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- EMDB-26212: mTORC2 complex with Akt -

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Basic information

Entry
Database: EMDB / ID: EMD-26212
TitlemTORC2 complex with Akt
Map data
Sample
  • Complex: apostate of mTORC2 complex, composed of mTOR, Rictor, mLST8 and mSin1
Keywordscomplex / SIGNALING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.44 Å
AuthorsYu Z / Chen J / Pearce D
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: J Biol Chem / Year: 2022
Title: Interactions between mTORC2 core subunits Rictor and mSin1 dictate selective and context-dependent phosphorylation of substrate kinases SGK1 and Akt.
Authors: Zanlin Yu / Junliang Chen / Enzo Takagi / Feng Wang / Bidisha Saha / Xi Liu / Lydia-Marie Joubert / Catherine E Gleason / Mingliang Jin / Chengmin Li / Carlos Nowotny / David Agard / Yifan ...Authors: Zanlin Yu / Junliang Chen / Enzo Takagi / Feng Wang / Bidisha Saha / Xi Liu / Lydia-Marie Joubert / Catherine E Gleason / Mingliang Jin / Chengmin Li / Carlos Nowotny / David Agard / Yifan Cheng / David Pearce /
Abstract: Mechanistic target of rapamycin complex 2 (mTORC2) is a multi-subunit kinase complex, central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1, distinguish it from the ...Mechanistic target of rapamycin complex 2 (mTORC2) is a multi-subunit kinase complex, central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1, distinguish it from the related mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously; however, important questions remain, particularly regarding the structural determinants mediating substrate specificity and context-dependent activity. Here, we used cryo-EM to obtain high-resolution structures of the human mTORC2 apo-complex in the presence of substrates Akt and SGK1. Using functional assays, we then tested predictions suggested by substrate-induced structural changes in mTORC2. For the first time, we visualized in the apo-state the side chain interactions between Rictor and mTOR that sterically occlude recruitment of mTORC1 substrates and confer resistance to the mTORC1 inhibitor rapamycin. Also in the apo-state, we observed that mSin1 formed extensive contacts with Rictor via a pair of short α-helices nestled between two Rictor helical repeat clusters, as well as by an extended strand that makes multiple weak contacts with Rictor helical cluster 1. In co-complex structures, we found that SGK1, but not Akt, markedly altered the conformation of the mSin1 N-terminal extended strand, disrupting multiple weak interactions while inducing a large rotation of mSin1 residue Arg-83, which then interacts with a patch of negatively charged residues within Rictor. Finally, we demonstrate mutation of Arg-83 to Ala selectively disrupts mTORC2-dependent phosphorylation of SGK1, but not of Akt, supporting context-dependent substrate selection. These findings provide new structural and functional insights into mTORC2 specificity and context-dependent activity.
History
DepositionFeb 16, 2022-
Header (metadata) releaseMar 8, 2023-
Map releaseMar 8, 2023-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26212.png

Downloads & links

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Map

FileDownload / File: emd_26212.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 324 pix.
= 356.4 Å		1.1 Å/pix.
x 324 pix.
= 356.4 Å		1.1 Å/pix.
x 324 pix.
= 356.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.18
Minimum - Maximum-9.61613 - 16.498259000000001
Average (Standard dev.)0.032793317 (±1.1239145)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 356.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : apostate of mTORC2 complex, composed of mTOR, Rictor, mLST8 and mSin1

EntireName: apostate of mTORC2 complex, composed of mTOR, Rictor, mLST8 and mSin1
Components
  • Complex: apostate of mTORC2 complex, composed of mTOR, Rictor, mLST8 and mSin1

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Supramolecule #1: apostate of mTORC2 complex, composed of mTOR, Rictor, mLST8 and mSin1

SupramoleculeName: apostate of mTORC2 complex, composed of mTOR, Rictor, mLST8 and mSin1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state3D array

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
GridModel: Quantifoil / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 22 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 78725
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD

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