Epu 2

Manufactured by Thermo Fisher Scientific

Sourced in United States

EPU 2 software is a data collection and processing platform for transmission electron microscopy (TEM) instruments. It provides an intuitive user interface for automated data acquisition and processing workflows. The software's core function is to facilitate the efficient collection and management of high-quality TEM images and data.

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Lab products found in correlation

25 protocols using epu 2

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 Analysis of Murine GLB1-CTSA Complex

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Concentrated murine GLB1 and CTSA were mixed in a 1:2 molar ratio—the initially presumed stoichiometry of the complex—and diluted to 0.5 mg/ml total with buffer [50 mM sodium acetate (pH 4.5) and 50 mM NaCl]. After 1 hour at 4°C, 4 μl of sample was applied to a glow-discharged nanofabricated gold-coated copper-rhodium grid with 2.5-μm holes (59 (link)). The grid was frozen in liquid ethane at liquid nitrogen temperature using a Vitrobot Mark IV (FEI) at 4°C and 100% humidity with 1.5-s blotting. Grids were screened for ice thickness and particle distribution on a Talos L120C electron microscope equipped with a lanthanum hexaboride (LaB₆) emitter and operating at 120 kV. Images were collected at a nominal magnification of 57,000 using a charge-coupled detector camera (BM-Ceta) with a magnified pixel size of 2.45 Å. Data were collected on a Titan Krios G3 electron microscope with a Falcon 4 detector using EPU 2 (Thermo Fisher Scientific) (table S1). The sample exhibited preferential orientation in the thin layer of vitreous ice (first three classes in fig. S1B), so a 40° tilted dataset was collected in addition to the nontilted one. Optimal ice thickness was found in the center of each hole, so only one image per hole was recorded.

Gorelik A., Illes K., Hasan S.M., Nagar B, & Mazhab-Jafari M.T. (2021). Structure of the murine lysosomal multienzyme complex core. Science Advances, 7(20), eabf4155.

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Cryo-EM Structure of Sr35-AvrSr35 Complex

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The Sr35–AvrSr35 complex grids were prepared for cryo-EM analysis. Holy carbon grids (Quantifoil Au 1.2/1.3, 300 mesh) were glow-discharged for 30 s at medium level in HarrickPlasma after 2 min evacuation. The purified Sr35–AvrSr35 protein was concentrated to approximately 0.5 mg ml^–1 and 3 µl of sample were applied to the grid. The grids were blotted for 2–3 s using a pair of filter papers (55 mm, Ted Pella Inc.) at 8 °C with 100% humidity and flash-frozen in liquid ethane using a FEI Vitrobot Marked IV. Stacks of Sr35–AvrSr35 cryo-EM samples were collected by a Titan Krios microscope operated at 300 kV, equipped with a K3 Summit direct electron detection camera (Gatan) using EPU 2 (Thermo Fisher Scientific, 2.8.1.10REL) at Zhengzhou University. Micrographs were recorded at 81,000× magnification corresponding to 1.1 Å per pixel. The defocus ranged from −1.5 µm to −2.0 µm. Each image stack contains 32 frames recorded every 0.11 s for an accumulated dose of approximately 50 e− per Å² and a total exposure time of 3.5 s. A second dataset from an independent protein purification was recorded at EMBL Heidelberg with the following parameters: Titan Krios microscope operated at 300 kV, equipped with a K3 Summit direct electron detection camera (Gatan), 50 e per Å², 40 frames per stack.

Förderer A., Li E., Lawson A.W., Deng Y.N., Sun Y., Logemann E., Zhang X., Wen J., Han Z., Chang J., Chen Y., Schulze-Lefert P, & Chai J. (2022). A wheat resistosome defines common principles of immune receptor channels. Nature, 610(7932), 532-539.

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Cryo-EM Structure of C5-UNbC5 Complex

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C5, purchased from Complement Technologies, was diluted to a final concentration of 1 μM and gently mixed with a 1.5 × molar excess of UNbC5-1 and UNbC5-2. The sample was diluted in PBS and incubated on ice for 20 min before freezing on glow discharged R1.2/1.3200 mesh Au holey carbon grids (Quantifoil). The grids were then plunge frozen in ethane using a Vitrobot Mark IV (Thermo Fisher Scientific), at 4 °C. Cryo-EM data were collected on a 200 kV Talos Arctica microscope (Thermo Fisher Scientific) equipped with a K2 summit detector (Gatan) and a post column 20 eV energy filter. Movies were collected in EPU 2 (Thermo Fisher Scientific, https://www.thermofisher.com/nl/en/home/electron-microscopy/products/software-em-3d-vis/epu-software.html) at a magnification of 130,000× with a pixel size of 1.04 Å/pix. Two datasets were collected in two collection sessions with similar settings of 40 frames with a total exposure of 54 and 57 e−/Å², and a defocus range of −0.8 to 2.6 μm.

Struijf E.M., De la O Becerra K.I., Ruyken M., de Haas C.J., van Oosterom F., Siere D.Y., van Keulen J.E., Heesterbeek D.A., Dolk E., Heukers R., Bardoel B.W., Gros P, & Rooijakkers S.H. (2023). Inhibition of cleavage of human complement component C5 and the R885H C5 variant by two distinct high affinity anti-C5 nanobodies. The Journal of Biological Chemistry, 299(8), 104956.

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Cryo-EM of Cytochrome bc-aa3 Supercomplex

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About 3 µl of purified supercomplex at a concentration of 10 mg ml⁻¹ with 20 µM stigmatellin (SMA) (Fluka 85865) and 2.5 mM sodium azide (AZI), was applied to a glow-discharged C-flat CF-2/2 Au-50 300 mesh holey-carbon grid. Subsequently, grids were plunge-frozen as for the native sample. Image data were acquired using the aforementioned microscope operated at an acceleration voltage of 300 kV, equipped with an FEI Falcon3 direct electron detector camera. Automated data collection was carried out using EPU 2 (Thermo Fisher Scientific) with a magnification of 75,000 × (pixel size = 0.853 Å) at a dose rate of 1.1 e⁻Å⁻² s⁻¹. Six movies were taken per hole, and each movie had a total accumulated exposure of 40.0 e⁻Å⁻² fractioned into 30 frames. A dataset of 2833 movies was acquired for the stigmatellin-azide-treated cyt bcc–aa₃ supercomplex in a single session using a defocus range between −1.0 and −3.0 µm.

Kao W.C., Ortmann de Percin Northumberland C., Cheng T.C., Ortiz J., Durand A., von Loeffelholz O., Schilling O., Biniossek M.L., Klaholz B.P, & Hunte C. (2022). Structural basis for safe and efficient energy conversion in a respiratory supercomplex. Nature Communications, 13, 545.

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Cryo-EM Imaging of S-Fer and SΔC-Fer Particles

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Samples were diluted to a final concentration of ~0.4 mg/mL for both the S-Fer and SΔC-Fer particles after purification. Three microliters of the samples were applied onto glow-discharged 200-mesh R2/1 Quantifoil grids coated with continuous carbon. The grids were blotted for 2 s and rapidly cryocooled in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) at 4°C and 100% humidity. Samples were screened using a Talos Arctica cryo-electron microscope (Thermo Fisher Scientific) operated at 200 kV. The spikeΔC ferritin sample was imaged in a Titan Krios cryo-electron microscope (Thermo Fisher Scientific) operated at 300 kV with GIF energy filter (Gatan) at a magnification of 130,000× (corresponding to a calibrated sampling of 1.06 Å per pixel). Micrographs were recorded with EPU 2.6 (Thermo Fisher Scientific) with a Gatan K2 Summit direct electron detector; each image was composed of 30 individual frames with an exposure time of 6 s and an exposure rate of 7.8 electrons per second per Å². A total of 3,684 movie stacks was collected.

Powell A.E., Zhang K., Sanyal M., Tang S., Weidenbacher P.A., Li S., Pham T.D., Pak J.E., Chiu W, & Kim P.S. (2020). A single immunization with spike-functionalized ferritin vaccines elicits neutralizing antibody responses against SARS-CoV-2 in mice. bioRxiv.

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Cryo-EM Analysis of Spike-Ferritin Particles

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Samples were diluted to a
final concentration of ∼0.4 mg/mL for both the S-Fer and SΔC-Fer
particles after purification. The samples (3 μL) were applied
onto glow-discharged 200-mesh R2/1 Quantifoil grids coated with continuous
carbon. The grids were blotted for 2 s and rapidly cryocooled in liquid
ethane using a Vitrobot Mark IV instrument (Thermo Fisher Scientific)
at 4 °C and 100% humidity. Samples were screened using a Talos
Arctica cryoelectron microscope (Thermo Fisher Scientific) operated
at 200 kV. The spikeΔC ferritin sample was imaged in a Titan
Krios cryoelectron microscope (Thermo Fisher Scientific) operated
at 300 kV with GIF energy filter (Gatan) at a magnification of 130 000×
(corresponding to a calibrated sampling of 1.06 Å per pixel).
Micrographs were recorded with EPU 2.6 (Thermo Fisher Scientific)
with a Gatan K2 Summit direct electron detector; each image was composed
of 30 individual frames with an exposure time of 6 s and an exposure
rate of 7.8 electrons per second per Å². A total of
3684 movie stacks was collected.

Powell A.E., Zhang K., Sanyal M., Tang S., Weidenbacher P.A., Li S., Pham T.D., Pak J.E., Chiu W, & Kim P.S. (2021). A Single Immunization with Spike-Functionalized Ferritin Vaccines Elicits Neutralizing Antibody Responses against SARS-CoV-2 in Mice. ACS Central Science, 7(1), 183-199.

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Cryo-EM of ATTR Fibril Structure

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For cryo-EM, aliquots of 4.0 μM of purified ATTR fibrils were applied to glow-discharged holey carbon grids (Quantifoil Cu R1.2/1.3, 200 mesh), blotted with filter paper, and plunge-frozen in liquid ethane using an FEI Vitrobot Mark IV. The cryo-EM images were collected on a Thermo Fisher Titan Krios microscope operated at 300 kV at a magnification of ×130,000 (1.041 Å/pixel) equipped with a BioQuantum-filtered (20 eV slit width) Gatan K2 Summit detector. Images were collected as 20-frame movies for a total accumulated dose of 28.4 electrons per Å². In total 17,863 images were recorded during two separate data collections using the EPU 2.7.0 software (Thermo Fisher) with defocus values ranging from –0.7 to –1.6 μm and in steps of 0.3 μm in both datasets.

Iakovleva I., Hall M., Oelker M., Sandblad L., Anan I, & Sauer-Eriksson A.E. (2021). Structural basis for transthyretin amyloid formation in vitreous body of the eye. Nature Communications, 12, 7141.

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Cryo-EM data collection on Titan Krios

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The cryo-EM data set is automatically collected by EPU-2.7.0 software (Thermo Fisher Scientific) on a 300 kV Titan Krios (Thermo Fisher Scientific) equipped with X-FEG electron source. The data were collected by a K3 Summit detector (with GIF Bio-Quantum Energy Filters, Gatan) operating in super-resolution mode. The raw movie stacks were recorded at a nominal magnification of 81,000×, corresponding to a pixel size of 1.061 Å/pixel (super resolution 0.5305 Å/pixel). The defocus range was set to −1.5 to −2.25 μm and the slit width of Energy Filters was set to 20 eV. Forty frames of non-gain normalized tiff stacks were recorded with a dose rate of ~23 e⁻/Å² per second and the total exposure time was set to 2.15 s, resulting in an accumulated dose of ~50 e⁻/Å² (~1.25 e⁻/Å² per frame).

Chiu Y.C., Yeh M.C., Wang C.H., Chen Y.A., Chang H., Lin H.Y., Ho M.C, & Lin S.M. (2023). Structural basis for calcium-stimulating pore formation of Vibrio α-hemolysin. Nature Communications, 14, 5946.

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Cryo-EM Data Acquisition with Titan Krios

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Cryo-EM grids were first checked on a Talos transmission electron microscope equipped with a Falcon III detector (Thermo Fisher Scientific) operated in linear mode. The images were recorded at a nominal magnification of 120,000x, corresponding to a pixel size of 0.86 Å/pixel, with a defocus setting of -3.0 μm. Suitable cryo-EM grids were recovered for further data collection on a 300 kV Titan Krios transmission electron microscope hosting a K3 detector (with GIF Bio-Quantum Energy Filters, Gatan) operating in super-resolution mode and using EPU-2.7.0 software (Thermo Fisher Scientific). The raw movie stacks were recorded at a magnification of 105,000×, corresponding to a pixel size of 0.83 Å/pixel (super-resolution 0.415 Å/pixel). The defocus range was set to −1.5 to -2.5 μm and the slit width of energy filters was set to 20 eV. Forty frames of non-gain-normalized tiff stacks were recorded with a dose rate of ~16 e-/Å2 per second and the total exposure time was set to 2.5 s, resulting in an accumulated dose of ~40 e-/Å2 (~1.0 e-/Å2 per frame). The parameters for cryo-EM data acquisition are summarized in Table 1.

Liao C.C., Wang Y.S., Pi W.C., Wang C.H., Wu Y.M., Chen W.Y, & Hsia K.C. (2023). Structural convergence endows nuclear transport receptor Kap114p with a transcriptional repressor function toward TATA-binding protein. Nature Communications, 14, 5518.

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