Four microliters of the OM core complex diluted to 0.01 mg/ml in the gel filtration buffer (see above) was applied on glow-discharged carbon-coated copper grids (400 mesh grid copper, Agar Scientific). After incubation for 2 min, the sample was washed twice with 10 μl water and then stained for 1 min with 10 μl 2 % uranyl acetate. Then the grids were blotted to remove excess stain, dried, and kept for the microscope sessions. The data were collected on a F20 microscope (FEI) operating at 200 kV at a magnification of 45,500xg using a low dose mode (~ 30 e Å2) and a defocus range of −0.7 to −2.0 μm. Images were recorded on a Gatan UltraScan 4000 CCD camera (Gatan) with a calibrated pixel size of 3.3 Å. 60 micrographs were collected. Quality was assessed visually and through CTF estimation. Images with distorted Thon rings and drift were not considered for the further processing.
Ultrascan 4000 ccd
Manufactured by Ametek
Sourced in Germany
The UltraScan 4000 CCD is a laboratory instrument that utilizes charge-coupled device (CCD) technology to capture and analyze images. It is designed for high-resolution imaging and data acquisition in various scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Jeol2100f field, by JEOL (2 mentions)
Uranyl formate, by Polysciences (2 mentions)
F20 microscope, by Thermo Fisher Scientific (1 mentions)
Tecnai g2 spirit, by Ametek (1 mentions)
Tecnai g2 polara, by Thermo Fisher Scientific (1 mentions)
Glow discharged 300 mesh copper grids, by Ted Pella (1 mentions)
Tecnai spirit 120 kv electron, by Ametek (1 mentions)
No 1 filter paper, by Cytiva (1 mentions)
Whatman, by Cytiva (1 mentions)
8 protocols using ultrascan 4000 ccd
1
Negative Staining Electron Microscopy of OM Core Complex
2
TEM Tomographic Specimen Preparation
3
Structural Characterization of Fab-Trimer Complexes
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IgG was purified from serum with protein A and then digested into Fab with papain. Purified Fab was added to BG505 SOSIP D664 MD39 mC trimer and complexes were purified using a GE Superose 6 column (GE HealthCare). Complexes were added to 400 mesh carbon-coated grids and stained with 2% uranyl formate. Grids of complexes made of Fab from serum collected at week 10 post immunization were imaged on a Tecnai F20 at 200 KeV using a 4k × 4k Gatan Ultrascan 4000 CCD. Complexes from week 18 post immunization were collected on a Tecnai Spirit at 120 KeV using a 4k × 4k TemCam F416 camera. Micrographs were collected with Leginon62 (link) and processed with Appion63 (link). Particles were chosen using DoGpicker64 and stacked, and initial 2D classes were made with MSA/MRA65 . After removing classes with double particles or those not resembling trimers or protomers, unbinned particles were put into Relion66 , where 3D classification was performed to obtain maps of trimer and Fab complexes. Clean maps were then refined and put into Chimera67 to segment and compare.
4
Negative Staining and Electron Microscopy Imaging
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Protein preparations were applied to glow-discharged continuous carbon EM grids and negatively stained with 2% uranyl formate. Grids were imaged by transmission electron microscopy using an FEI Tecnai G2 Spirit at 120kV acceleration voltage and a Gatan Ultrascan 4000 CCD using the Leginon software package (Suloway et al., 2009 (link)). Micrographs were collected at a nominal 67,000x magnification (pixel size 1.6 Å). GCTF was used for contrast transfer function (CTF) estimation, and Relion for particle picking and 2D classification (Scheres, 2012 (link); Zhang, 2016 (link); Zivanov et al., 2018 (link)).
5
Cryo-Electron Tomography Protocol for Cellular Structures
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Projection images were acquired at a range of tilt angles using an FEI Tecnai G2 Polara electron microscope, operating at 300 kV and equipped with a post-column energy filter and a Gatan Ultrascan 4000 CCD camera. The SerialEM software package [47] (link) was used for automated tilt series acquisition.
All tilt series were acquired with 1° tilt increments and with the total electron dose split evenly across all images of the series. The ribosome tomogram was taken at a nominal magnification of 50,000×, with a defocus of −6 µm, zero-loss energy filtering with a slit width of 20 eV, and a total electron dose of 180 e−/Å2. For the liposome tomogram conditions were the same, except that the nominal magnification was 41,000×, the defocus was set to −10 µm, and the total electron dose was 150 e−/Å2. The tomogram of C. crescentus cells was collected at a magnification of 22,500×, with a defocus of −6 µm, a total electron dose of 110 e−/Å2, and no energy filtering.
The tilt series were aligned and processed using the IMOD software package [48] (link), and reconstructions were generated using the SIRT algorithm implemented in Tomo3D [49] (link). Anisotropic nonlinear diffusion was carried out using TomoAND [50,51] . Line profiles were generated using ImageJ [52] (link), and volume rendering of tomograms was performed using PyMOL [53] .
All tilt series were acquired with 1° tilt increments and with the total electron dose split evenly across all images of the series. The ribosome tomogram was taken at a nominal magnification of 50,000×, with a defocus of −6 µm, zero-loss energy filtering with a slit width of 20 eV, and a total electron dose of 180 e−/Å2. For the liposome tomogram conditions were the same, except that the nominal magnification was 41,000×, the defocus was set to −10 µm, and the total electron dose was 150 e−/Å2. The tomogram of C. crescentus cells was collected at a magnification of 22,500×, with a defocus of −6 µm, a total electron dose of 110 e−/Å2, and no energy filtering.
The tilt series were aligned and processed using the IMOD software package [48] (link), and reconstructions were generated using the SIRT algorithm implemented in Tomo3D [49] (link). Anisotropic nonlinear diffusion was carried out using TomoAND [50,51] . Line profiles were generated using ImageJ [52] (link), and volume rendering of tomograms was performed using PyMOL [53] .
6
Salipro Lipid-Only Disc Preparation and Characterization
7
Cryo-EM Imaging of Purified Protein Complexes
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Three μL of purified sample of each F or G construct was applied to glow discharged 300 mesh copper grids (Ted Pella) covered with evaporated continuous carbon for 1 min before blotting away excess liquid with Whatman no. 1 filter paper. Grids were stained twice with 3 μL of 2% (w/v) uranyl formate and imaged using an FEI Tecnai Spirit 120 kV electron microscope equipped with a Gatan Ultrascan 4000 CCD camera. The pixel size at the specimen level was 1.60 Å. Data collection was performed using Leginon (75 (link)) and processing using cryoSPARC (76 (link)).
8
Salipro Lipid-Only Disc Preparation and Characterization
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Salipro lipid-only discs: Carbon coated copper grids (400 mesh) were glow discharged in low pressure air to render them hydrophilic for 20 sec before application of 4 μl sample for 30 sec. Washing was done with three drops of water before staining with 4 μl of uranyl formate (1%, Polysciences, USA) for 30 sec. The grids were air dried after removal of excess stain solution by torn filter paper. Negative stain image data were collected with a JEOL2100F field emission gun transmission electron microscope at an accelerating voltage of 200kV and a final magnification of 69500 on a CCD camera (4K × 4K, Tiez Video and Image Processing System, GmbH Gauting, Germany) with 15 mm pixel size (corresponding to 2.16 Å on the specimen level).
Salipro-T2 samples were stained with uranyl acetate and imaged on an FEI TECNAI Polara TEM at 100 kV at a magnification of 59kx and a pixel size of 1.91 A on a UltraScan 4000 CCD (Gatan, Inc.). Individual particles were windowed semi-automatically with the swarm option of e2boxer (EMAN2 package50 ). After normalization, 33000 particles were subjected to reference-free 2D classification and class-averaging using the EMAN2 routines (e2refine2d).
Salipro-T2 samples were stained with uranyl acetate and imaged on an FEI TECNAI Polara TEM at 100 kV at a magnification of 59kx and a pixel size of 1.91 A on a UltraScan 4000 CCD (Gatan, Inc.). Individual particles were windowed semi-automatically with the swarm option of e2boxer (EMAN2 package50 ). After normalization, 33000 particles were subjected to reference-free 2D classification and class-averaging using the EMAN2 routines (e2refine2d).
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