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- EMDB-3336: Cryo electron microscopy of a complex of Tor-1175RFP and Lst8 -

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Basic information

Entry
Database: EMDB / ID: EMD-3336
TitleCryo electron microscopy of a complex of Tor-1175RFP and Lst8
Map dataReconstruction of a complex of variant Tor-1175RFP and Lst8.
Sample
  • Sample: Complex of Tor-1175RFP with Lst8
  • Protein or peptide: Target of rapamycin (Tor)
  • Protein or peptide: Lst8
  • Protein or peptide: Red fluorescent protein (RFP)
Keywordscryo-EM / Tor / Lst8 / mTOR / kinase / PIKK / S/T protein kinase / TORC1 / mTORC1 / RFP
Function / homologyTORC1 complex / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / bioluminescence / generation of precursor metabolites and energy / Red fluorescent protein drFP583
Function and homology information
Biological speciesKluyveromyces marxianus (yeast) / Discosoma sp. (sea anemone)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.3 Å
AuthorsBaretic D / Berndt A / Ohashi Y / Johnson CM / Williams RL
CitationJournal: Nat Commun / Year: 2016
Title: Tor forms a dimer through an N-terminal helical solenoid with a complex topology.
Authors: Domagoj Baretić / Alex Berndt / Yohei Ohashi / Christopher M Johnson / Roger L Williams /
Abstract: The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor-Lst8 ...The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor-Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor-Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor-Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended 'railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit.
History
DepositionFeb 16, 2016-
Header (metadata) releaseFeb 24, 2016-
Map releaseApr 13, 2016-
UpdateApr 13, 2016-
Current statusApr 13, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3336.png

Downloads & links

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Map

FileDownload / File: emd_3336.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of a complex of variant Tor-1175RFP and Lst8.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 300 pix.
= 402. Å		1.34 Å/pix.
x 300 pix.
= 402. Å		1.34 Å/pix.
x 300 pix.
= 402. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.10567734 - 0.25205806
Average (Standard dev.)0.00097085 (±0.01605722)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 402.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z402.000402.000402.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-300-64
NX/NY/NZ6161129
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1060.2520.001

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Supplemental data

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Sample components

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Entire : Complex of Tor-1175RFP with Lst8

EntireName: Complex of Tor-1175RFP with Lst8
Components
  • Sample: Complex of Tor-1175RFP with Lst8
  • Protein or peptide: Target of rapamycin (Tor)
  • Protein or peptide: Lst8
  • Protein or peptide: Red fluorescent protein (RFP)

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Supramolecule #1000: Complex of Tor-1175RFP with Lst8

SupramoleculeName: Complex of Tor-1175RFP with Lst8 / type: sample / ID: 1000 / Oligomeric state: Dimer of Tor-1175RFP/Lst8 heterodimers / Number unique components: 3
Molecular weightTheoretical: 738 KDa

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Macromolecule #1: Target of rapamycin (Tor)

MacromoleculeName: Target of rapamycin (Tor) / type: protein_or_peptide / ID: 1 / Name.synonym: Tor / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Kluyveromyces marxianus (yeast)
Molecular weightTheoretical: 277 KDa
Recombinant expressionOrganism: Kluyveromyces marxianus (yeast)
SequenceGO: TORC1 complex

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Macromolecule #2: Lst8

MacromoleculeName: Lst8 / type: protein_or_peptide / ID: 2 / Name.synonym: Lethal with SEC13 protein 8 / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Kluyveromyces marxianus (yeast)
Molecular weightTheoretical: 34 KDa
Recombinant expressionOrganism: Kluyveromyces marxianus (yeast)

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Macromolecule #3: Red fluorescent protein (RFP)

MacromoleculeName: Red fluorescent protein (RFP) / type: protein_or_peptide / ID: 3 / Name.synonym: RFP, dsRed, FP583
Details: The tandem RFP was inserted between residues T1175 and K1176 of K. marxianus Target of rapamycin (Tor) polypeptide chain.
Number of copies: 4 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Discosoma sp. (sea anemone) / synonym: Coral anemones
Molecular weightTheoretical: 54 KDa
Recombinant expressionOrganism: Kluyveromyces marxianus (yeast)
SequenceUniProtKB: Red fluorescent protein drFP583

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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

Concentration2.5 mg/mL
BufferpH: 7.4
Details: 50 mM Hepes pH 7.4 (23 deg C), 75 mM KCl, 250 mM NaCl, 0.3 % (v/v) CHAPS, 1 mM TCEP
GridDetails: Quantifoil Au R 1.2/1.3, 300 mesh grids, blotted for 11-13 s at 4 deg C
VitrificationCryogen name: ETHANE / Instrument: OTHER / Method: 11-13 s at 4 deg C

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateJan 14, 2016
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 465 / Average electron dose: 40 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 105263 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 78000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsProcessed with RELION. CTF corrected each particle.
CTF correctionDetails: Each particle (Gctf)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 10.3 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 5253
Final angle assignmentDetails: RELION
FSC plot (resolution estimation)

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