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- EMDB-30597: Cryo-EM map of POT1 bound by TPP1 in extended conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-30597
TitleCryo-EM map of POT1 bound by TPP1 in extended conformation
Map dataExtended Conformation
Sample
  • Complex: Complex of POT1 with TPP1
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.6 Å
AuthorsSmith EW / Liu ZB / Lattmann S / Ahsan B / Rhodes D
Funding support Singapore, 1 items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore) Singapore
CitationJournal: PLoS One / Year: 2022
Title: Insights into POT1 structural dynamics revealed by cryo-EM.
Authors: Emmanuel W Smith / Simon Lattmann / Zhehui Barry Liu / Bilal Ahsan / Daniela Rhodes /
Abstract: Telomeres are protein-DNA complexes that protect the ends of linear eukaryotic chromosomes. Mammalian telomeric DNA consists of 5'-(TTAGGG)n-3' double-stranded repeats, followed by up to several ...Telomeres are protein-DNA complexes that protect the ends of linear eukaryotic chromosomes. Mammalian telomeric DNA consists of 5'-(TTAGGG)n-3' double-stranded repeats, followed by up to several hundred bases of a 3' single-stranded G-rich overhang. The G-rich overhang is bound by the shelterin component POT1 which interacts with TPP1, the component involved in telomerase recruitment. A previously published crystal structure of the POT1 N-terminal half bound to the high affinity telomeric ligand 5'-TTAGGGTTAG-3' showed that the first six nucleotides, TTAGGG, are bound by the OB1 fold, while the adjacent OB2 binds the last four, TTAG. Here, we report two cryo-EM structures of full-length POT1 bound by the POT1-binding domain of TPP1. The structures differ in the relative orientation of the POT1 OB1 and OB2, suggesting that these two DNA-binding OB folds take up alternative conformations. Supporting DNA binding studies using telomeric ligands in which the OB1 and OB2 binding sites were spaced apart, show that POT1 binds with similar affinities to spaced or contiguous binding sites, suggesting plasticity in DNA binding and a role for the alternative conformations observed. A likely explanation is that the structural flexibility of POT1 enhances binding to the tandemly arranged telomeric repeats and hence increases telomere protection.
History
DepositionOct 2, 2020-
Header (metadata) releaseOct 13, 2021-
Map releaseOct 13, 2021-
UpdateApr 27, 2022-
Current statusApr 27, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0041
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0041
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30597.png

Downloads & links

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Map

FileDownload / File: emd_30597.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationExtended Conformation
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.0041 / Movie #1: 0.0041
Minimum - Maximum-0.0071709715 - 0.017071413
Average (Standard dev.)8.3546394e-05 (±0.0007722431)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 220.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z220.000220.000220.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ336336336
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0070.0170.000

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Supplemental data

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Sample components

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Entire : Complex of POT1 with TPP1

EntireName: Complex of POT1 with TPP1
Components
  • Complex: Complex of POT1 with TPP1

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Supramolecule #1: Complex of POT1 with TPP1

SupramoleculeName: Complex of POT1 with TPP1 / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Baculovirus

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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

BufferpH: 7.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 9.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 32488

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