Epu automated data acquisition software for single particle analysis

Manufactured by Thermo Fisher Scientific

The EPU Automated Data Acquisition Software is a tool for single particle analysis. It is designed to automate the process of data acquisition for cryo-electron microscopy experiments.

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9 protocols using epu automated data acquisition software for single particle analysis

Cryo-EM Reconstitution of TnsC-TniQ Filaments

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For the reconstitution of TnsC filaments bound to TniQ, wild-type TnsC protein was diluted to a final concentration of 15 μM in a buffer containing 20 mM HEPES-KOH pH 7.5, 100 mM KCl, 10 mM MgCl₂, 1 mM DTT and 1 mM ATP and mixed at a ratio of 1:25 (TnsC:DNA) with a double-stranded 69-bp duplex DNA oligonucleotide (Table S3) and at a 1:2 ratio (TnsC:TniQ) with TniQ in a 22.4 μL reaction volume. For preparation of cryo-EM grids, 3.0 μL of sample was applied to glow-discharged 200-mesh copper 2 nm C R1.2/1.3 cryo-EM grids (Quantifoil Micro Tools), blotted 1 s at 100% humidity, 4°C, plunge frozen in liquid ethane (using a Vitrobot Mark IV plunger, FEI) and stored in liquid nitrogen. Cryo-EM data collection was performed on a FEI Titan Krios G3i microscope (University of Zurich, Switzerland) operated at 300 kV equipped with a Gatan K3 direct electron detector in super-resolution counting mode. A total of 10,436 movies were recorded at a calibrated magnification of 130,000× resulting in super-resolution pixel size of 0.325 Å. Each movie comprised 36 subframes with a total dose of 66.036 e⁻ Å⁻². Data acquisition was performed with EPU Automated Data Acquisition Software for Single Particle Analysis (ThermoFisher Scientific) with three shots per hole at −1.0 μm to −2.4 μm defocus (0.2 μm steps).

Schmitz M., Querques I., Oberli S., Chanez C, & Jinek M. (2022). Structural basis for the assembly of the type V CRISPR-associated transposon complex. Cell, 185(26), 4999-5010.e17.

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Isolation and Purification of Native Human α2-Macroglobulin for Structural Analysis

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Wild-type authentic native (hα₂M)₄ was isolated from thawed frozen plasma from healthy human donors, which was de-identified prior to use in this study. The protein was purified, assessed for peptidase-inhibition competence as described (15 (link), 26 (link), 49 (link)), and verified to be equivalent to protein purified from fresh plasma in functional and physiological assays. Aliquots of pure protein (5 μL) were diluted to 0.1 mg/mL, applied to R2/2 300 mesh acetone vapor-treated copper grids, and vitrified using a Leica EM CPC cryofixation unit. Data were collected on FEI Titan Krios electron microscopes operated at 300 kV, and images were recorded with Gatan K2-summit cameras in counting mode using the EPU Automated Data Acquisition Software for Single Particle Analysis (Thermo Fisher Scientific). The total number of recorded movies, nominal magnification, calibrated pixel size at the specimen level, total exposure, exposure per frame, and defocus range for each specimen are described in SI Appendix, Table 1.

Luque D., Goulas T., Mata C.P., Mendes S.R., Gomis-Rüth F.X, & Castón J.R. (2022). Cryo-EM structures show the mechanistic basis of pan-peptidase inhibition by human α2-macroglobulin. Proceedings of the National Academy of Sciences of the United States of America, 119(19), e2200102119.

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Cryo-EM of Purified TRICi Fraction 4

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Purified TRICi fraction 4 sample (5 µL) was applied onto R2/2 300 mesh Quantifoil Cu/Rh grids and vitrified using a ThermoFisher Scientific Vitrobot Mark IV automatic plunger. Data were collected on an FEI Talos Arctica electron microscope operated at 200 kV, and images were recorded on an FEI Falcon III detector. A total of 2,369 movies were recorded with a total exposure dose of 40.8 e^- Å⁻² divided into 40 frames and a calibrated pixel size of 1.37 Å on the specimen. Data acquisition was performed with EPU Automated Data Acquisition Software for Single Particle Analysis (ThermoFisher Scientific) at −0.70 to −3.0 µm defocus.

Vetter J., Papa G., Tobler K., Rodriguez J.M., Kley M., Myers M., Wiesendanger M., Schraner E.M., Luque D., Burrone O.R., Fraefel C, & Eichwald C. (2024). The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication. mBio, 15(4), e00499-24.

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Cryo-EM Imaging of Purified EV-E

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Purified EV-E samples in PBS (3 μL) were applied onto 400-mesh lacey grids coated in a 3 nm carbon film (Agar Scientific, UK). The sample was left to adsorb for 30 s before most of the sample was blotted away manually and this process was repeated 4 times. The grids were vitrified using a Leica EM GP freezing device (Leica Microsystems). Chamber conditions were set at 4 °C and 95% relative humidity. Grids were glow discharged for 30 seconds prior to application of the samples. Data were collected on a FEI Titan Krios electron microscope operated at 300 kV and images recorded on a FEI Falcon III detector operating in linear mode. A total of 8,785 movies were recorded at a calibrated magnification of 75,000x, yielding a pixel size of 1.065 Å on the specimen. Each movie comprises 39 frames with an exposure rate of 1.27 e^- Å^-2 per frame, with a total exposure time of 1 s and an accumulated exposure of 49.5 e^- Å^-2. Data acquisition was performed with EPU Automated Data Acquisition Software for Single Particle Analysis (ThermoFisher) at -0.75 μm to -3.5 μm defocus.

Chandler-Bostock R., Mata C.P., Bingham R.J., Dykeman E.C., Meng B., Tuthill T.J., Rowlands D.J., Ranson N.A., Twarock R, & Stockley P.G. (2020). Assembly of infectious enteroviruses depends on multiple, conserved genomic RNA-coat protein contacts. PLoS Pathogens, 16(12), e1009146.

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Cryo-EM of SARS-CoV-2 Spike Variants

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Purified SARS-CoV-2 spike samples in 10mM Hepes pH7.2 and 150mM NaCl (3 μL at 0.62–0.75mg/ml) were kept at 4°C until their application onto QUANTIFOIL R 1.2/1.3 Cu:300-mesh grids (QUANTIFOIL). The grids were vitrified using a Leica GP automatic vitrification robot (Leica). Chamber conditions were set at 10°C and 95% relative humidity. Grids were glow discharged for 30 seconds prior to application of the samples. Data were collected on a FEI Talos electron microscope operated at 200 kV and images recorded on a FEI Falcon III detector operating in electron counting mode. A total of 4,841 and 2,492 movies were recorded for [S:A222V + S:D614G] and S:D614G, respectively; at a calibrated magnification of 120,000x, yielding a pixel size of 0.85 Å on the specimen. Each movie comprises 60 frames with an exposure rate of 0.54 e-/Å² per frame, with a total exposure time of 20 s and an accumulated exposure of 32.4 e-/Å². Data acquisition was performed with EPU Automated Data Acquisition Software for Single Particle Analysis (Thermo Fisher Scientific) at -0.3 μm to -3.5 μm defocus.

Ginex T., Marco-Marín C., Wieczór M., Mata C.P., Krieger J., Ruiz-Rodriguez P., López-Redondo M.L., Francés-Gómez C., Melero R., Sánchez-Sorzano C.Ó., Martínez M., Gougeard N., Forcada-Nadal A., Zamora-Caballero S., Gozalbo-Rovira R., Sanz-Frasquet C., Arranz R., Bravo J., Rubio V., Marina A., Geller R., Comas I., Gil C., Coscolla M., Orozco M., Llácer J.L, & Carazo J.M. (2022). The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: The case of the spike A222V mutation. PLoS Pathogens, 18(7), e1010631.

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HBV pgRNA Cryo-EM Imaging

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Lacey carbon 400-mesh copper grids coated with a <3 nm continuous carbon film (Agar Scientific, UK) were glow-discharged in air (10 mA, 30 s) before applying one 3 μL aliquot of pgRNA containing HBV, reassembled and purified as described above for X-Ray Footprinting. Grids were blotted and vitrified in liquid nitrogen-cooled liquid ethane using a LEICA EM GP plunge freezing device (Leica Microsystems). Chamber conditions were set at 4 °C and 95% relative humidity. Grids were stored in liquid nitrogen prior to imaging with an FEI Titan Krios transmission electron microscope (ABSL, University of Leeds) at 300 kV, at a magnification of ×75,000 and a calibrated object sampling of 1.065 Å/pixel. Images were recorded on a FEI Falcon III detector operating in integrating mode. Each movie comprises 59 frames with an exposure rate of 0.9 e⁻ Å⁻² per frame, with a total exposure time of 1.5 s and an accumulated exposure of 53.1 e⁻ Å⁻². Data acquisition was performed with EPU Automated Data Acquisition Software for Single Particle Analysis (ThermoFisher) at −0.7 µm to −2.5 µm defocus.

Patel N., Clark S., Weiß E.U., Mata C.P., Bohon J., Farquhar E.R., Maskell D.P., Ranson N.A., Twarock R, & Stockley P.G. (2021). In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus. Communications Biology, 4, 1407.

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Cryo-EM Sample Preparation for Protein Complexes

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The complex of S (see the
section on Production of Proteins) with
compound 2 (Na salt) was prepared by incubating 5 min
at 25 °C 0.5 mg/mL S and 0.3 mM compound 2 in 0.5
mM, Hepes pH 7.2, 150 mM NaCl. Then, the mixture was placed at 4 °C,
and 3 μL aliquots were placed on cryo-EM grids (QUANTIFOIL R
1.2/1.3 Au:300-mesh grids) that had been made hydrophilic by glow
discharge (30 s using a Leica EM ACE600 device). The grids were then
placed in the blotting chamber of a Leica EM GP2 at 10 °C and
95% ambient humidity and immediately vitrified in liquid-N₂-cooled liquid ethane.
Transmission cryoEM images were collected
automatically (EPU Automated Data Acquisition Software for Single
Particle Analysis; Thermo Fisher Scientific) on a FEI Talos electron
microscope operated at 200 kV under low-dose conditions and images
recorded on a FEI Falcon III detector operating in electron counting
mode, at −1 to −2.5 μm defocus. A total of 3500
movies were recorded at a calibrated magnification of 120,000×,
yielding a pixel size of 0.85 Å on the specimen. Each movie comprises
60 frames with an exposure rate of 0.55 e^–/Å² per frame, with a total exposure time of 28 s and an accumulated
exposure of 30 e^–/Å².

Gargantilla M., Francés C., Adhav A., Forcada-Nadal A., Martínez-Gualda B., Martí-Marí O., López-Redondo M.L., Melero R., Marco-Marín C., Gougeard N., Espinosa C., Rubio-del-Campo A., Ruiz-Partida R., Hernández-Sierra M.D., Villamayor-Belinchón L., Bravo J., Llacer J.L., Marina A., Rubio V., San-Félix A., Geller R, & Pérez-Pérez M.J. (2023). C-2 Thiophenyl Tryptophan Trimers Inhibit Cellular Entry of SARS-CoV-2 through Interaction with the Viral Spike (S) Protein. Journal of Medicinal Chemistry, 66(15), 10432-10457.

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Cryo-EM Structure of BabSPARTA-gRNA Complex

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Purified BabSPARTA was mixed with a 5′-phosphorylated RNA guide (oBK084, 5′-UGACGGCUCUAAUCUAUUAGU-3′) in assembly buffer (20 mM HEPES pH 7.5, 125 mM KCl, 2 mM MgCl₂). The final sample contained 20 μM BabSPARTA and 30 μM gRNA (1:1.5 molar ratio) in a total volume of 80 μl. The volume was incubated at 50°C for 1 hour, centrifuged at 18000 rpm for 10′ at room temperature and directly used for cryo-EM grid preparation.
3.5 μl of sample was applied to a freshly glow discharged 200-mesh Au R1.2/1.3 grid (Quantifoil Micro Tools), incubated for 5 s, blotted for 6 s at 100% humidity, 4 °C, plunge frozen in liquid ethane (using a Vitrobot Mark IV plunger, FEI) and stored in liquid nitrogen. cryo-EM data collection was performed on a FEI Titan Krios G3i microscope (University of Zurich, Switzerland) operated at 300 kV and equipped with a Gatan K3 direct electron detector in super-resolution counting mode. A total of 14 332 movies were recorded at 130 000× magnification, resulting in a super-resolution pixel size of 0.325 Å. Each movie comprised 36 subframes with a total dose of 58.005 e⁻/Å². Data acquisition was performed with EPU Automated Data Acquisition Software for Single Particle Analysis (ThermoFisher Scientific) with three shots per hole at -1.0 mm to -2.4 mm defocus (0.2 mm steps).

Finocchio G., Koopal B., Potocnik A., Heijstek C., Westphal A.H., Jinek M, & Swarts D.C. (2024). Target DNA-dependent activation mechanism of the prokaryotic immune system SPARTA. Nucleic Acids Research, 52(4), 2012-2029.

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Cryo-EM analysis of TnsC-DNA complex

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Purified ShTnsC was diluted to a final concentration of 6.4 μM in a buffer containing 20 mM HEPES-KOH pH 7.5, 200 mM KCl, 10 mM MgCl2, 1 mM DTT and mixed with a 92 nt dsDNA oligonucleotide containing two mismatched regions (Supplementary Table 1) at a ratio of 5:1 (TnsC:DNA) in a 21.4 μL reaction volume and incubated for 10 min at 25 °C. Upon addition of AMPPNP at a final concentration of 1 mM, the sample was further incubated for 20 min at 37 °C. Sample vitrification was performed with a Leica EM GP2 plunge freezer at 10 °C and at 70 % humidity. 3.5 μL sample were applied onto a freshly glow-discharged 200-mesh copper 2 nm C R1.2/1.3 grids (Quantifoil Micro Tools). The grids were blotted for 1 s and plunge-vitrified in liquid ethane. Cryo-EM data collection was performed on a FEI Titan Krios G3i microscope (University of Zurich, Switzerland) operated at 300 kV equipped with a Gatan K3 direct electron detector in super-resolution counting mode. A total of 5,055 micrographs were recorded at a calibrated magnification of 130,000 × resulting in super-resolution pixel size of 0.325 Å. Each movie comprised 36 subframes with a total dose of 66.39 e− Å⁻². Data acquisition was performed with EPU Automated Data Acquisition Software for Single Particle Analysis (ThermoFisher) with three shots per hole at −1.0 μm to −2.4 μm defocus (0.2 μm steps).

Querques I., Schmitz M., Oberli S., Chanez C, & Jinek M. (2021). Target site selection and remodeling by type V CRISPR-transposon systems. Nature, 599(7885), 497-502.

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