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- EMDB-5197: Cryo-EM reconstruction of human mTORC1 -

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Basic information

Entry
Database: EMDB / ID: EMD-5197
TitleCryo-EM reconstruction of human mTORC1
Map dataDensity map of human mTORC1 resolution filtered to 26 Angstroms
Sample
  • Sample: Purified human mTORC1
  • Protein or peptide: mTOR
  • Protein or peptide: raptor
  • Protein or peptide: mLST8
  • Protein or peptide: PRAS40
KeywordsKinase complex / metabolic role
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 26.0 Å
AuthorsYip CK / Murata K / Walz T / Sabataini DM / Kang SA
CitationJournal: Mol Cell / Year: 2010
Title: Structure of the human mTOR complex I and its implications for rapamycin inhibition.
Authors: Calvin K Yip / Kazuyoshi Murata / Thomas Walz / David M Sabatini / Seong A Kang /
Abstract: The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth in response to the nutrient and energy status of the cell, and its deregulation is common in human cancers. Little is known ...The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth in response to the nutrient and energy status of the cell, and its deregulation is common in human cancers. Little is known about the overall architecture and subunit organization of this essential signaling complex. We have determined the three-dimensional (3D) structure of the fully assembled human mTORC1 by cryo-electron microscopy (cryo-EM). Our analyses reveal that mTORC1 is an obligate dimer with an overall rhomboid shape and a central cavity. The dimeric interfaces are formed by interlocking interactions between the mTOR and raptor subunits. Extended incubation with FKBP12-rapamycin compromises the structural integrity of mTORC1 in a stepwise manner, leading us to propose a model in which rapamycin inhibits mTORC1-mediated phosphorylation of 4E-BP1 and S6K1 through different mechanisms.
History
DepositionApr 28, 2010-
Header (metadata) releaseMay 5, 2010-
Map releaseJun 10, 2010-
UpdateJul 17, 2013-
Current statusJul 17, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5197_1.tif

Downloads & links

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Map

FileDownload / File: emd_5197.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDensity map of human mTORC1 resolution filtered to 26 Angstroms
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.1 Å/pix.
x 128 pix.
= 524.8 Å		4.1 Å/pix.
x 128 pix.
= 524.8 Å		4.1 Å/pix.
x 128 pix.
= 524.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-0.26783767 - 2.0916965
Average (Standard dev.)0.01507967 (±0.1707899)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 524.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.14.14.1
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z524.800524.800524.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-26-72
NX/NY/NZ6953145
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.2682.0920.015

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Supplemental data

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Sample components

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Entire : Purified human mTORC1

EntireName: Purified human mTORC1
Components
  • Sample: Purified human mTORC1
  • Protein or peptide: mTOR
  • Protein or peptide: raptor
  • Protein or peptide: mLST8
  • Protein or peptide: PRAS40

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Supramolecule #1000: Purified human mTORC1

SupramoleculeName: Purified human mTORC1 / type: sample / ID: 1000 / Oligomeric state: dimer of heterotetramer / Number unique components: 4
Molecular weightTheoretical: 1.0 MDa

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Macromolecule #1: mTOR

MacromoleculeName: mTOR / type: protein_or_peptide / ID: 1 / Name.synonym: mTOR / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HEK-293T / Location in cell: cytoplasm
Molecular weightTheoretical: 289 KDa
Recombinant expressionOrganism: Mammalian cells

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Macromolecule #2: raptor

MacromoleculeName: raptor / type: protein_or_peptide / ID: 2 / Name.synonym: raptor / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HEK-293T / Location in cell: cytoplasm
Molecular weightTheoretical: 150 KDa
Recombinant expressionOrganism: Mammalian cells

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Macromolecule #3: mLST8

MacromoleculeName: mLST8 / type: protein_or_peptide / ID: 3 / Name.synonym: mLST8 / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: HEK-293T / Location in cell: cytoplasm
Molecular weightExperimental: 36 KDa / Theoretical: 36 KDa
Recombinant expressionOrganism: Mammalian cells

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Macromolecule #4: PRAS40

MacromoleculeName: PRAS40 / type: protein_or_peptide / ID: 4 / Name.synonym: PRAS40 / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: HEK-293T / Location in cell: cytoplasm
Molecular weightExperimental: 27 KDa / Theoretical: 27 KDa
Recombinant expressionOrganism: Mammalian cells

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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

Concentration0.02 mg/mL
BufferpH: 7.5 / Details: 50mM HEPES, 150mM NaCl
GridDetails: 400 mesh Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
DateMar 12, 2008
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 21 µm / Number real images: 323
Tilt angle min0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 51159 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.4 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 52000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN / Tilt angle max: 45
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsManual picking
CTF correctionDetails: each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: FREALIGN / Details: final map was resolution filtered to 26 Angstroms / Number images used: 28325
Final angle assignmentDetails: SPIDER:theta 45 degrees, phi 45 degrees
Final two d classificationNumber classes: 200

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Atomic model buiding 1

Initial modelPDB ID:

3emh


Chain - Chain ID: A
DetailsPDBEntryID_givenInChain. Protocol: manual docking. model was manually docked into density map using Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: rigid body

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