A total of 10 μL of purified PSI trimer at a protein concentration of 37 μM (40 mg/mL), 1 μL of purified Ga-Fd in concentration of 400 μM (4.4 mg/mL), and 2 μL of purified Cyt c6 in concentration of 75 μM (0.75 mg/mL) were mixed together at ice temperature to a final concentration of 30 mg/mL PSI trimer. After incubation on ice in the dark for 2 h, an aliquot of 2.6 μL was applied to a glow-discharged (JEC-3000 FC, 30 s, carbon support film facing up) Quantifoil cryo-EM grid (R 1.2/1.3 Cu 300 mesh) and plunge frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA) fitted with Whatman #1 filter paper for blotting at 4 °C and 100% humidity for 3.5 s at blot force 0. Frozen grids were transferred to liquid nitrogen for storage. Screening was performed using a Talos Arctica (Thermo Fisher Scientific) transmission cryo-electron microscope operated at 200 kV. Cryo-grids exhibiting appropriate particle distribution and ice thickness were taken out of the Talos Arctica and further used for high-resolution data collection.
Talos arctica
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Talos Arctica is a high-performance cryo-transmission electron microscope (cryo-TEM) designed for structural biology research. It features a stable and efficient cryogenic system, advanced optics, and a powerful digital camera for obtaining high-resolution images of biological samples.
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Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (54 mentions)
Titan krios, by Thermo Fisher Scientific (27 mentions)
Epu software, by Thermo Fisher Scientific (12 mentions)
K2 summit, by Ametek (11 mentions)
Whatman, by Cytiva (8 mentions)
Falcon 3ec, by Thermo Fisher Scientific (8 mentions)
Falcon 3 detector, by Thermo Fisher Scientific (8 mentions)
Titan krios microscope, by Thermo Fisher Scientific (7 mentions)
K3 direct electron detector, by Ametek (6 mentions)
Vitrobot, by Thermo Fisher Scientific (6 mentions)
Vitrobot mk 4, by Thermo Fisher Scientific (5 mentions)
Falcon 3, by Thermo Fisher Scientific (5 mentions)
K2 direct electron detector, by Ametek (4 mentions)
K2 summit direct electron detector, by Ametek (4 mentions)
Solarus plasma cleaner, by Ametek (4 mentions)
Vitrobot 4, by Thermo Fisher Scientific (4 mentions)
400 mesh carbon coated copper grid, by Ted Pella (3 mentions)
Epu 2, by Thermo Fisher Scientific (3 mentions)
Ultraau foil r1 2 1, by Electron Microscopy Sciences (3 mentions)
K3 camera, by Ametek (3 mentions)
184 protocols using talos arctica
1
Cryo-EM Sample Preparation for PSI
2
Cryo-TEM Imaging of Lamellin-3K and E. coli
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For 3K-PBS, 5 μL of 125 μM lamellin-3K was applied to freshly plasma-cleaned TEM grids (Quantifoil, Cu, 300-mesh, R2/1) and vitrified into liquid ethane using Automatic Plunge Freezer EM GP2 from Leica Microsystems (8 °C, 100% rel. humidity, 300 s waiting time, 3.5 s blotting time). The grids were subsequently mounted into the Autogrid cartridges and loaded to Talos Arctica (ThermoScientific) transmission electron microscope for imaging. The microscope was operated at 200 kV. The micrographs were collected on Falcon3 direct electron detection camera at 92,000x nominal magnification with an underfocus in the range of 2−3 μm and an overall dose of ~40 e/Å2.
For 2K-PBS and samples with E. coli, 4μL of sample was applied to freshly plasma-cleaned TEM grids (Quantifoil, Cu, 300 or 200 mesh, R2/1) and vitrified into liquid ethane using ThermoScientific Vitrobot Mark IV (4 °C, 100% rel. humidity, 30 s waiting time (10 s for bacteria), 6 s blotting time (3 s for bacteria)). The grids were subsequently mounted into the Autogrid cartridges and loaded to Talos Arctica (ThermoScientific) transmission electron microscope for imaging. The microscope was operated at 200 kV. Cryo-TEM micrographs were collected on Ametek K2 direct electron detection camera at the 49,000x and 79,000x nominal magnification with the underfocus in the range 2–5 μm and the overall dose of 20 to 40 e/Å2.
For 2K-PBS and samples with E. coli, 4μL of sample was applied to freshly plasma-cleaned TEM grids (Quantifoil, Cu, 300 or 200 mesh, R2/1) and vitrified into liquid ethane using ThermoScientific Vitrobot Mark IV (4 °C, 100% rel. humidity, 30 s waiting time (10 s for bacteria), 6 s blotting time (3 s for bacteria)). The grids were subsequently mounted into the Autogrid cartridges and loaded to Talos Arctica (ThermoScientific) transmission electron microscope for imaging. The microscope was operated at 200 kV. Cryo-TEM micrographs were collected on Ametek K2 direct electron detection camera at the 49,000x and 79,000x nominal magnification with the underfocus in the range 2–5 μm and the overall dose of 20 to 40 e/Å2.
3
Cryo-EM Sample Preparation of PSI Complexes
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A total of 10 μL of purified PSI trimer at a protein concentration of 37 μM (40 mg/mL), 1 μL of purified Ga-Fd in concentration of 400 μM (4.4 mg/mL) and 2 μL of purified Cyt c6 in concentration of 75 μM (0.75 mg/mL) were mixed together at ice temperature to a final concentration of 30 mg/mL PSI trimer. After incubation on ice in the dark for two hours, an aliquot of 2.6 μL was applied to a glow-discharged (JEC-3000 FC, 30 s, carbon support film facing up) Quantifoil cryo-EM grid (R 1.2/1.3 Cu 300 mesh) and plunge frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA) fitted with Whatman #1 filter paper for blotting at 4 ℃ and 100% humidity for 3.5 s at blot force 0. Frozen grids were transferred to liquid nitrogen for storage. Screening was performed using a Talos Arctica (Thermo Fisher Scientific) transmission cryo electron microscope operated at 200 kV. Cryo-grids exhibiting appropriate particle distribution and ice thickness were taken out of the Talos Arctica and further used for high resolution data collection.
4
Cryo-EM Structure Determination Protocol
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High-resolution cryo-EM data were acquired on Titan Krios operated at 300 keV with a FalconIII detector in counting mode with 20 frames per movie and nominal magnification of 96000× (0.845 Å pixel size). The electron dose was 33.6 e/Å2. For the sprayed apoferritin dataset the defocus range was −0.5 to −3.5 μm, while the vitrobot apoferritin dataset had a range of −0.5 to −2.5 μm. The larger defocus was due to the slightly thicker ice in sprayed samples. Movies were motion-corrected by using MotionCor2 with 5 by 5 patches76 (link) and CTF estimation was done by CTFfind4 on dose-weighted micrographs77 (link). All particle picking was done by crYOLO using a model trained in-house78 (link). Subsequent image processing was performed in Scipion and RELION-3, using PDB 1AEW79 (link), filtered to 40 Å as a reference80 (link),81 (link). CSN5H138A-SCF-N8Skp2/Cks1 complexes were purified as described39 ,82 (link), and data for 2D classification collected on a Talos Arctica (Thermo-Fisher Scientific, Waltham, MA) operating at 200 keV and with a Falcon III direct electron detector in linear mode with 10 frames per movie, 6.5 e/Å2, 1.61 Å pixel size. Movies were aligned using MotionCor2 and CTF estimation was done by gCTF83 (link). Particle picking was done by crYOLO using an in-house trained model78 (link) followed by 2D classification in cryoSPARC 2.14.284 (link).
5
Cryo-EM Imaging and Analysis of Tau Fibril Polymorphs
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Tilt series of the S42Y fibril sample were collected on a Talos Arctica cryo-electron microscope (Thermo Fisher Scientific, Waltham, MA, USA) operated at 200 kV, equipped with a post-column BioQuantum energy filter (the slit was set to 20 eV) and a K2 direct electron detector. Automated data collection was performed using SerialEM under the following conditions: 63,000 × microscope magnification, spot size 7, 100-μm condenser aperture, and defocus range of 6 ~ 3 µm. The image pixel size was 2.134 Å/pixel. Tilt series ranged from − 60° to 60° at 3° step increments. A total of 35 tilt series were collected in counting mode with a cumulative dose of 80–100 e-/Å2. For the WT preformed fibrils, we collected tilt series at 49,000 × microscope magnification, the spot size of 8, and defocus of – 4 μm. All other imaging parameters were the same as described for the S42Y data collection. Tilt series alignment and tomogram reconstruction were performed using the latest EMAN2 tomography workflow [10 (link)]. Measurements of the fibril species were done using the EMAN2 measurement functionality. Subtomogram analysis and visualization of the tomogram were done using Chimera (University of California, San Francisco) [62 (link)].
6
Cryo-EM Structure Determination of YnaI
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Three-ul of purified YnaI in LMNG at ~5 mg/ml were mixed with 3 mM fluorinated Fos-Choline-8 (Anatrace) right before applying to a plasma-cleaned C-flat holy carbon grids (1.2/1.3, 400 mesh). The grid was prepared with a Vitrobot Mark IV (Thermo Fisher Scientific) with the environmental chamber set at 100% humidity and 4 °C. It was blotted for 2 s and then flash frozen in liquid ethane cooled by liquid nitrogen. Cryo-EM data were collected on a Talos Arctica (Thermo Fisher Scientific) operated at 200 keV and equipped with a K3 direct detector (Gatan). Totally, 4622 movies were recorded at 45,000x magnification with a calibrated pixel size of 0.864 Å using Leginon37 (link). Defocus range was set from −1.2 um to −2.3 um. The parallel illumination set up on the Arctica gave a dose rate of ~24 electrons per pixel per second. Each movie was dose-fractionated to 50 frames with a total exposure of 2 s, leading to a total dose of ~64 electrons/Å2.
7
Visualizing Aβ-enriched Extracellular Vesicles
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Freshly prepared 10 000g (large) Aβ-EV pellets resuspended in saline were plunge frozen in liquid ethane using a Vitrobot Mk IV (Thermo Fisher Scientific). Images of the vitrified specimen were acquired using a Talos Arctica transmission electron microscope (Thermo Fisher Scientific). See also Supplementary material .
8
Cryo-EM Sample Preparation of Synechococcus Phage
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QuantiFoil holey carbon R 1.2/1.3 300-mesh grids were glow discharged on a PELCO easiGlow system for 45 s with 15 mA current. Grid freezing was then performed on an FEI Vitrobot Mark IV with the chamber humidity set to 100% and the temperature set to 4°C. Three μL of sample (4 µM Synechococcus phage S-CBP4 α subunit in 50 mM HEPES pH 7.6, 150 mM NaCl, 7.55 mM MgCl2, 200 µM TTP, 200 µM GDP, 1 mM TCEP, 1% v/v glycerol) was applied onto the grid. The sample was blotted for 4 s and then immediately plunged into liquid ethane cooled by liquid nitrogen.
Data collection was performed at the Cornell Center for Materials Research (CCMR) on a Talos Arctica (Thermo Fisher Scientific) operating at 200 keV with a Gatan K3 direct electron detector and BioQuantum energy filter at a nominal magnification of ×79,000 (1.07 Å pixel–1). A total of 856 movies was collected with a nominal defocus range from –0.6 to –2.0 μm and a total dose of 50 e- Å–2 over 50 frames (2.164 s total exposure time, 0.0435 s frame time, 26.96 e- Å–2 s–1 dose rate).
Data collection was performed at the Cornell Center for Materials Research (CCMR) on a Talos Arctica (Thermo Fisher Scientific) operating at 200 keV with a Gatan K3 direct electron detector and BioQuantum energy filter at a nominal magnification of ×79,000 (1.07 Å pixel–1). A total of 856 movies was collected with a nominal defocus range from –0.6 to –2.0 μm and a total dose of 50 e- Å–2 over 50 frames (2.164 s total exposure time, 0.0435 s frame time, 26.96 e- Å–2 s–1 dose rate).
9
Cryo-EM Sample Preparation for DGAT1 Structure
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To prepare the cryo-EM sample for structure determination, 2–3 µL of purified DGAT1 in PMAL-C8 with 5 µM inhibitor was applied to Quantifoil holey carbon grid (Cu R1.2/1.3; 400 mesh) glow discharged for 30 s. Optimal particle distribution was obtained with a protein concentration of 4–5 mg/mL. The grids were blotted with a Whatman #1 filter paper for 5 s with ~95% humidity at 4 °C and plunged frozen in liquid ethane using an FEI Vitrobot Mark IV system (Thermo Fisher Scientific). Cryo-EM data were collected on a Talos Arctica or a Titan Krios electron microscope (Thermo Fisher Scientific). Images were recorded using SerialEM43 (link). Refer to Supplementary Table 1 for detailed information about microscope type and data collection parameters.
10
Cryo-EM Sample Preparation and Processing
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An amount of 4 µl of marked fractions from size exclusion chromatography (asterisk, Figure 3 ) diluted 40 times (in 50 mM HEPESpH ≈ 8, 130 mM NaCl, 0.01% DDM-CL) were loaded on freshly prepared graphene oxide coated grids (prepared using methods described in [52 (link)]) and plunge frozen into liquid ethane using a Vitrobot Mark IV unit (Thermo Fisher Scientific) with wait time of 2 s after sample application and blotting time of 2 s. 1081 movies acquired on a TALOS Arctica (Thermo Fisher Scientific) microscope with K2 direct electron detector (Gatan) in linear mode (at GW4 facility, University of Bristol).
Patch motion correction was carried out using cryoSPARC v2.15 + 200728 4.4 within the cryoSPARC web interface [53–56 (link)]. The remainder of the processing was conducted within the Scipion v2.0 framework [43 (link)] and included use of: CTFFIND4 [57 (link),58 (link)], Xmipp v3.0 [45 (link),59 (link)] and Relion v3.0 [46 (link),47 (link),51 (link),60 (link)].
Patch motion correction was carried out using cryoSPARC v2.15 + 200728 4.4 within the cryoSPARC web interface [53–56 (link)]. The remainder of the processing was conducted within the Scipion v2.0 framework [43 (link)] and included use of: CTFFIND4 [57 (link),58 (link)], Xmipp v3.0 [45 (link),59 (link)] and Relion v3.0 [46 (link),47 (link),51 (link),60 (link)].
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