour LevelBy AUTHOR: 0.331 / Movie #1: 0.331
Minimum - Maximum-2.0510957 - 3.9702332
Average (Standard dev.)0.0060537243 (±0.1030544)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 431.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0781.0781.078
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z431.200431.200431.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-2.0513.9700.006

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Supplemental data

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Sample components

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Entire : Decameric assembly of human CST complex with telomeric single-str...

EntireName: Decameric assembly of human CST complex with telomeric single-stranded DNA
Components
  • Complex: Decameric assembly of human CST complex with telomeric single-stranded DNA
    • Protein or peptide: Homo sapiens CST complex subunit CTC1
    • Protein or peptide: Homo sapiens CST complex subunit STN1
    • Protein or peptide: Homo sapiens CST complex subunit TEN1
    • DNA: 3xTEL

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Supramolecule #1: Decameric assembly of human CST complex with telomeric single-str...

SupramoleculeName: Decameric assembly of human CST complex with telomeric single-stranded DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
Molecular weightExperimental: 2 MDa

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Macromolecule #1: Homo sapiens CST complex subunit CTC1

MacromoleculeName: Homo sapiens CST complex subunit CTC1 / type: protein_or_peptide / ID: 1 / Details: 2XFLAG-CTC1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDYKDDDDKD YKDDDDKAAG RAQVPSSEQA WLEDAQVFIQ KTLCPAVKEP NVQLTPLVID CVKTVWLSQG RNQGSTLPLS YSFVSVQDLK THQRLPCCSH LSWSSSAYQA WAQEAGPNGN PLPREQLLLL GTLTDLSADL EQECRNGSLY VRDNTGVLSC ELIDLDLSWL ...String:
MDYKDDDDKD YKDDDDKAAG RAQVPSSEQA WLEDAQVFIQ KTLCPAVKEP NVQLTPLVID CVKTVWLSQG RNQGSTLPLS YSFVSVQDLK THQRLPCCSH LSWSSSAYQA WAQEAGPNGN PLPREQLLLL GTLTDLSADL EQECRNGSLY VRDNTGVLSC ELIDLDLSWL GHLFLFPRWS YLPPARWNSS GEGHLELWDA PVPVFPLTIS PGPVTPIPVL YPESASCLLR LRNKLRGVQR NLAGSLVRLS ALVKSKQKAY FILSLGRSHP AVTHVSIIVQ VPAQLVWHRA LRPGTAYVLT ELRVSKIRGQ RQHVWMTSQS SRLLLLKPEC VQELELELEG PLLEADPKPL PMPSNSEDKK DPESLVRYSR LLSYSGAVTG VLNEPAGLYE LDGQLGLCLA YQQFRGLRRV MRPGVCLQLQ DVHLLQSVGG GTRRPVLAPC LRGAVLLQSF SRQKPGAHSS RQAYGASLYE QLVWERQLGL PLYLWATKAL EELACKLCPH VLRHHQFLQH SSPGSPSLGL QLLAPTLDLL APPGSPVRNA HNEILEEPHH CPLQKYTRLQ TPSSFPTLAT LKEEGQRKAW ASFDPKALLP LPEASYLPSC QLNRRLAWSW LCLLPSAFCP AQVLLGVLVA SSHKGCLQLR DQSGSLPCLL LAKHSQPLSD PRLIGCLVRA ERFQLIVERD VRSSFPSWKE LSMPGFIQKQ QARVYVQFFL ADALILPVPR PCLHSATPST PQTDPTGPEG PHLGQSRLFL LCHKEALMKR NFCVPPGASP EVPKPALSFY VLGSWLGGTQ RKEGTGWGLP EPQGNDDNDQ KVHLIFFGSS VRWFEFLHPG QVYRLVAPGP ATPMLFEKDG SSCISRRPLE LAGCASCLTV QDNWTLELES SQDIQDVLDA NKSLPESSLT DLLSDNFTDS LVSFSAEILS RTLCEPLVAS LWMKLGNTGA MRRCVKLTVA LETAECEFPP HLDVYIEDPH LPPSLGLLPG ARVHFSQLEK RVSRSHNVYC CFRSSTYVQV LSFPPETTIS VPLPHIYLAE LLQGGQSPFQ ATASCHIVSV FSLQLFWVCA YCTSICRQGK CTRLGSTCPT QTAISQAIIR LLVEDGTAEA VVTCRNHHVA AALGLCPREW ASLLDFVQVP GRVVLQFAGP GAQLESSARV DEPMTMFLWT LCTSPSVLRP IVLSFELERK PSKIVPLEPP RLQRFQCGEL PFLTHVNPRL RLSCLSIRES EYSSSLGILA SSC

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Macromolecule #2: Homo sapiens CST complex subunit STN1

MacromoleculeName: Homo sapiens CST complex subunit STN1 / type: protein_or_peptide / ID: 2 / Details: 6xHIS-STN1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MHHHHHHQPG SSRCEEETPS LLWGLDPVFL AFAKLYIRDI LDMKESRQVP GVFLYNGHPI KQVDVLGTVI GVRERDAFYS YGVDDSTGVI NCICWKKLNT ESVSAAPSAA RELSLTSQLK KLQETIEQKT KIEIGDTIRV RGSIRTYREE REIHATTYYK VDDPVWNIQI ...String:
MHHHHHHQPG SSRCEEETPS LLWGLDPVFL AFAKLYIRDI LDMKESRQVP GVFLYNGHPI KQVDVLGTVI GVRERDAFYS YGVDDSTGVI NCICWKKLNT ESVSAAPSAA RELSLTSQLK KLQETIEQKT KIEIGDTIRV RGSIRTYREE REIHATTYYK VDDPVWNIQI ARMLELPTIY RKVYDQPFHS SALEKEEALS NPGALDLPSL TSLLSEKAKE FLMENRVQSF YQQELEMVES LLSLANQPVI HSASSDQVNF KKDTTSKAIH SIFKNAIQLL QEKGLVFQKD DGFDNLYYVT REDKDLHRKI HRIIQQDCQK PNHMEKGCHF LHILACARLS IRPGLSEAVL QQVLELLEDQ SDIVSTMEHY YTAF

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Macromolecule #3: Homo sapiens CST complex subunit TEN1

MacromoleculeName: Homo sapiens CST complex subunit TEN1 / type: protein_or_peptide / ID: 3 / Details: 6xHIS-TEN1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSYYHHHHHH DYDIPTTENL YFQGAMGSMS QLVVDICKLN QVLLNLNERS KEIFTNSSRR FRVVAQLLDI QPYDDEENEG FLCKLIVGNL PDFHAQILDD VAHFTQVKNV RLELYVSESL YRSTFDINCS NRTPELYDAV DLTVAIWNNG FINIKCEVID IEILNLEEVN ...String:
MSYYHHHHHH DYDIPTTENL YFQGAMGSMS QLVVDICKLN QVLLNLNERS KEIFTNSSRR FRVVAQLLDI QPYDDEENEG FLCKLIVGNL PDFHAQILDD VAHFTQVKNV RLELYVSESL YRSTFDINCS NRTPELYDAV DLTVAIWNNG FINIKCEVID IEILNLEEVN QLREFIASPM GQEFLQLSNS TGDYSQPNT

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Macromolecule #4: 3xTEL

MacromoleculeName: 3xTEL / type: dna / ID: 4
Details: Three repeats of TTAGGG single-stranded DNA molecule
Classification: DNA
Source (natural)Organism: Homo sapiens (human)
SequenceString:
TTAGGGTTAG GGTTAGGG

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration10 mg/mL
BufferpH: 7.5
Details: 50 mM HEPES-NaOH pH 7.5, 500 mM NaCl, 15 mM imidazole, 1 mM TCEP
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: LEICA EM GP
Details3xTELssDNA molecules were added at 1.2-fold excess of CST monomers and incubated on ice for 2 hr before applying to EM grids.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 28053 / Average electron dose: 60.0 e/Å2
Details: Two datasets of 0 deg (20,826 movies) and 30 deg (7,227 movies) stage-tilt were collected on the FEI Titan Krios. Collection was done with super-resolution CDS mode at 0.539 angstroms/pixel
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6983835
CTF correctionSoftware - Name: Gctf
Details: CTF correction were separately done for the 0 and 30 deg. tilt datasets.
Startup modelType of model: OTHER / Details: Generated from CryoSparc2 ab initio program
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: D5 (2x5 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Details: Homologous refinement in CryoSparc2 / Number images used: 309576
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.7)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.7)
FSC plot (resolution estimation)

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