Computational protocol: Cryo-EM structure of Saccharomyces cerevisiae target of rapamycin complex 2

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Protocol publication

[…] Single-particle cryo-EM data were collected using a FEI TITAN Krios microscope operated at 300 kV at EMBL Heidelberg. The data were recorded on a FEI Falcon II DED using automated data acquisition software (FEI EPU). A total of 4189 dose-fractionated movies each containing 40 frames with an accumulated total dose of 50 e/Å2 were recorded at a nominal magnification of ×59,000 corresponding to a pixel size of 1.38 Å and a total exposure time of 2.3 s. Images were acquired with a defocus range of −2 μm to −4 μm in steps of 0.1 μm. A second data set was collected from the same microscope using a K2 Summit detector (Gatan) with an energy filter. A total of 2847 dose-fractionated movies each containing 40 frames (0.5 s per frame) with a accumulated total dose of 47 e/Å2 were recorded semi-automatically using SerialEM at a nominal magnification of ×105,000 in super-resolution mode. Images were recorded with a defocus range of −1.5 μm to −3.5 μm. Subsequently, cropping in Fourier space resulted in a pixel size of 1.35 Å.The movie frames from the Falcon II DED were aligned, dose-filtered and summed in Unblur1.0.2. The individual movie frames from the K2 Summit detector were gain-corrected, aligned with the “patch” option, dose-weighted and summed using the MotionCor2 program. The CTF parameters for both data sets were estimated from CTFFIND4, integrated in Relion1.4.Automated particle picking was carried out in e2boxer.py from EMAN2 with Gauss option with the box size of 320 pixels. All automatically picked particles were subjected to two rounds of 2D classification in Relion resulting in a pool of 111,196 good quality particles for data set 1 (from Falcon II). The TORC2 negative stain reconstruction (EMDB: 2990; ref. ) was filtered to 60 Å and used as initial reference for 3D classification. At every step of classification, the data were grouped into two volumes (Supplementary Fig. ). After three rounds of successive 3D classification with K = 2, a stable class with 16,190 particles yielded a well-defined volume (Supplementary Fig. ). The second data set (from K2 Summit) consisting of 71,519 particles was sorted according to the same protocol and gave rise to additional 10,663 particles. The two data sets were combined after rescaling the second data set to a pixel size of 1.38 Å. The volume was further refined with a molecular shape mask (5 pixel Gaussian fall-off) and post-processed in Relion with C2 symmetry imposed. The resolution of the final volume was determined to be 7.9 Å based on the Fourier shell correlation (FSC) = 0.143 criterion (Supplementary Fig. ). During post-processing, the volume was corrected for the modulation transfer function of the detector and the effect of mask was checked by phase randomization. The final map was sharpened with a B-factor value of −130 Å2. The local resolution analysis of the map was carried out using the program ResMap. [...] The atomic model derived from the 6.1 Å map of the Km Tor–Lst8 complex was used as a template structure to obtain the homology model for S. cerevisiae Tor2. Homology modelling was performed in the Phyre2 homology modelling server. The model was adjusted to fit into the map manually using COOT with the TORC2 map contoured at 7.5 sigma where individual helices are clearly visible as tubes (Supplementary Fig.  and Fig. ). The remaining unfilled density was examined to fit other subunits (Avo1, Avo2 and Avo3). The secondary structure prediction of Avo1 indicates that this TORC subunit is largely unstructured, with the exception of the 13 kDa PH domain (aa 1076–1176; 3ulb; ref. ) and the 16-kDa CRIM domain (aa 638–786; 2rvk; ref. ). SwissModel was used to build a homology model for Avo1_CRIM based on the NMR structure of the Sin1 CRIM domain from fission yeast (2rvk; ref. ). The Avo1_CRIM model (residues 647–792) fitted into the density between the Tor2 FRB domain and Lst8 (at 3.5 sigma level; cc of 0.87 in Chimera). The N-terminal part of Avo2 has been predicted to contains five ankyrin repeats (residues 4–171). The homology model of the N-terminal part of Avo2 (aa 4–163) was generated in Phyre2 using the pdb structure 1n11 (chain A) as template. The Avo2 model fits well into the density previously assigned to Avo2 connecting the Tor2 spiral domain on one side and the Tor2 C-terminal domain (cc of 0.94). For Avo3, a set of 17 helices of average length of 20 amino acids could be modelled into EM density contoured at 7.5 sigma. Secondary structure prediction suggests that the middle part of Avo3 contains an armadillo-like helical domain. Accordingly, we assign these alpha helices to the Avo3 armadillo-like domain. Moreover, the localization of these helices agrees with the predicted central position of Avo3 deduced from XL-MS and from negative stain EM domain localization experiments. The partial model of TORC2 was refined against the density in real_space using the “phenix.real_space_refine” program. The cross-correlation values for the docked atomic coordinates into the corresponding subunit EM map are listed in Supplementary Table  and the overall fitting was assessed by calculating the FSC between the atomic model and the corresponding parts of the map and the half maps using REFMAC (Supplementary Fig. ). The refined model was used for all the interpretation. The figures were prepared in Chimera and PyMol (http://www.pymol.org/) (DeLano Scientific, San Carlos, CA, USA). […]

Pipeline specifications

Software tools SerialEM, Unblur, MotionCor2, CTFFIND, RELION, e2boxer.py, EMAN, ResMap, Phyre, Coot, PHENIX, PyMOL
Databases DED
Applications cryo-EM, Protein structure analysis
Organisms Saccharomyces cerevisiae, Homo sapiens
Chemicals Sirolimus