Freshly glow-discharged grids (300 mesh holey carbon Au Quantifoil R1.2/1.3) were used for vitrification in a Mark IV Vitrobot (FEI), using 100% relative humidity and 4-s blot time, and plunge frozen in liquid nitrogen–cooled ethane. The grids were loaded onto an FEI Titan Krios G3 electron microscope operating at 300 kV high tension, equipped with a K3 BioQuantum imaging system using a 20-eV energy slit. Imaging was performed in nanoprobe mode using a 50-μm C2 aperture using a ∼1.3-μm parallel illuminated beam diameter and no objective aperture. The nominal magnification postenergy filter ( EFTEM) magnification was 105,000×, corresponding to a calibrated super-resolution pixel size on the specimen of 0.4175 Å ⋅ pix−1. The dose rate was 8 e− ⋅ pix−1 ⋅ s−1 with a total exposure time of 5.9 s resulting in an accumulated fluence of 68 e− ⋅ Å−2 fractionated evenly across 117 frames, using correlated double sampling. Movies were acquired semiautomatedly with SerialEM (42 (link)) using 3 × 3 hole beam-image shift, using a nominal under-focus range of 0.7 to 1.5 μm with focus targeting once per nine shots. The movies were drift corrected and dose weighted using University of California, San Francisco (UCSF) MotionCor2 (43 (link)) and 2× Fourier binned to a pixel size of 0.835 Å ⋅ pix−1.
R1 2 1
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The R1.2/1.3 is a laboratory equipment product from Thermo Fisher Scientific. It is designed for general laboratory use. The core function of the R1.2/1.3 is to perform a specific task or set of tasks within a laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (3 mentions)
Pdc 32g 2, by Harrick (1 mentions)
Vitrobot 4, by Thermo Fisher Scientific (1 mentions)
Talos arctica microscope, by Thermo Fisher Scientific (1 mentions)
Titan krios, by Thermo Fisher Scientific (1 mentions)
Tecnai t20 electron, by Thermo Fisher Scientific (1 mentions)
6 protocols using r1 2 1
1
Cryo-EM Imaging of Macromolecular Complexes
2
Cryo-EM Sample Preparation and Data Analysis
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Samples for cryo-EM study were prepared essentially as described15 (link),52 (link) (Supplementary Table 4 ). All EM grids were evacuated for 2 min and glow-discharged for 30 s using a plasma cleaner (Harrick PDC-32G-2). Four microliters of spike protein (0.8 mg ml−1) was mixed with the same volume of Fabs (1 mg ml−1 each), and the mixture was immediately applied to glow-discharged holy-carbon gold grids (Quantifoil, R1.2/1.3) in an FEI Vitrobot IV (4 °C and 100% humidity). Data collection was performed using either a Titan Krios G3 equipped with a K3 direct detection camera, or a Titan Krios G2 with a K2 camera, both operating at 300 kV. Data processing was carried out using cryoSPARC (v3.2.1)53 (link). After 2D classification, particles with good qualities were selected for global 3D reconstruction and then subjected to homogeneous refinement. To improve the density surrounding the RBD–Fab region, UCSF Chimera (v1.16)54 (link) and Relion (v3.1)55 (link) were used to generate the masks, and local refinement was then performed using cryoSPARC (v3.2.1). Coot (v0.8.9.2)56 (link) and Phenix (v1.20)57 (link) were used for structural modelling and refinement. Figures were prepared using USCF ChimeraX (v1.3)58 (link) and Pymol (v2.4.0, Schrödinger, LLC.).
3
Cryo-EM Sample Preparation for TAF-DNA Complex
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For cryo-EM sample preparation, purified TAF complex was stabilized using the GraFix method [29 (link),30 (link)] and DNA was added in 2x molar excess and incubated for 15 min at room temperature for the TAFs–DNA complex. Then, 4 µL of each protein sample was applied to a Quantifoil R1.2/1.3 holey carbon grid and rapidly frozen in liquid ethane using a Vitrobot IV (FEI). The sample-containing grid was then loaded into the Titan Krios FEG–TEM 300 kV instrument equipped with a K2 direct electron detector (Gatan) and imaged under low-dose conditions (10–15 e/Å2). Collected movies were motion and CTF corrected using SIMPLE [31 (link),32 (link)], and particles were manually picked using EMAN2 [33 (link)]. Data processing was performed using SIMPLE [31 (link),32 (link)], and depictions of 3D reconstructed models were generated using UCSF Chimera [34 (link)].
4
Cryo-EM Sample Preparation Protocol
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Complexes stored at -80°C were thawed on ice. A glow discharged Quantifoil R1.2/1.3 300 M Cu holey carbon grid was placed inside the FEI Vitrobot Mark IV humidity chamber at 100% humidity. Then, 2μl of thawed sample was applied to the grid followed by 0.5μL of 1 M acetate pH 5.5. The grids were then blotted and plunge frozen in liquid ethane. Frozen grids were stored in liquid nitrogen prior to use.
5
Cryo-EM Structural Analysis of PVC Samples
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Negative staining was used to assess the sample homogeneity and concentration. Aliquots of 4 μl of PVC samples were applied to glow-discharged copper grids, washed, and stained with 2% uranyl acetate. The negative-stained grids were examined on an FEI Tecnai T20 electron microscope operated at 120 kV.
For cryo-grid preparation, aliquots of 4 μl of samples were applied to glow-discharged holey-carbon copper grids (Quantifoil, Germany, R1.2/1.3, 400 mesh) inside the chamber of an FEI Vitrobot IV (4°C and 100% humidity). The cryo-grids were screened using an FEI Talos Arctica microscope operated at 200 kV. Data acquisition was performed using an FEI Titan Krios operated at 300 kV. Images were recorded using a GIF K2 camera (Gatan, USA) with SerialEM (50 (link)) in the superresolution counting and movie mode, at a nominal magnification of ×105,000, corresponding to a calibrated pixel size of 0.678 Å at object scale (superresolution), with defocus ranging from 0.7 to 2.5 μm. A total of 32 frames were collected for each movie stack. The dose rate was 4.9 e− Å−2 s−1 with a total exposure time of 6.4 s.
For cryo-grid preparation, aliquots of 4 μl of samples were applied to glow-discharged holey-carbon copper grids (Quantifoil, Germany, R1.2/1.3, 400 mesh) inside the chamber of an FEI Vitrobot IV (4°C and 100% humidity). The cryo-grids were screened using an FEI Talos Arctica microscope operated at 200 kV. Data acquisition was performed using an FEI Titan Krios operated at 300 kV. Images were recorded using a GIF K2 camera (Gatan, USA) with SerialEM (50 (link)) in the superresolution counting and movie mode, at a nominal magnification of ×105,000, corresponding to a calibrated pixel size of 0.678 Å at object scale (superresolution), with defocus ranging from 0.7 to 2.5 μm. A total of 32 frames were collected for each movie stack. The dose rate was 4.9 e− Å−2 s−1 with a total exposure time of 6.4 s.
6
Cryo-EM Structure Determination of Protein Complex
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For grid preparation, 3 μL of the sample was applied to glow-discharged electron microscopy grids (Quatifoil R1.2/1.3) and plunge-frozen in liquid ethane using the FEI Vitrobot Mark IV at 4.5 °C. 6784 movies were collected using the Titan Krios electron microscope equipped with the Falcon II electron detector with the pixel size of 1.107 Å. 25 frames were recorded per exposure with the dose of 4e/Å2 per movie frame. Motion correction, CTF estimation, and particle picking were performed in Warp52 (link). Particles were exported to Relion53 (link) for 2D classification. Initial model generation, consecutive 3D classification/refinement with no symmetry imposed (C1) and with C7 symmetry were performed in cisTEM54 (link) resulting in two classes representing different states, each containing approximately 42.000 particles. Particles from both classes were imported to cryoSPARC55 (link), refined against structures from cisTEM without symmetry and with a C7 symmetry applied. Resolution of 3.4 Å (C7) and 4.0 Å (C1) for the “wide” structure and 3.4 Å (C7) 4.2 Å (C1) for the “tight” structure was estimated. Finally, both C1 and C7 structures were analysed for local resolution variations and locally sharpened in cryoSPARC. 3D Variability analysis56 (link) was performed to analyse the mode of apical domain’s motion with the following parameters: low pass filtering = 6 Å, 3 modes.
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