Falcon 4 direct electron detector

Manufactured by Thermo Fisher Scientific

Sourced in Netherlands

The Falcon 4 direct electron detector is a high-performance camera designed for cryo-electron microscopy (cryo-EM) applications. It captures images of samples at the atomic level with high resolution and low noise. The Falcon 4 directly detects electrons, enabling efficient data collection and analysis for structural biology research and applications.

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19 protocols using falcon 4 direct electron detector

Cryo-EM Structure of A2BR-Gs Complexes

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Samples (3 μl) of A_2BR–NECA–G_s and A_2BR–BAY60-6583–G_s were applied to Au grids (Quantifoil, 300 mesh AU R1.2/1.3) that were glow-discharged for 40 s using Gatan Solarus (950). Grids were subsequently plunge-frozen by Vitrobot Mark IV (Thermo Fisher Scientific) at 4°C and 100% humidity.
Cryo-EM datasets were collected on the Krios G4 Cryo–Transmission Electron Microscope equipped with the Falcon 4 Direct Electron Detector (Thermo Fisher Scientific) in superresolution mode operating at 300 kV accelerating voltage with 10-eV slit width. Movies were taken under Energy-filtered transmission electron microscopy (EFTEM) nanoprobe mode, with 50-μm C2 aperture and calibrated magnification of ×130,000, corresponding to a pixel size of 0.96 Å. Each movie was taken in electron-event representation (EER) format with a total dose of 60 electrons per Å². EPU software (Thermo Fisher Scientific, v.1.11.0) was used to collect data with a defocus range of −1.2 to −1.8 μm.

Chen Y., Zhang J., Weng Y., Xu Y., Lu W., Liu W., Liu M., Hua T, & Song G. (2022). Cryo-EM structure of the human adenosine A2B receptor–Gs signaling complex. Science Advances, 8(51), eadd3709.

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Cryo-EM Structure Determination of 4F2hc-LAT2

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After the final protein purification step by SEC, 3 µL of the 4F2hc-LAT2 sample at ≈3 mg/mL in SEC-buffer were adsorbed on glow discharged (20 s, 10 mA, 0.25 mbar) cryo-EM grids (Cu-R1.2/1.3 grids 300 Mesh; Quantifoil, Germany) for 3 s. The grids were plunge frozen in liquid ethane after blotting off excess buffer for 3 s using a Vitrobot Mark IV apparatus operated at approximately 100% humidity and cooled to 4–5 °C. The grids were stored in liquid nitrogen until further use. Cryo-EM data were collected using a 300 kV Thermo Fisher Scientific Titan Krios G4 electron microscope equipped with a Falcon 4 direct electron detector in an aberration-free image shifting and fringe-free illumination mode. The data were collected at a magnification of 96,000× corresponding to 0.83 Å/pix on the camera and at defocus ranges of −0.9 to −2.2 µm. Images were recorded in an automated fashion using the EPU 2 software (Thermo Fisher Scientific, Hillsboro, OR, USA) in counting mode for 4.6 s with a dose rate of 8.7 e⁻/Å²/s, resulting in a total accumulated dose on the specimen level of approximately 40 e⁻/Å² per exposure.

Jeckelmann J.M, & Fotiadis D. (2020). Sub-Nanometer Cryo-EM Density Map of the Human Heterodimeric Amino Acid Transporter 4F2hc-LAT2. International Journal of Molecular Sciences, 21(19), 7094.

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Electron-counted MicroED Data Collection

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Electron-counted MicroED data were collected on a Titan Krios 3Gi TEM (Thermo Fisher) operated at 300 kV as described previously (Martynowycz et al., 2022 (link)). Briefly, the TEM was set up for low exposure data collection using a 50 μm C2 aperture, spot size 11, and a beam diameter of 25 μm. A 100 μm SA aperture was used, corresponding to an area of 2 μm diameter on the specimen. Crystalline lamellae were continuously rotated over a range of 84° at a rotation speed of 0.2°/s over 420 s with a total exposure of approximately 0.64 e^-.Å⁻² per dataset. Data were recorded on a Falcon 4 direct electron detector (Thermo Fisher) in electron-counting mode operating at an internal frame rate of 250 Hz. Data from 16 crystal lamellae were integrated using XDS (Kabsch, 2010 ) and scaled and merged in AIMLESS (Evans and Murshudov, 2013 (link)). The structure was phased ab initio by placing a three-residue idealized α-helical fragment using PHASER (McCoy et al., 2007 (link)) followed by density modification in ACORN (Foadi et al., 2000 (link)). The entire structure was built automatically using BUCCANEER (Cowtan, 2006 (link)) and refined in REFMAC5 (Murshudov et al., 2011 (link)) using electron scattering factors.

Clabbers M.T., Martynowycz M.W., Hattne J, & Gonen T. (2022). Hydrogens and hydrogen-bond networks in macromolecular MicroED data. Journal of Structural Biology: X, 6, 100078.

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cryo-EM structure of hMPV F-Fab complex

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Purified DS-CavEs2-IPDS was combined with a 1.5-fold molar excess of MPV467 Fab incubated at room temperature for 10 min before being moved to ice. Just before freezing, sample was diluted to a concentration of 0.66 mg/mL hMPV F in 2 mM Tris pH 8.0, 200 mM NaCl, and 0.02% NaN₃ buffer. Then 1 µL of 0.5% amphipol A8-35 was combined with 10 µL of diluted sample, and 4 µL of this sample was applied to a gold 1.2/1.3 300 mesh grid (Protochips Au-Flat) that had been plasma-cleaned for 180 s using a Solarus 950 plasma cleaner (Gatan) with a 4:1 ratio of O₂/H₂. Grids were plunge-frozen using a Vitrobot Mark IV (Thermo Fisher) with a 10 °C, 100% humidity chamber. Blotting settings were 5 s of wait followed by 4 s of blotting with −2 force before plunging into nitrogen-cooled liquid ethane. Using a Glacios (Thermo Scientific) equipped with a Falcon 4 direct electron detector (Thermo Scientific), a single grid was imaged to collect a total of 1,458 images. Data were collected at a 30° tilt with magnification of 150,000× corresponding to a calibrated pixel size of 0.94 Å/pix and a total exposure of 40 e⁻/Å². Data collection statistics are listed in SI Appendix, Table S2.

Banerjee A., Huang J., Rush S.A., Murray J., Gingerich A.D., Royer F., Hsieh C.L., Tripp R.A., McLellan J.S, & Mousa J.J. (2022). Structural basis for ultrapotent antibody-mediated neutralization of human metapneumovirus. Proceedings of the National Academy of Sciences of the United States of America, 119(25), e2203326119.

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Cryo-EM of Protein Samples at Varied Concentrations

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Two datasets were collected using the same settings, but different concentrations. Samples were prepared as previously described^{69 (link)}. Specifically, the sample was concentrated to 1.4 mg/ml for the first and 2.8 mg/ml for the second dataset. C-flat grids (Protochips; CF-1.2/1.3–3Cu-50) were prepared by glow-discharging them with a PELCO easiGlow at 15 mA for a duration of 45 s. A total of 3 µL of the sample were promptly applied to the grids and immediately plunge-frozen in liquid ethane with the use of a vitro bot Mark IV (Thermo Fischer). This process was conducted at 4 °C with a relative humidity of 100%. Data was collected on a Glacios microscope (Thermo Fischer), operating at 200 kV and equipped with a Selectris energy filter (Thermo Fischer) with a slit with of 10 eV. Movies were recorded using a Falcon 4 direct electron detector (Thermo Fischer) at a nominal magnification of 130,000 which is equal to a calibrated pixel size of 0.924 Å per pixel. The dose rate for collection was set to 5.22 e- per pixel with a total dose of 50 e- per Å^{2 (link)}. 46896 movies were automatically gathered using EPU software (v.2.9, Thermo Fischer) with a defocus range of −0.8 µm to −2.0 µm and stored in EER (electron-event representation) format.

Köck Z., Schnelle K., Persechino M., Umbach S., Schihada H., Januliene D., Parey K., Pockes S., Kolb P., Dötsch V., Möller A., Hilger D, & Bernhard F. (2024). Cryo-EM structure of cell-free synthesized human histamine 2 receptor/Gs complex in nanodisc environment. Nature Communications, 15, 1831.

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Cryo-EM Detergent Screening Protocol

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To screen various commonly used detergents during cryo-EM data collection, the Thermo Scientific™ VitroEase™ Buffer Screening Kit was used. The cryo-EM sample solutions with a final concentration of 0.5% (w/v) detergents were prepared via mixing 9 μL ~ 6.5 mg/mL spikes and 1 μL one of the following detergents (CTAB, CHAPS, OG, Tween-20, or FOM) from the kit. DDM was also tested. For each sample, 3 μL sample was deposited on an UltrAuFoil R1.2/1.3 300 mesh grid that had been glow-discharged for 30 s in a GloQube Plus Glow Discharge System. Plunge freezing was performed with a Vitrobot Mark IV using a blot force 0 and blot time 5 s at 100% humidity and 4 °C. All frozen grids were imaged with a Thermo Scientific Krios G4 Cryo-Transmission Electron Microscope (Cryo-TEM) operated at a fixed 300 kV, and images were recorded with a Falcon4 Direct Electron Detector in EER format. Movies were acquired using EPU Multigrid software in one day. Approximately 500 micrographs were automatically collected for each sample with a defocus range between 0.6 and 1.1 μm, a pixel size of 1.05 Å/pixel and a total dose ~40 e/Å². The micrographs were processed with cryoSPARC Live during data collection^{34 (link)}.

Egri S.B., Wang X., Díaz-Salinas M.A., Luban J., Dudkina N.V., Munro J.B, & Shen K. (2023). Detergent modulates the conformational equilibrium of SARS-CoV-2 Spike during cryo-EM structural determination. Nature Communications, 14, 2527.

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Cryo-EM Imaging of LLOV NP-RNA Complexes

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For the LLOV NP(full)–RNA complex, an aliquot of a 2.5 µl purified NP–RNA complex solution at a concentration of 1 mg/ml was applied to Quantifoil R1.2/1.3 200 mesh grids and blotted for 7 s, with a humidity of 100% at 4°C, before being rapidly frozen with liquid ethane using the Vitrobot Mark IV system (Thermo Fisher Scientific). Images of the frozen sample stored at insert temperature were acquired as movies on a Titan Krios cryo-TEM (Thermo Fisher Scientific), operated at 300 kV and equipped with a Falcon3EC direct electron detector (Thermo Fisher Scientific), and a Cs corrector (CEOS GmbH) at the Institute for Protein Research, Osaka University.
For the LLOV NP(1–450)–RNA complex, an aliquot of 3 µl purified NP-RNA complex solution at a concentration of 0.3 mg/ml was applied to Quantifoil R1.2/1.3 300 mesh grids and blotted for 6 s, with a humidity of 100% at 4°C, before being rapidly frozen in liquid ethane using the Vitrobot Mark IV system. Images were acquired using a Glacios cryo-TEM (Thermo Fisher Scientific) operated at 200 kV, with a Falcon4 direct electron detector (Thermo Fisher Scientific) in the electron counting mode, at the Institute for Life and Medical Sciences, Kyoto University.
The detailed imaging conditions for both the samples are described in Table S1.

Hu S., Fujita-Fujiharu Y., Sugita Y., Wendt L., Muramoto Y., Nakano M., Hoenen T, & Noda T. (2023). Cryoelectron microscopic structure of the nucleoprotein–RNA complex of the European filovirus, Lloviu virus. PNAS Nexus, 2(4), pgad120.

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Cryo-EM Sample Preparation and Data Collection

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Sample quality was inspected by negative-stain electron microscopy as previously described^{50 (link)}. Micrographs of the negatively-stained sample were recorded manually on a JEM2100plus transmission electron microscope (Jeol), operating at 200 kV and equipped with a Xarosa CMOS (Emsis) camera at a nominal magnification of 30,000, corresponding to a pixel size of 3.12 Å per pixel.
For cryo-EM, the sample was concentrated to 10 mg/ml. C-flat grids (Protochips; CF-1.2/1.3-3Cu-50) were glow-discharged, using a PELCO easiGlow device at 15 mA for 45 s and 3 µl of the concentrated sample were immediately applied and plunge frozen in liquid ethane, using a Vitrobot Mark IV (Thermo Fisher) at 100% relative humidity, 4 °C. The dataset was collected using a Glacios microscope (Thermo Fisher), operating at 200 kV and equipped with a Selectris energy filter (Thermo Fisher) with a slit of 10 eV. Movies were recorded with a Falcon 4 direct electron detector (Thermo Fisher) at a nominal magnification of 130,000 corresponding to a calibrated pixel size of 0.924 Å per pixel, and the data was saved in the electron-event representation (EER) format. The dose rate was set to 5.22 e^- per pixel per second and a total dose of 50 e^- per Å². 13,604 movies were collected automatically, using EPU software (v.2.9, Thermo Fisher) with a defocus range of −0.8 to −2.0 µm.

Schäfer J.H., Körner C., Esch B.M., Limar S., Parey K., Walter S., Januliene D., Moeller A, & Fröhlich F. (2023). Structure of the ceramide-bound SPOTS complex. Nature Communications, 14, 6196.

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Cryo-EM Structural Analysis of Glycoprotein Complexes

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2.5 μl of purified gp15:gp16-N at 2 mg/ml or gp16-C:2N2:PG at 1 mg/mL were adsorbed for 5-10 sec to 300-mesh Quantifoil R 1.2/1.3 holey carbon grids (EMS) glow discharged for 30 sec at 25 mA using an easiGlow (PELCO). The samples were blotted and frozen in liquid ethane using a Vitrobot Mark IV (Thermo Scientific). 7,818 movies for gp15:gp16-N samples were collected on a Titan Krios microscope (Thermo Scientific) at NIH-NCI-NCEF, operated at 300 kV, and equipped with a K3 direct electron detector camera (Gatan). Latitude S (Gatan) was used for data collection in super-resolution mode with an image pixel size of 0.54 Å and a nominal magnification of 81,000x. 3,306 movies for gp16-C:2N2:PG samples were collected on a Glacios (Thermo Scientific) equipped with a Falcon 4 direct electron detector (Thermo Scientific). EPU (Thermo Scientific) was used for data collection with an image pixel size of 0.74 Å and a nominal magnification of 190,000x. Further collection parameters are shown in Table 1.

Swanson N.A., Lokareddy R.K., Li F., Hou C.F., Leptihn S., Pavlenok M., Niederweis M., Pumroy R.A., Moiseenkova-Bell V.Y, & Cingolani G. (2021). Cryo-EM Structure of the Periplasmic Tunnel of T7 DNA-Ejectosome at 2.7 Å resolution. Molecular cell, 81(15), 3145-3159.e7.

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Cryo-EM Imaging of Cross-Linked Protein Complex

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The freshly reconstituted protein complex was cross-linked with 1 mM bis-sulfosuccinimidyl suberate (Thermo Fisher Scientific) on ice for 30 min. The reaction was quenched by 50 mM tris (pH 8.0) for an additional 10 min. Four microliters of aliquots of samples at 0.1 to 0.15 mg/ml was applied to graphene oxide–coated Quantifoil R1.2/1.3 gold 400-mesh grids (Electron Microscopy Sciences). The grids were then blotted and vitrified in liquid ethane using Vitrobot Mark IV (Thermo Fisher Scientific). Vitrified grids were screened in Talos F200C (Thermo Fisher Scientific) and Tecnai F20 (FEI) transmission electron microscopes to optimize the freezing conditions.
Cryo-EM data were collected in Glacios (Thermo Fisher Scientific) equipped with Falcon-4 direct electron detector operated at 200 kV in electron counting mode. Movies were collected at a nominal magnification of 150,000× and a pixel size of 0.92 Å in EER format. A total dose of 40 e⁻/Å² per movie was used with a dose rate of 5 to 6 e⁻/Å² per second. A total of 11,803 movies were recorded by automated data acquisition with EPU (Thermo Fisher Scientific).

Ito F., Alvarez-Cabrera A.L., Liu S., Yang H., Shiriaeva A., Zhou Z.H, & Chen X.S. (2023). Structural basis for HIV-1 antagonism of host APOBEC3G via Cullin E3 ligase. Science Advances, 9(1), eade3168.

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