Falcon 3 direct electron detector

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Falcon III direct electron detector is a high-performance imaging device designed for cryo-electron microscopy (cryo-EM) applications. It features a direct electron detection system that captures and converts electrons directly into digital signals, providing high-resolution images with low noise and exceptional contrast. The Falcon III is capable of fast frame rates and is optimized for single-particle analysis and other cryo-EM techniques.

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Falcon III (44 citations) Falcon III detector (30 citations) Falcon-3 direct detector (5 citations) FEI Falcon 3 direct electron detector (4 citations) Falcon III direct electron detector 30 (2 citations)

39 protocols using falcon 3 direct electron detector

Structural Analysis of RuBisCO and α-Carboxysomes

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For the structural characterisation of RuBisCO, 3 µL aliquots of purified α-carboxysomes at a concentration of ~1 mg mL -1 were applied to Graphene Oxide coated, 300 mesh, 2/2 µm hole/spacing, holey carbon grids (EMR). A Leica EM GP Automatic Plunge Freezer (Leica) was used to plunge freeze the sample, blotting for 3-6 s. Cryo-EM data was collected with a 300 kV Titan Krios TEM, equipped with a Falcon 3 direct electron detector (Thermo Fisher) operated in linear mode. 4593 micrographs were collected using the EPU software (Thermo Fisher) with a pixel size of 1.11 Å pix -1 , a total dose rate of 30 e -Å -2 , and 44 fractions per micrograph. The defocus range was -0.5 to -1.5 µm.
For structural characterisation of the intact α-carboxysome complex, 3 µL aliquots of purified sample at a concentration of 3 mg mL -1 were applied to Graphene Oxide coated grids, 300 mesh, 2/2 µm hole/spacing, holey carbon grids (EMR). A Leica EM GP Automatic Plunge Freezer (Leica) was used to plunge freeze, blotting for 6 s. Cryo-EM data were collected with a 300 kV Titan Krios TEM with a Falcon 3 direct electron detector (FEI) operated in counting mode. 5429 micrographs were collected using EPU software (Thermo Fisher) with a pixel size of 2.23 Å pix -1 with a total dose rate of 29.7 e -Å -2 with 33 frames per micrograph. The defocus range was -1.0 to -2.2 µm.

Evans S.L., Al-Hazeem M.M.J., Mann D., Smetacek N., Beavil A.J., Sun Y., Chen T., Dykes G.F., Liu L., & Bergeron J.R.C. (2022). Single-particle cryo-EM analysis of the shell architecture and internal organization of an intact α-carboxysome.

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Cryo-EM Imaging of PRV Capsids

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A 3 μL aliquot of purified PRV samples was applied to freshly glow-discharged holey carbon Quantifoil Cu grids (R2/2, 200 mesh, Quantifoil Micro Tools), and then blotted for 6 s before plunge-freezing the grids into liquid ethane cooled by liquid nitrogen inside a Vitrobot Mark IV (Thermo Fisher Scientific) at 100% humidity and 4 °C. Cryo-EM images of the PRV capsids were acquired with the FEI Tecnai F30 TEM (Thermo Fisher Scientific) with a Falcon 3 direct electron detector (Thermo Fisher Scientific) at a nominal 93,000× magnification, corresponding to a calibrated physical pixel size of 1.117 Å. Each movie contained 39 frames, with a total dose of 30 e⁻/Å² at an exposure time of 1 s. Data were automatically collected with Thermo Fisher EPU software.

Wang G., Zha Z., Huang P., Sun H., Huang Y., He M., Chen T., Lin L., Chen Z., Kong Z., Que Y., Li T., Gu Y., Yu H., Zhang J., Zheng Q., Chen Y., Li S, & Xia N. (2022). Structures of pseudorabies virus capsids. Nature Communications, 13, 1533.

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Cryo-EM of ALB1 Nucleosome-FoxA1 Complex

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Aliquots (2.5 µl) of the purified ALB1 nucleosome mixed with the human FoxA1 deletion mutant, FoxA1(170–472), which contains both the DNA-binding and histone-binding domains, were applied to Quantifoil holey carbon grids (R1.2/1.3 200-mesh Cu), which were freshly cleaned using a Solarus Plasma Cleaner (Gatan, Pleasanton, USA) for 15 s at 20 W in a 23% H₂, 77% O₂ gas mix. The grids were blotted for 3 s at 16°C and 100% relative humidity, and then immediate plunge-frozen in liquid ethane with a Vitrobot Mark IV (Thermo Fisher, Hillsboro, USA). Cryo-EM data were collected using the EPU automation software on a Talos Arctica microscope (Thermo Fisher, Hillsboro, USA), operating at 200 kV at a calibrated magnification of 100 000× (pixel size of 1.40 Å), with defocus ranging from −1.5 to −3.0 µm. Digital micrographs were recorded with 2-second exposure times on a Falcon 3 direct electron detector (Thermo Fisher, Hillsboro, USA) in the linear mode, at a dose rate of approximately 40 electrons per Å² per second with 25 ms per frame time, retaining a total of 79 frames with an accumulated total dose of approximately 80 electrons per Å².

Takizawa Y., Tanaka H., Machida S., Koyama M., Maehara K., Ohkawa Y., Wade P.A., Wolf M, & Kurumizaka H. (2018). Cryo-EM structure of the nucleosome containing the ALB1 enhancer DNA sequence. Open Biology, 8(3), 170255.

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Cryo-ET of Human Ribosomes

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Tilt series of human ribosome samples were collected on grids vitrified under the same conditions as the grids used for single-particle data collection. Data sets for both ribosomes vitrified on a standard holey carbon grid and ribosomes vitrified on an ssDNA-coated grid were collected using a ThermoFisher Arctica electron microscope equipped with a Falcon 3 direct electron detector (ThermoFisher Scientific) in integrating mode. Tilt series were collected using SerialEM (Schorb et al., 2019 ▸ ), with a tilt range of −56° to +56° and a step increment of 2°, using the dose-symmetric acquisition scheme (Turoňová et al., 2020 ▸ ). The tilt series were collected with a total exposure of 180 e⁻ Å⁻² and a defocus of −6 µm. The magnification was set to 28 000×, which resulted in a pixel size of 5.19 Å. Acquired tilt series were aligned in IMOD 4.11 (Kremer et al., 1996 ▸ ) by cross-correlation between subsequent tilts. Tomogram positioning was performed manually. The tomograms were reconstructed using a weighted back-projection algorithm. Tomograms were visualized using 3dmod (Kremer et al., 1996 ▸ ).

Hrebík D., Gondová M., Valentová L., Füzik T., Přidal A., Nováček J, & Plevka P. (2022). Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules. Acta Crystallographica. Section D, Structural Biology, 78(Pt 11), 1337-1346.

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Cryo-ET of Virus Structures

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For cryo-ET of the viruses, perforated carbon film–covered microscopical 200 mesh grids (R1/4 batch of Quantifoil, MicroTools GmbH) were cleaned with chloroform and hydrophilized by 60 s glow discharging at 8 W in a BALTEC MED 020 device (Leica Microsystems), before 4 μl aliquots of the virus solution were applied to the grids. The samples were vitrified by automatic blotting and plunge freezing with a FEI VitrobotMark IV (Thermo Fisher Scientific) using liquid ethane as cryogen. The vitrified specimens were transferred under liquid nitrogen into the autoloader of a FEI TALOS ARCTICA electron microscope (Thermo Fisher Scientific). This microscope is equipped with a high-brightness field-emission gun operated at an acceleration voltage of 200 kV. Single-axis tilt series (±64° at 2° angular increment) were recorded with the Falcon 3 direct electron detector (Thermo Fisher Scientific) using a Volta Phase Plate at 28 K primary magnification with a total dose lower than 100 e−/Å². Tomogram reconstruction was performed using Thermo Fisher Inspect3D software.

Zhang X., Abel T., Su S., Herrmann A., Ludwig K, & Veit M. (2022). Structural and functional analysis of the roles of influenza C virus membrane proteins in assembly and budding. The Journal of Biological Chemistry, 298(3), 101727.

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Cryo-FIB Lamella Preparation and Cryo-ET Data Collection

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Cryo-FIB milling of the specimen was performed as described previously (10 (link)) using a Zeiss Crossbeam 550 FIB-SEM microscope and generated 200-nm thick lamellae. Subsequently, the EM grids were transferred to a Titan Krios transmission electron microscope operating at 300 kV, equipped with a Falcon 3 direct electron detector (Thermo Fisher Scientific) for cryo-ET data collection. Tilt series images were acquired bidirectionally using the SerialEM software (50 (link)) at 18,000× or 22,500× magnification (pixel sizes 4.6 Å or 3.7 Å, respectively) with a defocus of −4 μm, ±60° oscillation, 1° increments with a total final dose of 100 e⁻/Å².

von Kügelgen A., van Dorst S., Yamashita K., Sexton D.L., Tocheva E.I., Murshudov G., Alva V, & Bharat T.A. (2023). Interdigitated immunoglobulin arrays form the hyperstable surface layer of the extremophilic bacterium Deinococcus radiodurans. Proceedings of the National Academy of Sciences of the United States of America, 120(16), e2215808120.

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Lipid Vesicle Permeabilization Assay

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LUVs of different lipid compositions were prepared from MLVs as described above. Experiments with LLO only were performed at different incubation times (30 min, 2 h and 6 h), at 20 °C or 37 °C and pH 6.5. For experiments with LLO and LmPC-PLC preincubation, all the incubation steps were performed at 37 °C. 2.5 mM LUVs were preincubated with 5 μM LmPC-PLC for 30 minutes. After that, LLO was added to a final concentration of 5 μM and the sample mixtures were further incubated for 60 minutes. After incubation, 3 μl of each sample was transferred to glow-discharged (GloQube® Plus, Quorum, UK) Quantifoil R1.2-1.3 grids (Quantifoil, Germany) and blotted under 100% humidity using Mark IV Vitrobot (Thermo Fisher Scientific). Blot force used was 3 and blot time was 6.5 s. Micrographs were collected on cryo-transmission electron microscope Glacios (Thermo Fisher Scientific) operated at 200 kV and equipped with Falcon 3 direct electron detector (Thermo Fisher Scientific), at a nominal magnification of 5,300x and 73,000x and defocus range of –3 µm to –4 µm.

Petrišič N., Adamek M., Kežar A., Hočevar S.B., Žagar E., Anderluh G, & Podobnik M. (2023). Structural basis for the unique molecular properties of broad-range phospholipase C from Listeria monocytogenes. Nature Communications, 14, 6474.

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Cryo-EM Imaging of hnRNPDL-2 Amyloid Fibrils

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For cryo-EM, sample vitrification was carried out using a Mark IV Vitrobot (Thermo Fisher Scientific). 3 μl hnRNPDL-2 amyloid fibrils diluted in MQ water at a final concentration of 0.25 mg/mL were applied to a C-Flat 1.2/1.3-3Cu-T50 grid (Protochips) previously glow-discharged at 30 mA for 30 s in a GloQube (Quorum Technologies). Sample was incubated on grid for 60 s at 4 °C and 100% humidity, blotted and plunge-frozen into liquid ethane. Vitrified samples were transferred to a Talos Arctica transmission electron microscope (Thermo Fisher Scientific) operated at 200 kV and equipped with a Falcon 3 direct electron detector (Thermo Fisher Scientific) and EPU 2.8 (Thermo Fisher Scientific) software. A total of 1114 movies were collected using EPU 2.8 (Thermo Fisher Scientific) in electron counting mode with an applied dose of 40 e^-/Å² divided in 40 frames at a magnification of 120 kx. All the micrographs were acquired with a pixel size of 0.889 Å/pixel and a defocus range of −1.0 to −2.2 μm.

Garcia-Pardo J., Bartolomé-Nafría A., Chaves-Sanjuan A., Gil-Garcia M., Visentin C., Bolognesi M., Ricagno S, & Ventura S. (2023). Cryo-EM structure of hnRNPDL-2 fibrils, a functional amyloid associated with limb-girdle muscular dystrophy D3. Nature Communications, 14, 239.

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Cryo-EM Structure Determination Protocol

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Two μL of the sample solution was applied to a glow-discharged QUANTIFOIL R1.2/1.3 Cu 300 mesh grid (Quantifoil Micro Tools GmbH, Germany). After blotting the excess solution on the grid with filter paper, the samples were rapidly frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). The frozen grids were screened to check sample conditions such as the ice thickness and particle dispersity using a Talos Arctica cryo-transmission electron microscope (cryo-TEM) operating at 200 keV and equipped with a Falcon 3 direct electron detector (Thermo Fisher Scientific) at Institute for Protein Research, Osaka University. Cryo-EM data collection was performed on a Titan Krios cryo-TEM equipped with a Cs corrector (Thermo Fisher Scientific) operating at 300 keV in EFTEM nanoprobe mode at Institute for Protein Research, Osaka University. Images were acquired as movies using Gatan BioQuantum energy filter (slit width of 20 eV) and K3 direct detection camera (Gatan, Inc., USA) in electron counting mode. A total of 10,241 movies were collected at a dose rate of 12.9 e -/pixel/s, a pixel size of 0.675 Å 2 , and a total dose of 75 e -/Å ) was used for automated data collection using a 3x3-hole pattern beam-image shift scheme with a nominal defocus range of -0.7 to -1.5 μm.

Suno R., Sugita Y., Morimoto K., Takazaki H., Tsujimoto H., Hirose M., Suno-Ikeda C., Nomura N., Hino T., Inoue A., Iwasaki K., Kato T., Iwata S, & Kobayashi T. (2022). Structural insights into the G protein selectivity revealed by the human EP3-G_i signaling complex. Cell reports, 40(11).

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Graphene-Coated Grids for Cryo-EM Imaging

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ILY–CD59 nanodisc complexes were imaged using holey carbon grids coated with graphene oxide. To coat R1.2/1.3 Quantifoil grids with graphene oxide, grids were first glow-discharged for 1 min, then, 0.2 mg/ml of a graphene oxide solution (Sigma) in water was applied to the glow-discharged, top face of the grid, followed by blotting by filter paper on the bottom face of the grid. This process was repeated twice, followed by two washes of the top face of the grid with 20 µl of water. Grids were left to dry, and used within one hour of graphene oxide coating. Immediately following concentration, 2.5 µl of the early prepore-locked ILY–CD59 oligomers on nanodiscs was adsorbed on graphene oxide-coated grids and blotted for 2.5 s at “blot force” 3 and plunge frozen in liquid ethane cooled to liquid nitrogen temperatures with a Vitrobot mark IV (Thermo Fisher Scientific). Electron micrograph movies were collected on a 300 keV Titan Krios (Thermo Fisher Scientific) fitted with a Falcon III direct electron detector (Thermo Fisher Scientific) in linear mode with image acquisition software EPU (Thermo Fisher scientific). Specific collection details for all three datasets are summarized in Supplementary Table 3.

Shah N.R., Voisin T.B., Parsons E.S., Boyd C.M., Hoogenboom B.W, & Bubeck D. (2020). Structural basis for tuning activity and membrane specificity of bacterial cytolysins. Nature Communications, 11, 5818.

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