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- EMDB-4095: Negative stain EM-structure of Checkpoint point kinase Tel1 -

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Basic information

Entry
Database: EMDB / ID: EMD-4095
TitleNegative stain EM-structure of Checkpoint point kinase Tel1
Map dataNegative stain structure of dimeric Tel1.
Sample
  • Complex: Tel1 protein
Biological speciesSaccharomyces cerevisiae S288c (yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 22.7 Å
AuthorsDarbari VC / Sawicka M / Wanrooij PH / Hailemariam S / Zhang X / Burgers PM
CitationJournal: J Biol Chem / Year: 2016
Title: The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization.
Authors: Marta Sawicka / Paulina H Wanrooij / Vidya C Darbari / Elias Tannous / Sarem Hailemariam / Daniel Bose / Alena V Makarova / Peter M Burgers / Xiaodong Zhang /
Abstract: The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play ...The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form.
History
DepositionAug 8, 2016-
Header (metadata) releaseAug 17, 2016-
Map releaseAug 17, 2016-
UpdateAug 2, 2017-
Current statusAug 2, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4095.png

Downloads & links

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Map

FileDownload / File: emd_4095.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain structure of dimeric Tel1.
Voxel sizeX=Y=Z: 4.64 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.055703584 - 0.25362128
Average (Standard dev.)0.00078898005 (±0.009466247)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-50-50-50
Dimensions100100100
Spacing100100100
CellA=B=C: 464.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.644.644.64
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z464.000464.000464.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-50-50-50
NC/NR/NS100100100
D min/max/mean-0.0560.2540.001

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Supplemental data

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Sample components

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Entire : Tel1 protein

EntireName: Tel1 protein
Components
  • Complex: Tel1 protein

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Supramolecule #1: Tel1 protein

SupramoleculeName: Tel1 protein / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae S288c (yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: BJ2168 / Recombinant plasmid: pBL602
Molecular weightTheoretical: 640 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
40.0 mMC8H18N2O4SHEPES
700.0 mMKClPotassium Chloride
10.0 mMK2HPO4/KH2PO4Potassium phosphate
10.0 %C3H8O3Glycerol
1.0 mMC4H10O2S2DTT
1.0 mMC10H16N2O8EDTAEthylenediaminetetraacetic acid
0.5 mMC14H24N2O10EGTA
0.1 %C58H114O26Tween
0.01 %NP-40
StainingType: NEGATIVE / Material: 2% Uranyl Acetate
GridModel: TAAB / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 38000
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 5.0 e/Å2

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Image processing

Particle selectionNumber selected: 7350
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: IMAGIC (ver. 4)
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Angular sampling: 5.0 degrees
Software - Name: IMAGIC
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 22.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: IMAGIC / Number images used: 7350
FSC plot (resolution estimation)

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