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Epu software package

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The EPU software package is a comprehensive solution for data acquisition and processing in cryo-electron microscopy (cryo-EM) experiments. It provides a user-friendly interface for controlling the microscope, automating data collection, and managing the workflow of cryo-EM projects.

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Lab products found in correlation

Vitrobot mark 4, by Thermo Fisher Scientific (16 mentions) Titan krios electron microscope, by Thermo Fisher Scientific (3 mentions) K2 summit direct detector, by Ametek (2 mentions) R1.2 1 3 gold grids, by Quantifoil (2 mentions) Titan krios g2 transmission electron microscope, by Thermo Fisher Scientific (2 mentions) Titan krios g3 electron microscope, by Thermo Fisher Scientific (2 mentions) Glacios microscope, by Thermo Fisher Scientific (2 mentions) K3 energy filter, by Thermo Fisher Scientific (2 mentions) K3 camera, by Ametek (1 mentions) Solarus 950 plasma cleaner, by Ametek (1 mentions) Cu r2 1 200 mesh, by Quantifoil (1 mentions) Microscopy suite, by Ametek (1 mentions) S cerevisiae, by Merck Group (1 mentions) Bioquantum, by Ametek (1 mentions) Titan krios g3, by Thermo Fisher Scientific (1 mentions) K2 summit, by Ametek (1 mentions) K2 summit direct electron detector, by Ametek (1 mentions) Titan krios microscope, by Thermo Fisher Scientific (1 mentions) Titan krios, by Thermo Fisher Scientific (1 mentions) K2 summit, by Thermo Fisher Scientific (1 mentions)

14 protocols using epu software package

1

Cryo-EM Data Collection on PIC-cMed

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Cryo-EM data collection was performed on R1.2/1.3 gold grids (Quantifoil, Großlöbichau, Germany). Grids were glow-discharged for 45 seconds before application of 5 µL concentrated PIC-cMed sample, blotted for 5 seconds and vitrified by plunging into liquid ethane with a Vitrobot Mark IV (FEI Company, Hillsboro, US) operated at 4°C and 100% humidity. Cryo-EM data were acquired on a FEI Titan Krios G2 transmission electron microscope (FEI, Hillsboro, USA) operated in EFTEM mode at 300 kV and equipped with a K2 Summit direct detector (Gatan, Pleasanton, USA). Automated data acquisition was carried out using the FEI EPU software package at a nominal magnification of 105,000x (1.37 Å/pix). A total of 14,000 image stacks were collected at a defocus range from -0.5 µM to -5.0 µM. Each stack contained 40 frames that were acquired over a 10 seconds exposure time window in the counting mode of the camera. A dose rate of 4.2 e-/Å2s was applied, resulting in a total dose of 42 e-/Å2.
Schilbach S., Hantsche M., Tegunov D., Dienemann C., Wigge C., Urlaub H, & Cramer P. (2017). Structures of transcription pre-initiation complex with TFIIH and Mediator. Nature, 551(7679), 204-209.
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2

Cryo-EM Data Collection on PIC-cMed

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Cryo-EM data collection was performed on R1.2/1.3 gold grids (Quantifoil, Großlöbichau, Germany). Grids were glow-discharged for 45 seconds before application of 5 µL concentrated PIC-cMed sample, blotted for 5 seconds and vitrified by plunging into liquid ethane with a Vitrobot Mark IV (FEI Company, Hillsboro, US) operated at 4°C and 100% humidity. Cryo-EM data were acquired on a FEI Titan Krios G2 transmission electron microscope (FEI, Hillsboro, USA) operated in EFTEM mode at 300 kV and equipped with a K2 Summit direct detector (Gatan, Pleasanton, USA). Automated data acquisition was carried out using the FEI EPU software package at a nominal magnification of 105,000x (1.37 Å/pix). A total of 14,000 image stacks were collected at a defocus range from -0.5 µM to -5.0 µM. Each stack contained 40 frames that were acquired over a 10 seconds exposure time window in the counting mode of the camera. A dose rate of 4.2 e-/Å2s was applied, resulting in a total dose of 42 e-/Å2.
Schilbach S., Hantsche M., Tegunov D., Dienemann C., Wigge C., Urlaub H, & Cramer P. (2017). Structures of transcription pre-initiation complex with TFIIH and Mediator. Nature, 551(7679), 204-209.
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3

Cryo-EM Imaging of CIV Complexes

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GDN-purified CIV (3 µl) at a concentration of ~4 mg ml−1 was applied to holey carbon film coated copper EM grids (C flat 2/2 3 C T50) that had been glow-discharged in air (120 s, 20 mA using PELCO easiGlow). Grids were blotted for 3 s at 4 °C and 100 % humidity before rapid freezing in liquid ethane with a Vitrobot Mark IV (Thermo Fisher Scientific). Cryo-EM data were collected using a Titan Krios G3 electron microscope (Thermo Fisher Scientific) operated at 300 kV, equipped with a Gatan BioQuantum energy filter and a K3 Summit direct electron detector (AMETEK). Automated data collection was done with the EPU software package (Thermo Fisher Scientific). A dataset of 9289 movies, each consisting of 40 exposure fractions was collected at a nominal magnification of 105,000x, corresponding to a calibrated pixel size of 0.86 Å. The camera exposure rate and the total exposure of the specimen were 13.0 e− pixel−1 s−1 and ~49 e− Å−2, respectively (Table 2).
Moe A., Ädelroth P., Brzezinski P, & Näsvik Öjemyr L. (2023). Cryo-EM structure and function of S. pombe complex IV with bound respiratory supercomplex factor. Communications Chemistry, 6, 32.
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4

Cryo-EM Grid Preparation Protocol

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Glow-discharging of holey carbon grids (Cu R2/1; 200 mesh; Quantifoil) was conducted for 20 s under oxygen atmosphere using a Solarus 950 plasma cleaner (Gatan). Using a Vitrobot Mark IV (Thermo Fisher), EM-grids were prepared at ambient temperature and humidity by applying 4 μl of sample onto the grids, blotting with Whatman filter paper Nr.1 for 3 s (blot force = 0), and plunging them into liquid ethane cooled by liquid nitrogen.
Two datasets were acquired using the EPU software package (Thermo Fisher) on a Titan Krios TEM (Thermo Fisher/FEI). Data was acquired in dose fractionation mode at 300 kV using an energy-filtered K3 camera (Gatan) operated by the Gatan Microscopy Suite (version 3.32). The defocus range was set to −0.5 μm to −1.5 μm at an object pixel size of 1.07 Å/px. Micrograph movie stacks contained 30 frames with a cumulative dose of 48 e/A2 and 45.4 e/A2 for the first and second dataset, respectively. In each selected hole, micrograph movies stacks were acquired at four pre-defined positions after the defocus was automatically adjusted.
Cerullo F., Filbeck S., Patil P.R., Hung H.C., Xu H., Vornberger J., Hofer F.W., Schmitt J., Kramer G., Bukau B., Hofmann K., Pfeffer S, & Joazeiro C.A. (2022). Bacterial ribosome collision sensing by a MutS DNA repair ATPase paralog. Nature, 603(7901), 509-514.
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5

Cryo-EM Structural Determination of Respiratory Supercomplexes

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Purified supercomplexes (3 µL) at a concentration of ~8 mg mL–1, supplemented with S. cerevisiae cyt. c (Sigma-Aldrich; aerobically grown S. cerevisiae contains 95% isoform-1 cyt. c) (83 (link)) at a ∼1:12 molar ratio, was applied to holey carbon film coated copper EM grids (C flat 2/2 3C T50) that had been glow-discharged in air (120 s, 20 mA using PELCO easiGlow). Grids were blotted for 3 s at 4 °C and 100% humidity before rapid freezing in liquid ethane with a Vitrobot Mark IV (Thermo Fisher Scientific). Cryo-EM data were collected using a Titan Krios G3 electron microscope (Thermo Fisher Scientific) operated at 300 kV, equipped with a Gatan BioQuantum energy filter and a K3 Summit direct electron detector (AMETEK). Automated data collection was done with the EPU software package (Thermo Fisher Scientific). A dataset of 10,347 movies, each consisting of 40 exposure fractions was collected at a nominal magnification of 105,000×, corresponding to a calibrated pixel size of 0.85 Å. The camera exposure rate and the total exposure of the specimen were 15.2 e–/pixel/s and ~41 e–/Å2, respectively (SI Appendix, Table S1).
Moe A., Dimogkioka A.R., Rapaport D., Öjemyr L.N, & Brzezinski P. (2023). Structure and function of the S. pombe III–IV–cyt c supercomplex. Proceedings of the National Academy of Sciences of the United States of America, 120(46), e2307697120.
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6

Cryo-EM Imaging of Biomolecular Complexes

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Individual cryo-EM images were collected on a Titan Krios G3 transmission electron microscope (Thermo Fisher Scientific) operated at 300 kV with a K2 Summit direct electron detector and Bioquantum energy filter (Gatan). All images were collected using the EPU software package (Thermo Fisher Scientific) with an applied defocus range of -3.5 µM and a magnified pixel size of 1.059 Å for the largest magnification of 130000x. The lower magnification grid overview and grid square images were collected at 3500- and 8000-fold magnification, respectively.
Hohle M.M., Lammens K., Gut F., Wang B., Kahler S., Kugler K., Till M., Beckmann R., Hopfner K.P, & Jung C. (2022). Ice thickness monitoring for cryo-EM grids by interferometry imaging. Scientific Reports, 12, 15330.
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7

Cryo-EM Imaging of DpK2 Protein Complex

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Purified DpK2 protein samples were diluted to 0.4 mg/ml in TBS and prepared for cryo-electron microscopy (cryo-EM) as described previously (73 (link)). Data were collected on a Glacios microscope (Thermo Fisher Scientific) operated at an accelerating voltage of 200 kV with a 70-μm C2 aperture at an indicated magnification of ×120,000 in nanoprobe energy-filtered TEM (EFTEM) mode, spot size 4. A Falcon 3 direct electron detector was used to acquire dose-fractionated images of the DpK2 complex. Movies were recorded as uncompressed gain-normalized .MRC files yielding a physical pixel size of 0.895 Å/pixel. An exposure time of 40 s amounting to a total dose of 43 e-/Å2 was fractionated into 38 subframes. Defocus was varied in the range between −0.4 to −1.5 μm. Beam-image shift was used to acquire data from 9 surrounding holes, after which the stage was moved to the next collection area using the EPU software package (Thermo Fisher Scientific).
Dunstan R.A., Bamert R.S., Belousoff M.J., Short F.L., Barlow C.K., Pickard D.J., Wilksch J.J., Schittenhelm R.B., Strugnell R.A., Dougan G, & Lithgow T. (2021). Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage. Microbiology Spectrum, 9(1), 10.1128/spectrum.01023-21.
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8

High-Resolution Cryo-EM Imaging Protocol

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Three datasets were collected on the same FEI Titan Krios microscope (LMB Krios1), operating at 300 keV with the specimen at cryogenic temperatures (approximately −180°C), with images recorded at a defocus of between −1.5 and −3.5 μm. A total of 4,923 movies were acquired across two collections using the K2 Summit direct electron detector (Gatan) in electron counting mode with a GIF Quantum energy filter slit width of 20 eV, using a calibrated pixel size of 1.145 Å/pixel. These data were collected using the EPU software package (ThermoFisher) and the dose fractionated into 40 frames per movie, with an exposure time of 10 s to achieve a total dose of 39.8 e‐/Å2. An additional 2,400 movies were acquired using the Falcon III direct electron detector (ThermoFisher) in electron counting mode using a calibrated pixel size of 1.07 Å/pixel. 75 movie frames were recorded over 60 s to give a total dose of 37.5 e−/Å2.
Jones M.L., Baris Y., Taylor M.R, & Yeeles J.T. (2021). Structure of a human replisome shows the organisation and interactions of a DNA replication machine. The EMBO Journal, 40(23), e108819.
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9

Cryo-EM Imaging of DpK2 Protein Complex

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Purified DpK2 protein samples were diluted to 0.4 mg/ml in TBS and prepared for cryo-electron microscopy (cryo-EM) as described previously (73 (link)). Data were collected on a Glacios microscope (Thermo Fisher Scientific) operated at an accelerating voltage of 200 kV with a 70-μm C2 aperture at an indicated magnification of ×120,000 in nanoprobe energy-filtered TEM (EFTEM) mode, spot size 4. A Falcon 3 direct electron detector was used to acquire dose-fractionated images of the DpK2 complex. Movies were recorded as uncompressed gain-normalized .MRC files yielding a physical pixel size of 0.895 Å/pixel. An exposure time of 40 s amounting to a total dose of 43 e-/Å2 was fractionated into 38 subframes. Defocus was varied in the range between −0.4 to −1.5 μm. Beam-image shift was used to acquire data from 9 surrounding holes, after which the stage was moved to the next collection area using the EPU software package (Thermo Fisher Scientific).
Dunstan R.A., Bamert R.S., Belousoff M.J., Short F.L., Barlow C.K., Pickard D.J., Wilksch J.J., Schittenhelm R.B., Strugnell R.A., Dougan G, & Lithgow T. (2021). Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage. Microbiology Spectrum, 9(1), 10.1128/spectrum.01023-21.
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10

Cryo-EM Protocol for LIN28B-Nucleosome Complexes

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Cryo-EM grids were prepared using a 0.5mg/ml sample of LIN28B-nucleosome core particles (4μL) applied onto 200 mesh Quantifoil grids with 2μm holes and 2μm spacing (Quantifoil Micro Tools GrmbH). The sample was blotted using a Vitrobot Mark IV (Thermo Fisher Scientific) operated at 4°C with 2.5s blot time, -1 blot force, 10s wait time and 100% humidity before plunge freezing in liquid ethane. The frozen grids were stored in liquid nitrogen until imaging.
Cryo-EM data collection was performed at Birkbeck, University of London using a Titan Krios electron microscope (Thermo Fisher Scientific) operating at 300keV and equipped with a BioQuantum K3 energy filter (20eV slit width) and K3 summit direct electron detector. Data were collected using the K3 in super resolution mode at a nominal magnification of 130kx (0.335 Å per super resolution pixel). Movies composed of 50 frames (0.03s/frame) were collected at a dose rate of 16.814 e-/pixel/sec corresponding to a total accumulated dose of 56.7 e-/Å2. A dataset of 12892 movies was collected using defocus values ranging from -1.5 to -3.3μm with 6x exposures per hole (0.8μm illumination area) using the EPU software package (Thermo Fisher Scientific). Cryo-EM data collection statistics are summarized in Extended Data Fig. 4a. Cryo-EM data processing and model building are indicated in the Supplementary protocols.
Roberts G.A., Ozkan B., Gachulincová I., O’Dwyer M.R., Hall-Ponsele E., Saxena M., Robinson P.J, & Soufi A. (2021). Dissecting OCT4 defines the role of nucleosome binding in pluripotency. Nature cell biology, 23(8), 834-845.
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