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- EMDB-4503: Human substrate-free CCT -

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Basic information

Entry
Database: EMDB / ID: EMD-4503
TitleHuman substrate-free CCT
Map dataHuman substrate-free CCT
Sample
  • Complex: Human substrate-free CCT
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.5 Å
AuthorsCuellar J / Santiago C / Ludlam WG / Bueno-Carrasco MT / Valpuesta JM / Willardson BM
Funding support United States, Spain, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research InstituteEY012287 United States
Spanish Ministry of Science, Innovation, and UniversitiesBFU2016-75984 Spain
CitationJournal: Nat Commun / Year: 2019
Title: Structural and functional analysis of the role of the chaperonin CCT in mTOR complex assembly.
Authors: Jorge Cuéllar / W Grant Ludlam / Nicole C Tensmeyer / Takuma Aoba / Madhura Dhavale / César Santiago / M Teresa Bueno-Carrasco / Michael J Mann / Rebecca L Plimpton / Aman Makaju / Sarah ...Authors: Jorge Cuéllar / W Grant Ludlam / Nicole C Tensmeyer / Takuma Aoba / Madhura Dhavale / César Santiago / M Teresa Bueno-Carrasco / Michael J Mann / Rebecca L Plimpton / Aman Makaju / Sarah Franklin / Barry M Willardson / José M Valpuesta /
Abstract: The mechanistic target of rapamycin (mTOR) kinase forms two multi-protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. Two ...The mechanistic target of rapamycin (mTOR) kinase forms two multi-protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. Two of the subunits of these complexes are mLST8 and Raptor, β-propeller proteins that stabilize the mTOR kinase and recruit substrates, respectively. Here we report that the eukaryotic chaperonin CCT plays a key role in mTORC assembly and signaling by folding both mLST8 and Raptor. A high resolution (4.0 Å) cryo-EM structure of the human mLST8-CCT intermediate isolated directly from cells shows mLST8 in a near-native state bound to CCT deep within the folding chamber between the two CCT rings, and interacting mainly with the disordered N- and C-termini of specific CCT subunits of both rings. These findings describe a unique function of CCT in mTORC assembly and a distinct binding site in CCT for mLST8, far from those found for similar β-propeller proteins.
History
DepositionDec 27, 2018-
Header (metadata) releaseJul 3, 2019-
Map releaseJul 3, 2019-
UpdateNov 6, 2019-
Current statusNov 6, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0038
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0038
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4503.png

Downloads & links

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Map

FileDownload / File: emd_4503.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman substrate-free CCT
Voxel sizeX=Y=Z: 0.89 Å
Density
Contour LevelBy AUTHOR: 0.0038 / Movie #1: 0.0038
Minimum - Maximum-0.008884804 - 0.021280224
Average (Standard dev.)-0.00028310472 (±0.0029267685)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 249.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.890.890.89
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z249.200249.200249.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.0090.021-0.000

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Supplemental data

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Sample components

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Entire : Human substrate-free CCT

EntireName: Human substrate-free CCT
Components
  • Complex: Human substrate-free CCT

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Supramolecule #1: Human substrate-free CCT

SupramoleculeName: Human substrate-free CCT / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Details: we have characterized the eukaryotic chaperonin CCT (1 MDa; formed by two copies of eight homologous subunits)
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293T
Molecular weightExperimental: 1 MDa

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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

Concentration0.5 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R2/2 / Material: COPPER/RHODIUM / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
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 / Cs: 2.7 mm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3292 / Average exposure time: 8.0 sec. / Average electron dose: 42.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 504060
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: INSILICO MODEL / In silico model: ransac (Xmipp3) and Initial model (Eman2)
Initial angle assignmentType: COMMON LINE / Software - Name: RELION (ver. 2)
Final 3D classificationSoftware - Name: RELION (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2) / Number images used: 139819
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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