Images were recorded on a Titan Krios electron microscope (FEI) equipped with a Cs corrector and a K2 summit direct detector with a Quantum energy filter (Gatan) at 1.0961 A per pixel in counting mode using the Leginon software package33 (link). Pixel size was calibrated in-house using a proteasome test sample. Energy filter slit width of 30 eV was used during the collection and aligned automatically every hour using Leginon. The first data collection was performed using a dose of ~66.37 e-/Å2 across 50 frames (200 msec per frame) at a dose rate of ~8.0 e–/pix/sec, using a set defocus range of −1.9 μm to −2.0 μm. The second data collection was performed using a dose of ~ 63.61 e-/Å2 across 50 frames (200 msec per frame) at a dose rate of ~7.6 e–/pix/sec, using a set defocus range of −1.5 μm to −2.5 μm. 100 μm objective aperture was used. A total of 8,318 micrographs were recorded over two separate data collection sessions using an image beam shift data collection strategy34 (link).
Krios electron microscope
Manufactured by Thermo Fisher Scientific
The Krios electron microscope is a high-resolution cryo-electron microscope designed for structural biology research. It provides advanced imaging capabilities to enable the study of macromolecular structures at the atomic level.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (3 mentions)
K2 summit direct electron detector, by Ametek (3 mentions)
Mark 4, by Thermo Fisher Scientific (3 mentions)
Ultraufoil, by Quantifoil (3 mentions)
K2 summit, by Ametek (2 mentions)
Gif quantum energy filter, by Ametek (2 mentions)
K2 summit direct electron detector, by Thermo Fisher Scientific (2 mentions)
Falcon 1, by Thermo Fisher Scientific (2 mentions)
Cu r1 2 1, by Quantifoil (2 mentions)
Gp plunger system, by Leica (2 mentions)
Easyglow, by Ted Pella (1 mentions)
Fluorinated fos choline 8, by Anatrace (1 mentions)
Talos arctica microscope, by Thermo Fisher Scientific (1 mentions)
Ceta camera, by Thermo Fisher Scientific (1 mentions)
Solarus 950 plasma cleaner, by Ametek (1 mentions)
Whatman, by Cytiva (1 mentions)
Falcon 2 detector, by Thermo Fisher Scientific (1 mentions)
Falcon 2 direct electron detector, by Thermo Fisher Scientific (1 mentions)
Vitrobot mark 2, by Thermo Fisher Scientific (1 mentions)
Ms peg 12 methyl peg nhs ester, by Thermo Fisher Scientific (1 mentions)
29 protocols using krios electron microscope
1
Cryo-EM Imaging of Proteasome at High Resolution
2
Salmonella Minicell Electron Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Minicells of the Salmonella wild-type strain and the ΔfliH-fliI flhB(P28T) bypass mutant were prepared as described previously (19 (link)). Images of the minicells were collected at the liquid-nitrogen temperature using a Titan Krios electron microscope (FEI) operated at 300 kV and with a Falcon 4k × 4k direct electron detector (FEI) as described before (19 (link)). Images were generally binned 2-fold, and 3D reconstructions were calculated using the IMOD software package (51 (link)).
3
Structural Insights into Ribosome-Mediated Translation Termination
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Programmed CMV-stalled RNCs were prepared from a wheat germ in-vitro-translation extract as described by Bhushan et al. (2010) (link). Recombinant yeast eRF1, eRF3, and ABCE1 were overexpressed in E. coli or S. cerevisiae and affinity purified. For release assays, RNCs were incubated together with the ligands, and tagged nascent peptidyl tRNA or free peptide was analyzed by western blotting.
Termination complexes were formed by in vitro reconstitution with recombinant-purified factors. The complexes were vitrified, and data were collected on a Titan Krios electron microscope (FEI). Single-particle analysis followed by 3D reconstruction was performed using the SPIDER software package (Frank et al., 1996 (link)). For molecular interpretation of the Triticum aestivum 80S ribosome, we used an updated model (Gogala et al., 2014 (link)). Models of eRF1, eRF3, and ABCE1 were based on existing crystal structures. SeeSupplemental Experimental Procedures for a detailed description of the Experimental Procedures .
Termination complexes were formed by in vitro reconstitution with recombinant-purified factors. The complexes were vitrified, and data were collected on a Titan Krios electron microscope (FEI). Single-particle analysis followed by 3D reconstruction was performed using the SPIDER software package (Frank et al., 1996 (link)). For molecular interpretation of the Triticum aestivum 80S ribosome, we used an updated model (Gogala et al., 2014 (link)). Models of eRF1, eRF3, and ABCE1 were based on existing crystal structures. See
4
Cryo-EM Protocol for Thin Ice Formation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cryo-EM grids were prepared with the Vitrobot Mark IV (FEI) operated at 100% humidity at 4°C. Immediately prior to sample vitrification, Quantifoil 1.2/1.3-μm holy carbon grids were glow-discharged with Easyglow (TedPella), and fluorinated fos-choline-8 (Anatrace) was added to the protein sample to a final concentration of 3 mM, which was an essential step for producing good quality thin ice. For each grid, an aliquot of 3.5 μL was applied and incubated for 20 seconds inside the Vitrobot. Blotting time was set to 2.5 seconds with 2 seconds of drain time. The low pH sample was treated identically, except for incubation with 0.3 mM NAD before freezing (and that no fluorinated fos-choline-8 was added to obtain one of the pH 6.2 data sets). Cryo-EM data sets were collected on a Titan Krios electron microscope (FEI) operating at 300 keV with a Gatan K2 Summit direct electron detector attached to a Gatan imaging filter (GIF). Movies were recorded under super-resolution counting mode at a pixel size of 0.535 Å and a dose rate of 0.876 e/pixel/frame for a total of 60 frames. The total electron dose was 45 electrons per Å2 per movie for 9 seconds.
5
Cryo-EM Sample Preparation for PAPP-A Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Holy-carbon gold grids (Quantifoil, R1.2/1.3) were treated with Solarus 950 plasma cleaner (Gatan) with a 4:1 ratio of O2/H2 for 60 s for glow-discharge before cryo-EM sample preparation. 4 μL aliquots of freshly prepared PAPP-A·proMBP or PAPP-A·STC2 (0.2 mg/mL) complex were applied on the grids, blotted with filter paper (Whatman No. 1) with force set to –2 for 0.5 s at 4 °C and 100% humidity, and plunge-frozen in the liquid ethane using a Vitrobot Mark IV (FEI).
The cryo-grids were screened on a 200 kV Talos Arctica microscope equipped with an FEI Ceta camera and a K2 Summit direct electron detector (Gatan). Data collection was carried out with Titan Krios electron microscope (FEI) operated at 300 kV.
Images were recorded with a K2 Summit direct electron detector (Gatan) in the super-resolution mode at a nominal magnification of 130,000× and a dose rate of 8 e−/s/pixel. Movies were recorded semi-automatically using the SerialEM software62 (link). A GIF Quantum energy filter (Gatan), with a slit width of 20 eV was used at the end of the detector. The defocus range was set from –0.7 to –1.2 μm. The total exposure time was 8.32 s, and intermediate frames were recorded every 0.26 s. 32 frames per image were acquired. Statistics for data collection are summarized in Supplementary TableS1 .
The cryo-grids were screened on a 200 kV Talos Arctica microscope equipped with an FEI Ceta camera and a K2 Summit direct electron detector (Gatan). Data collection was carried out with Titan Krios electron microscope (FEI) operated at 300 kV.
Images were recorded with a K2 Summit direct electron detector (Gatan) in the super-resolution mode at a nominal magnification of 130,000× and a dose rate of 8 e−/s/pixel. Movies were recorded semi-automatically using the SerialEM software62 (link). A GIF Quantum energy filter (Gatan), with a slit width of 20 eV was used at the end of the detector. The defocus range was set from –0.7 to –1.2 μm. The total exposure time was 8.32 s, and intermediate frames were recorded every 0.26 s. 32 frames per image were acquired. Statistics for data collection are summarized in Supplementary Table
6
High-Resolution Cryo-EM Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Grids were transferred to a Titan Krios electron microscope (FEI), operating at 300 kV and equipped with a Gatan GIF Quantum energy filter (slit width 20 eV). 23,061 micrographs were recorded with the grid tilting at angles of 0, 30, 45 and 55 degrees using a K2 Summit detector (Gatan Company) in the super-resolution mode with a nominal magnification of 64,000×, resulting in a calibrated pixel size of 1.11 Å (Supplementary information, Table S1 ). The total dose followed a cosine alpha scheme where the dose is inversely proportional to the cosine alpha of the tilting angle. Within each stack, the exposure time for each frame and the dose rate were kept the same. With an exposure time of 0.5 s per frame and 40 frames, the total exposure time for each 0°-tilting (tilt0) stack is 20 s, amounting to a total dose of 52 e−/Å2 for each micrograph. AutoEMation53 (link) was used for fully automated data collection. All frames in each stack were first aligned and summed using MotionCorr,54 (link) with 2-fold binned to a pixel size of 2.22 Å. The output stacks from MotionCorr were further motion-corrected using MotionCor255 (link) and dose weighting was performed. The average defocus values were set between −1.0 and −3.5 μm and were estimated by Gctf.56 (link)
7
Cryo-EM Imaging of Ribosomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The specimens were transferred to a Titan Krios electron microscope (FEI) under cryo-conditions. The microscope was operated at 300 kV. Images were recorded using an FEI Falcon I direct electron detection camera with the Leginon program (35 (link)) at a calibrated magnification of ×59,000, yielding a pixel size of 1.09 Å. Defocus values for the contrast transfer function were calculated using CTFFIND3 (36 (link)) and were in the range of 1.5 to 3.5 μm in the final data set. A total of 8800 micrographs were collected, 6800 of which were used for particle selection after excluding those micrographs that had either poor power spectra or thick ice, using the method described by Jiang et al. (37 (link)). Program EMAN was used to select ribosome-sized single-particle images and then manually examine the quality of these images, yielding a data set of 380,000 single-particle images. The images were subjected to three-dimensional (3D) classifications and then map refinements, all performed using program RELION 1.2 (38 (link)). The process of hierarchical 3D classification is detailed in fig. S6. The refined maps were corrected for the modulation transfer function of the detector, and the high spatial frequencies were boosted using B-factor sharpening (39 (link)). Local resolutions of the maps were calculated using ResMap [fig. S7; (40 (link))].
8
Cryo-EM Structural Determination Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cryo‐EM, using a Vitrobot Mark IV (FEI), the SV sample was applied to a glow discharged, Quantifoil R1.2/1.3, 300 mesh grid (Electron Microscopy Sciences, Hatfield, PA, USA), blotted for 4–5 s and rapidly frozen in liquid ethane. Imaging was performed with a Titan Krios electron microscope (FEI) operated at 300 kV.
9
Cryo-EM Imaging of Purified RyR1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aliquots of 3 μl of purified RyR1 (∼5 mg/ml) were placed on glow-discharged 400-mesh R2.0/2.0 Quantifoil holy carbon grids (Quantifoil Micro Tools GmbH). Grids were blotted for 2 seconds and flash-frozen in liquid ethane using an FEI Mark IV Vitrobot plunger. Grids were transferred to an FEI Titan Krios electron microscope that was operating at 300 kV. Images were collected automatically using SerialEM55 (link) and recorded in video mode (17 frames/s) using an FEI Falcon-II detector at a nominal magnification of 59 000× g and a pixel size of 1.396 Å. A dose rate of 24 electrons per Å2 per second 24e−/(sec·Å2) and an exposure time of 2 s were used.
10
Structural Insights into Ribosome-Mediated Translation Termination
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!