Computational protocol: Phase-plate cryo-EM structure of a class B GPCR-G protein complex

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[…] Concentrated sample from Superdex 200 Increase 10/300 GL column was loaded onto a Superose 6 Increase 10/300 GL column (GE Healthcare). Eluted fractions were used to prepare specimens for EM imaging using conventional negative staining protocol. Negative-stained samples were imaged at room temperature with a Tecnai T12 (FEI) electron microscope operated at 120 kV. Images were recorded at magnification of 57,000× and a defocus value of −1 μm on a Gatan US4000 CCM camera. All images were binned to 2×2 pixels to obtain a pixel size of 4.16 Å.For cryo-EM, purified CTR Gs heterotrimer complex was diluted to 0.3mg/mL with 20mM HEPES pH 7.4, 100mM NaCl, 2mM MgCl2, 100nM sCT. Vitrified specimen was prepared by applying 5 μL of protein complexes onto a glow discharged 300 mesh copper Quantifoil R1.2/1.3 grid (Quantifoil Micro Tools), plunge-frozen in liquid ethane cooled by liquid nitrogen inside a Vitrobot Mark IV (FEI) with blotting time of 3 s and draining time of 0.5 s. Cryo-EM imaging was performed on a Titan Krios microscope operated at 300 kV (FEI) equipped with a Gatan Quantum energy filter, a Gatan K2 Summit direct electron camera (Gatan) and a Volta phase plate (FEI). 2780 movies were taken in EFTEM nanoprobe mode, with 50μm C2 aperture, at a calibrated magnification of 47170 corresponding to a magnified pixel size of 1.06 Å. Each movie comprises 50 sub frames with a total dose of 50 e−/Å2, exposure time between 11 and 13.75 s and a dose rate between 4 and 5 e−/pix/s on the detector. Data acquisition was done using SerialEM software and custom macros for automated single particle data acquisition with Volta phase plate at −500 nm defocus. [...] Image processing and three-dimensional reconstructions were performed as previously described. Dose fractionated image stacks were subjected to beam-induced motion correction, globally and locally, by MotionCor2. A sum of all frames, filtered according to exposure dose, in each image stack was used for further processing. CTF parameters for each micrograph were determined by CTFFIND4.Particle selection, two-dimensional classification and three-dimensional classification were performed on a binned dataset with a pixel size of 2.12 Å using RELION2. Semi-automated selected 1,213,995 particle projections were subjected to reference-free two-dimensional classification to discard false positive particles or particles categorized in poorly defined classes, resulting in 426,001 projections for further processing. An ab initio map generated by VIPER was used as initial reference model for maximum-likelihood-based three-dimensional classification. One stable class with detailed features accounting for 106,838 particles was then subjected to focused refinement with a soft mask including receptor and Gs protein and excluding the α-helical domain, produced the final map with global nominal resolution of 4.1 Å, and nominal resolution of 3.8-Å in the 7TM and G protein region.Reported resolutions are based on the “gold-standard” Fourier shell correlation (FSC) using the 0.143 criterion. All density maps were corrected for the modulation transfer function (MTF) of the K2 summit direct detector and then sharpened by applying temperature-factor that was estimated using post-processing in RELION. Local resolution was determined using ResMap with half-reconstructions as input maps. [...] The initial template of hCTR was derived from a homology based predicted model calculated by I-TASSER. Models of sCT and Gs heterotrimer were adopted from the NMR structure (PDB ID: 2GLH) and β2AR-Gs crystal structure (PDB ID: 3SN6), respectively. All models were visualized and docked into the density in Chimera, followed by manual adjustment and real-space refinement using COOT. Sequence assignment was guided by bulky residues such as Phe, Tyr, Trp and Arg. The final model was subjected to global refinement and minimization in real space using the module ‘phenix.real_space_refine’ in PHENIX. Due to lower local resolution for the peptide, its model was omitted from the deposited structure. Model overfitting was evaluated through its refinement against one cryo-EM half map. FSC curves were calculated between the resulting model and the half map used for refinement as well as between the resulting model and the other half map for cross-validation (). The final refinement statistics are provided in . […]

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