Em gp plunge freezer

Manufactured by Leica

Sourced in Germany

The EM GP plunge freezer is a cryogenic instrument used for the rapid freezing of biological specimens. It is designed to rapidly cool samples to cryogenic temperatures, typically liquid nitrogen, in order to preserve their structural integrity for subsequent analysis using electron microscopy.

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32 protocols using em gp plunge freezer

Cryo-EM Sample Preparation of SLFN11 Proteins

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Purified SLFN11^wt was dialyzed into dialysis buffer (20 mM HEPES pH 7.5, 60 mM NaCl, 2 mM MgCl₂, 1 mM DTT) and pre-incubated for 30 min with brewer′s yeast tRNAs mixture (Sigma-Aldrich). The sample was diluted in cryo-EM buffer (20 mM HEPES pH 7.5, 2 mM MgCl₂, 1 mM DTT) to a final concentration of 3.5 μM SLFN11 and 10 μM tRNA mixture. 4.5 μl was applied onto a glow discharged QUANTIFOIL® R2/1 + 2 nm carbon Cu200 grid. The sample was vitrified in liquid ethane using an EM GP plunge freezer (Leica, 10 °C and 90% humidity).
Freshly purified SLFN11^E209A was diluted in cryo-EM buffer (50 mM Glycine pH 9, 200 mM NaCl, 2 mM MgCl₂, 1 mM DTT) to a final concentration of 5 μM. 4.5 μl was applied onto a glow discharged QUANTIFOIL® R2/1 Cu200 grid. The sample was vitrified in liquid ethane using an EM GP plunge freezer (Leica, 10 °C and 90% humidity).
Freshly purified SLFN11^wt was pre-incubated for 30 min with 60 nt ssDNA and 1 mM ADP. The sample was diluted in cryo-EM buffer (100 mM Glycine pH 9, 2 mM MgCl₂, 1 mM DTT) to a final concentration of 5 μM SLFN11, 2 μM ssDNA, and 1 mM ADP. 4.5 μl was applied onto a glow discharged QUANTIFOIL® R2/1 Cu200 grid. The sample was vitrified in liquid ethane using an EM GP plunge freezer (Leica, 10 °C and 90% humidity).

Metzner F.J., Wenzl S.J., Kugler M., Krebs S., Hopfner K.P, & Lammens K. (2022). Mechanistic understanding of human SLFN11. Nature Communications, 13, 5464.

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Cryo-EM Imaging of Polymerized Tubulin

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Molybdenum R1.2/1.3 and R2/2 200-mesh grids with a holey carbon support film (Quantifoil, Jena, DE) were glow discharged for 40 s under high vacuum shortly before sample application. OdinTubulin protein (40 to 60 μM) was polymerized for 10 to 20 min at 37° and 80°C using an adapted protocol for eukaryotic tubulin (38 (link)) by adding temperature-equilibrated K-Pipes buffer and 2 mM GTP (Sigma-Aldrich). The polymerized sample (2.5 μl) was applied to the glow-discharged grids, blotted for 0.5 to 1.5 s, and plunge frozen with an EM GP plunge freezer (Leica Microsystems) operated at room temperature at 90% humidity.

Akıl C., Ali S., Tran L.T., Gaillard J., Li W., Hayashida K., Hirose M., Kato T., Oshima A., Fujishima K., Blanchoin L., Narita A, & Robinson R.C. (2022). Structure and dynamics of Odinarchaeota tubulin and the implications for eukaryotic microtubule evolution. Science Advances, 8(12), eabm2225.

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Cryo-EM Preparation of OdinTubulin Filaments

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Negatively stained GTP-induced tubules were observed using a Hitachi-H7600 transmission electron microscope (Institute for Advanced Research, Nagoya University). Thawed OdinTubulin protein was diluted to final concentration of 40-60 µM in pre-warmed polymerization K-PIPES buffer as previously described for eukaryotic tubulin (35) and polymerized for 10-20 min using 2 mM GTP (Sigma-Aldrich). 2.5 µl of the mixture was applied onto glow discharged grid STEM100Cu elastic carbon grids (Ohkenshoji co., Ltd) and absorbed for 1 min. The sample was blotted with filter paper and then 2% uranyl acetate solution (5 µl) was applied. After 1 min, the grids were blotted again and allowed to dry overnight. Cryo-EM grid preparation Molybdenum R1.2/1.3 and R2/2 200-mesh grids with a holey carbon support film (Quantifoil, Jena, DE) were glow discharged for 40 s under high vacuum shortly before sample application. OdinTubulin protein (40-60 µM) was polymerized for 10-20 minutes at 37 ˚C and 80 ˚C using an adapted protocol for eukaryotic tubulin (35) by adding temperature equilibrated K-PIPES buffer and 2 mM GTP (Sigma-Aldrich). 2.5 µL of the polymerized sample was applied to the glowdischarged grids, blotted for 0.5-1.5 s and plunge-frozen with an EM GP plunge freezer (Leica Microsystems) operated at room temperature at 90% humidity.

Akıl C., Ali S., Tran L., Gaillard J., Li W., Hayashida K., Hirose M., Kato T., Oshima A., Fujishima K., Blanchoin L., Narita A., & Robinson R. (2021). Structure and dynamics of Odinarchaeota tubulin and the implications for eukaryotic microtubule evolution.

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Cryo-EM Imaging of C. burnetii OM Vesicles

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Stationary phase C. burnetii samples were frozen on a glow discharged Quantifoil™ R 2/2 grid using a Leica EM GP plunge freezer (Leica Microsystems, Wetzlar, Germany) set to a relative humidity of 100%. Grids were mounted in an autogrid assembly and cryo 2D images collected using an FEI Titan Krios 300kV FEG-TEM and an FEI Falcon II CMOS direct electron detection camera. Low dose images of 13e/A² were captured at 6 μm defocus with a nominal magnification of 29 K which corresponds to a pixel size of 2.87 Å using a 70 μm objective aperture. OM vesiculation was quantified by imaging ≥50 bacterial cells per strain and is expressed as the percentage of bacteria with vesicles clearly attached to the bacterial OM.

Sandoz K.M., Moore R.A., Beare P.A., Patel A.V., Smith R.E., Bern M., Hwang H., Cooper C.J., Priola S.A., Parks J.M., Gumbart J.C., Mesnage S, & Heinzen R.A. (2020). β-Barrel Proteins Tether the Outer Membrane in Many Gram-Negative Bacteria. Nature microbiology, 6(1), 19-26.

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Cryo-EM Imaging of 4AuscFvpolII Tetramers

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A 3-μl drop of purified 4AuscFvpolII was applied to a glow-discharged, 400-mesh, copper R2/2 Quantifoil grids (Quantifoil Micro Tools GmbH, Jena, Germany). Grids were mounted on a Leica EM GP Plunge Freezer (Leica Microsystems, Wetzlar, Germany) and were blotted using five seconds of pre-blotting time, four seconds of blotting time and one second post-blotting time. Both blotting, and plunge-freezing in liquid ethane (−178°C) were performed at 22°C and 90% humidity.
Tilt-pair images were collected on a Thermo Scientific™ Tecnai F20 FEG transmission electron microscope operating at 200 kV. Images were recorded using a 4k x 4k K2 Summit direct detector (Gatan, Inc.) operating in the electron counting mode. Tilt pairs at 0° and −45° were recorded using the SerialEM (Mastronarde, 2005) software package at a magnification corresponding to a pixel size of 1.218 Å, at about 1.5 μm underfocus and total electron dose of 40 e⁻/Å². Four AuNPs grouped together were assigned as a polII tetrameric particle. Tilt-pair particles were boxed out using EMAN software package (Tang et al., 2007 (link)).

Jagota M., Townshend R.J., Kang L.W., Bushnell D.A., Dror R.O., Kornberg R.D, & Azubel M. (2021). Gold nanoparticles and tilt pairs to assess protein flexibility by cryo-electron microscopy. Ultramicroscopy, 227, 113302.

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Cryo-EM Preparation of Mitotic Chromosomes

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Purified HeLa mitotic chromosomes were diluted 100-fold in an aqueous solution containing 5 mM Tris-HCl (pH 7.5) and 2 mM KCl. The resulting sample, which contained swollen chromosomes, was mixed with 10 nm BSA Gold Tracer (EMS, Haltfield, PA, USA) at a 5:1 ratio (chromosome:gold), and a volume of 3 μL (containing 4×10⁻⁴A₂₆₀ units of mitotic chromosomes) was applied to glow-discharged, 200-mesh, gold R2/2 Quantifoil grids (Quantifoil Micro Tools GmbH, Jena, Germany). Grids were mounted in a Leica EM GP Plunge Freezer (Leica Microsystems, Wetzlar, Germany) and were blotted from the reverse side using two seconds of pre-blotting time, two seconds of blotting time, and no post-blotting time. Both blotting and plunge-freezing in liquid ethane (−178 °C) were performed at 22 °C and 90% relative humidity.

Beel A.J., Azubel M., Matteï P.J, & Kornberg R.D. (2021). Structure of Mitotic Chromosomes. Molecular cell, 81(21), 4369-4376.e3.

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Cryo-EM of Purified BAM Complex

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Purified BAM complex in TBS with 0.05% (w/v) n-dodecyl β-D-maltopyranoside (DDM) at a concentration of 13 mg ml⁻¹ was diluted 1:2 in the same buffer. A 4 μl aliquot of solution was applied to a quantifoil r3.5/1 EM grid (Quantifoil Micro Tools), allowed to incubate at room temperature and 95% humidity for 30 s, then manually blotted with a Whatman filter paper. A second 4 μl aliquot of BAM solution was added and the grid blotted and vitrified by plunging into liquid ethane using a Leica EM GP plunge freezer (Leica Microsystems).
The grids were imaged on a Titan Krios electron microscope (FEI Corporation) operating at 300 kV. Seven thousand and five hundred micrographs with a nominal defocus range of 1.5–3.5 μM were recorded with a Gatan K2 Summit energy-filtered direct detector (Gatan, Inc.) as 20 frame movies with a total electron dose of ∼40 e⁻ Å⁻². The sampling rate was 1.04 Å per pixel.

Iadanza M.G., Higgins A.J., Schiffrin B., Calabrese A.N., Brockwell D.J., Ashcroft A.E., Radford S.E, & Ranson N.A. (2016). Lateral opening in the intact β-barrel assembly machinery captured by cryo-EM. Nature Communications, 7, 12865.

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Isolating Mature 80S Ribosomes from HEK Expi293F Cells

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Mature 80S ribosomes were isolated using two different protocols from the HEK Expi293F cells as described elsewhere with modifications (Khatter et al. 2014 (link), 2015 (link); Bhaskar et al. 2020 (link)). The ribosomal pellet obtained in the final step was then resuspended in resuspension buffer (50 mM HEPES 7.6, 80 mM Potassium acetate, 5 mM Magnesium chloride, and 1 mM DTT) to obtain a final concentration of ribosomes at 0.5–0.7 mg/mL. A total of 4 µL of the sample was applied on the Quantifoil R2/2 Cu 300 mesh grids (Quantifoil, Micro Tools GmbH). The grids were blotted for 3 sec at 4°C and 80% humidity and subsequently plunge frozen in liquid ethane using Leica EM GP plunge freezer (Leica Microsystems). Alternatively, 4 µL of the sample was applied on the Quantifoil R2/2 Cu 300 mesh grids coated with 3 nm continuous carbon (Quantifoil, Micro Tools GmbH). The grids were blotted for 3 sec at 4°C and 80% humidity and subsequently plunge frozen in liquid ethane using Vitrobot (Thermo Fisher Scientific).

Bhaskar V., Desogus J., Graff-Meyer A., Schenk A.D., Cavadini S, & Chao J.A. (2021). Dynamic association of human Ebp1 with the ribosome. RNA, 27(4), 411-419.

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Cryo-EM Imaging of C. burnetii OM Vesicles

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Cryo-TEM Imaging of Brucella

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Overnight Brucella cultures were used to inoculate (at a starting OD₆₀₀ of ≈0.015) 2 ml of Brucella broth supplemented with or without 2 mM EDTA or ampicillin (5 μg ml⁻¹). After 4 hours of treatment at 37°C / 5% CO₂ / 220 rpm, cells were harvested by centrifugation (5000 x g / 10 min) and the pellets were resuspended in PBS + 4% formaldehyde to fix the cells. Cell killing was confirmed before sample removal for imaging. After 1 hour, cells were pelleted and resuspended in 500 μl EM buffer (20 mM Tris (pH 7.6), 50 mM glucose, 10 mM EDTA). Fixed Brucella cells were vitrified on glow-discharged 200 mesh copper EM-grids with extra thick R2/2 holey carbon film (Quantifoil). Per grid, 3 μl of the sample were applied and automatically blotted and plunged into liquid ethane with the Leica EM GP plunge-freezer. Images were collected on a Talos L120C TEM (Thermo Fischer) using the Gatan cryo-TEM (626) holder. The images were acquired at a defocus between 8–10 μm, with a pixel size of 0.458 nm.

Herrou J., Willett J.W., Fiebig A., Varesio L.M., Czyż D.M., Cheng J.X., Ultee E., Briegel A., Bigelow L., Babnigg G., Kim Y, & Crosson S. (2019). Periplasmic protein EipA determines envelope stress resistance and virulence in Brucella abortus. Molecular microbiology, 111(3), 637-661.

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