Titan krios transmission electron microscope

To prepare immune complexes, EV-D68 18947 virion, and 2H12 or 8F12 Fab were incubated at a molar ratio of 1:120 for 20 min at room temperature. 3 µL of EV-D68/2H12 or EV-D68/8F12 complex was placed onto a plasma-cleaned holey carbon grid (R1.2/1.3, 200 mesh; Quantifoil Micro Tools) or a continuous ultrathin carbon film covered lacey carbon grid (400 mesh; Ted Pella), respectively. The grids were blotted and plunged into liquid nitrogen-cooled liquid ethane with a Mark IV Vitrobot (Thermo Fisher Scientific). Cryo-EM movies of the samples were collected on a Titan Krios transmission electron microscope (Thermo Fisher Scientific) operated at an accelerating voltage of 300 kV. The movies were recorded using a K2 Summit direct electron detector (Gatan) in super-resolution counting mode (yielding a pixel size of 1.318 Å after two times binning), in an automatic manner using SerialEM^{48 (link)}. Each movie was dose-fractioned into 38 frames. The total electron dose was set to ~38 e⁻/Ų. The exposure time for each frame was set to 0.2 s. Defocus values ranged from −0.4 to −1.8 μm (Supplementary Table 2).

UltrAuFoil R 1.2/1.3, Au 300 grids were used for plunge-freezing. Prior to sample application, grids were plasma treated in a PELCO easiGlow glow discharge cleaning system (0.39 mBar, 15 mA, “glow” 25 s, “hold” 10 s). A Mark IV Vitrobot (Thermo Fisher Scientific) set to 100% humidity at 4 °C was used to plunge-freeze the grids in liquid ethane after applying 3 µl of protein sample to their gold-coated side using a blot time of 5 s, a blot force of 5, and a wait time of 15 s. The grids were stored in liquid nitrogen before imaging.
Images of frozen-hydrated particles of hTRPV6 in the presence of genistein were collected using Leginon^{105 (link)–107 (link)} software on a Titan Krios transmission electron microscope (Thermo Fisher Scientific) operating at 300 kV and equipped with a post-column GIF Quantum energy filter and a Gatan K3 Summit direct electron detection camera (Gatan, Pleasanton, CA, USA). 3630 micrographs were collected in counting mode with a raw image pixel size of 0.83 Å across the defocus range of −0.8 to −2.0 µm. The total dose of ~60 e⁻Å⁻² was attained by using the dose rate of ~16 e⁻pixel⁻¹ s⁻¹ across 50 frames during the 2.5-s exposure time.

Samples of mTORC2 were applied to holey carbon copper grids (R2/2 – Quantifoil) bearing an additional fine film of carbon. Grids were blotted for two seconds and then plunged directly into a mixture of liquid ethane (33%) and propane (67%) using a vitrobot mark 4 (Thermo Fisher Scientific, Waltham, MA, USA) at 4°C and 95% humidity. Data were recorded semi-automatically using SerialEM (Mastronarde, 2005 (link)) on a Titan Krios transmission electron microscope (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a K2 Summit direct electron detector (GATAN, San Diego, USA) at 300 kV, 47 100 fold magnification and with an applied defocus of between −1.0 and −3.0 μm, resulting in 3 997 images of 3 838 by 3 710 pixels with a pixel size of 1.06 Å on the object scale. Each image was recorded as forty separate frames in electron counting mode, comprising a total exposure of 80 e^-Å⁻², which were subsequently aligned, summed and weighted by dose according to the method of Grant and Grigorieff (Grant and Grigorieff, 2015 (link)) using Motioncor2 (Zheng et al., 2017 (link)) to obtain the final image.

Human 20S proteasomes were mixed with MonoUb-Cyclin B1-NT at a molar ratio of 1:5, incubated on ice, and frozen within 5 min. A volume of 2 µL sample was placed onto a glow discharged Quantifoil holey carbon grid (R2/1, 200 mesh, Quantifoil Micro Tools), which was freshly coated with graphene oxide (Sigma Aldrich). The grid was blotted with Vitrobot Mark IV (FEI company), and flash-frozen in liquid ethane. The 20S sample was handled similarly without added substrate and incubation. Images were taken by using a Titan Krios transmission electron microscope (Thermo Fisher) operated at 300 kV and equipped with a Cs corrector (Supplementary Fig. 6a and 7a). Images were collected using a K2 Summit direct electron detector (Gatan) in super-resolution mode at a nominal magnification of 18,000X, yielding a pixel size of 1.318 Å after two times binning (Supplementary Fig. 6 and Supplementary Table 3). Each movie was dose-fractioned into 38 frames with a dose rate of 8 e^–/pixel/s on the detector. The exposure time was 7.6 s with 0.2 s for each frame, generating a total dose of 38 e^–/Ų. Defocus value varies from −0.9 to −1.8 µm. All of the images were collected using the SerialEM automated data collection software package^{74 (link)}.

Cryo-EM grids were cleaned with a customized protocol^{46 (link)} prior to glow-discharge. The dialyzed RyR1-nanodisc preparations were incubated for 30 min with either 5 mM ACP/5 mM Mg²⁺ (~1 mM free Mg²⁺; ACP/LMg²⁺ dataset) or with 1 mM ACP/ 11.6 mM Mg²⁺ (~10 mM free Mg²⁺; ACP/HMg²⁺ dataset) before being plunge frozen. Aliquots of 1.25–1.5 μl RyR1-nanodisc were applied onto each side of glow-discharged C-Flat − 1.2/1.3 Au holey-carbon (Protochips, NC) or UltraAufoil −1.2/1.3 holey-gold (Quantifoil, Germany) with 300 TEM mesh. The grids were blotted with Whatman 540 filter paper for 1–1.5 s in a Vitrobot^TM Mark IV (Thermo Fisher Scientific) and rapidly plunged into liquid ethane. The sample quality was initially assessed on a Tecnai F20 (Thermo Fisher Scientific) electron microscope. Cryo-EM data was collected in multiple sessions on a Titan Krios transmission electron microscope (Thermo Fisher Scientific) operated at 300 kV under super-resolution (RyR1-ACP/LMg²⁺) or counting mode (RyR1-ACP/HMg²⁺), with a K2 or K3-Summit detector (Gatan) as shown in Supplementary Table 1. A Gatan Quantum Energy Filter (GIF) with a slit width of 20 eV was employed in all three datasets. Datasets were collected in automated mode with the Latitude program (Gatan) with a total electron dosage of 60–70 e⁻/Ų applied across 50–60 frames. Supplementary Table 1 summarizes the image acquisition parameters.

Data collection and preprocessing was the same as previously described^{11 (link)}. Briefly, data was collected using SerialEM^{37 (link)} on a 300 keV Titan Krios transmission electron microscope (Thermo Fischer Scientific) and a K3 direct electron detector (Gatan). Inelastically scattered electrons were filtered out prior to detection using a GIF quantum energy filter (Gatan) using a slit width of 20 eV. Images were acquired at a nominal magnification of 105,000x and a calibrated pixel size of 0.834 Å/pixel. Due to previously observed preferred orientation when imaging RdRp complexes^{10 (link)}, data was collected using a 30° tilt to obtain more particle orientations. 7043 raw micrographs were acquired in total and preprocessed on-the-fly in Warp for automatic contrast transfer function (CTF) estimation, averaging, motion correction, and particle prediction and extraction. 2.2 million individual RdRp particles were predicted and exported by Warp and imported into cryoSPARC and subjected to a Hetero Refinement job using five ‘Junk‘ classes and one class representing monomeric RdRp as described previously^{11 (link)}. The resulting particle set was used for a 3D homogeneous refinement to obtain refined poses and positions for each RdRp monomer.