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- PDB-6emk: Cryo-EM Structure of Saccharomyces cerevisiae Target of Rapamycin... -

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Basic information

Entry
Database: PDB / ID: 6emk
TitleCryo-EM Structure of Saccharomyces cerevisiae Target of Rapamycin Complex 2
DescriptorSerine/threonine-protein kinase TOR2
Target of rapamycin complex subunit LST8
(Target of rapamycin complex 2 subunit ...) x 3
KeywordsSIGNALING PROTEIN / target of rapamycin / torc2 / FRB domain / Tor2-Lst8 / kinases
Specimen sourceSaccharomyces cerevisiae (strain atcc 204508 / s288c) / yeast / Baker's yeast /
MethodElectron microscopy (7.9 Å resolution / Particle / Single particle)
AuthorsKaruppasamy, M. / Kusmider, B. / Oliveira, T.M. / Gaubitz, C. / Prouteau, M. / Loewith, R. / Schaffitzel, C.
CitationNat Commun, 2017, 8, 1729-1729

primary. Nat Commun, 2017, 8, 1729-1729 Yorodumi Papers
Cryo-EM structure of Saccharomyces cerevisiae target of rapamycin complex 2.
Manikandan Karuppasamy / Beata Kusmider / Taiana M Oliveira / Christl Gaubitz / Manoel Prouteau / Robbie Loewith / Christiane Schaffitzel

#1. Mol. Cell, 2015, 58, 977-988 Yorodumi Papers
Molecular Basis of the Rapamycin Insensitivity of Target Of Rapamycin Complex 2.
Gaubitz, C. / Oliveira, T.M. / Prouteau, M. / Leitner, A. / Karuppasamy, M. / Konstantinidou, G. / Rispal, D. / Eltschinger, S. / Robinson, G.C. / Thore, S. / Aebersold, R. / Schaffitzel, C. / Loewith, R.

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 2, 2017 / Release: Dec 6, 2017

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Structure visualization

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Assembly

Deposited unit
A: Serine/threonine-protein kinase TOR2
B: Target of rapamycin complex subunit LST8
C: Serine/threonine-protein kinase TOR2
D: Target of rapamycin complex subunit LST8
E: Target of rapamycin complex 2 subunit TSC11
F: Target of rapamycin complex 2 subunit TSC11
G: Target of rapamycin complex 2 subunit AVO2
H: Target of rapamycin complex 2 subunit AVO2
I: Target of rapamycin complex 2 subunit AVO1
J: Target of rapamycin complex 2 subunit AVO1


Theoretical massNumber of molelcules
Total (without water)1,041,14010
Polyers1,041,14010
Non-polymers00
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)16320
ΔGint (kcal/M)-69
Surface area (Å2)292380
MethodPISA

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Components

#1: Polypeptide(L)Serine/threonine-protein kinase TOR2 / Dominant rapamycin resistance protein 2 / Phosphatidylinositol 4-kinase TOR2 / PtdIns-4-kinase TOR2 / Target of rapamycin kinase 2 / Temperature-sensitive CSG2 suppressor protein 14


Mass: 281915.438 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (strain atcc 204508 / s288c) / yeast /
References: UniProt: P32600, EC: 2.7.1.67, EC: 2.7.11.1
#2: Polypeptide(L)Target of rapamycin complex subunit LST8 / TORC subunit LST8 / Lethal with SEC13 protein 8


Mass: 34077.879 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (strain atcc 204508 / s288c) / yeast /
References: UniProt: P41318
#3: Polypeptide(L)Target of rapamycin complex 2 subunit TSC11


Mass: 25804.662 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (strain atcc 204508 / s288c) / yeast /
#4: Polypeptide(L)Target of rapamycin complex 2 subunit AVO2 / TORC2 subunit AVO2 / Adheres voraciously to TOR2 protein 2


Mass: 47206.457 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (strain atcc 204508 / s288c) / yeast /
References: UniProt: Q04749
#5: Polypeptide(L)Target of rapamycin complex 2 subunit AVO1 / TORC2 subunit AVO1 / Adheres voraciously to TOR2 protein 1


Mass: 131565.453 Da / Num. of mol.: 2
Source: (gene. exp.) Saccharomyces cerevisiae (strain atcc 204508 / s288c) / yeast /
References: UniProt: Q08236

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: Target of rapamycin protein complex 2 / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5 / Source: RECOMBINANT
Molecular weightValue: 1.4 deg. / Units: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Source (recombinant)Organism: Saccharomyces cerevisiae
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 kelvins / Details: 2 - 3 sec blotting

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 kelvins / Temperature (min): 70 kelvins
Image recording

Imaging ID: 1

IDAverage exposure timeElectron doseDetector modeFilm or detector modelNumber of grids imagedNumber of real images
12.350INTEGRATINGFEI FALCON II (4k x 4k)44189
22047SUPER-RESOLUTIONGATAN K2 QUANTUM (4k x 4k)12847
Image scansDimension width: 3710 / Dimension height: 3838 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: REFMAC / Version: 5.8.0158 / Classification: refinement
EM software
IDNameVersionCategoryImaging IDImage processing IDFitting ID
2SerialEM3.6IMAGE ACQUISITION1
4CTFFIND4CTF CORRECTION1
7Coot0.8.8MODEL FITTING1
9RELION1.4INITIAL EULER ASSIGNMENT1
10RELION1.4FINAL EULER ASSIGNMENT1
12RELION1.4RECONSTRUCTION1
13EMAN2PARTICLE SELECTION2
15CTFFIND4CTF CORRECTION2
16RELION1.4INITIAL EULER ASSIGNMENT2
17RELION1.4FINAL EULER ASSIGNMENT2
19RELION1.4RECONSTRUCTION2
20REFMAC5.8.0158MODEL REFINEMENT1
Image processing
IDImage recording ID
11
22
CTF correction
IDEM image processing IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
IDImage processing IDNumber of particles selected
11350000
22200000
Symmetry
IDImage processing IDEntry IDPoint symmetry
116EMKC2
226EMKC2
3D reconstruction #1Resolution: 7.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 16190 / Symmetry type: POINT
3D reconstruction #2Resolution: 8 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 10663 / Symmetry type: POINT
Atomic model buildingRef protocol: FLEXIBLE FIT / Ref space: REAL
Atomic model buildingPDB-ID: 5FVM
Pdb chain residue range: 81-2474
Refine
Refine IDB iso meanAniso B11Aniso B12Aniso B13Aniso B22Aniso B23Aniso B33Correlation coeff Fo to FcR factor R workR factor obsHighest resolutionLowest resolutionNumber reflection obsPercent reflection obsOverall SU BOverall SU MLStereochemistry target valuesSolvent model details
1295.421-0.802.16-1.12-1.830.112.630.9870.121390.121398.00220.8030863100.00111.2930.794MAXIMUM LIKELIHOOD WITH PHASESPARAMETERS FOR MASK CACLULATION
ELECTRON MICROSCOPY
Number of atoms included #1Total: 48016
Refine LS restraints
Refine IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0130.02047333
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg2.0241.96061663
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg10.8765.0004749
ELECTRON MICROSCOPYr_dihedral_angle_2_deg40.82824.4012036
ELECTRON MICROSCOPYr_dihedral_angle_3_deg23.37615.0008088
ELECTRON MICROSCOPYr_dihedral_angle_4_deg16.61915.000268
ELECTRON MICROSCOPYr_chiral_restr0.1530.2007744
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.02130090
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it10.66830.43623745
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it19.39445.99026911
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it11.15330.10823588
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined47.64078872
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS shellHighest resolution: 8 Å / R factor R work: 0.377 / Lowest resolution: 8.207 Å / Number reflection R free: 0 / Number reflection R work: 2391 / Total number of bins used: 20 / Percent reflection obs: 1

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