Ultraufoil r1.2 1 3 300 mesh grid, by Electron Microscopy Sciences - Product details

1

Negative Staining and Cryo-EM of TRPML3

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For negatively-stained TRPML3, 4 μL of amphipol-solubilized protein (0.04 mg/mL) was applied to a freshly glow-discharged 400 Cu-Rh Maxtaform grid (Electron Microscropy Services) that had been coated with a thin layer of carbon. After incubation on grid for ~90s, excess solution was wicked away using Whatman #1 filter paper and 5 μL of ~1% (w/v) uranyl formate solution was immediately applied directly to the grid. After ~10s, excess stain was wicked away and the grid surface was 3x repeated washed with 25 μL 1% (w/v) UF solution to yield thorough embedding of the sample. Following the final wash the grid was blotted to dryness and allowed to air-dry in a fume hood.
For cryo-EM, 3 uL of purified amphipol-solubilized TRPML3 (0.5 mg/ml) was applied to a freshly plasma cleaned (75% argon/25% oxygen atmosphere, 15 Watts for 6 seconds in a Gatan Solarus) UltrAuFoil® R1.2/1.3 300-mesh grid (Electron Microscopy Services) and manually blotted using filter paper (Whatman No.1) for ~4 seconds (≥95% relative humidity, 4°C) immediately prior to plunge freezing in liquid ethane cooled by liquid nitrogen.

Hirschi M., Herzik MA J.r., Wie J., Suo Y., Borschel W.F., Ren D., Lander G.C, & Lee S.Y. (2017). Cryo-EM structure of the lysosomal Ca2+-permeable channel TRPML3. Nature, 550(7676), 411-414.

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2

TRPV3 K169A Cryo-EM Grid Preparation

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Cryo-EM grid preparation was performed similarly for each TRPV3 K169A specimen. 3 μl sample was dispensed on a freshly glow discharged (30 s) UltrAuFoil R1.2/1.3 300-mesh grid (Electron Microscopy Services), blotted for 3 s with Whatman No. one filter paper using the Leica EM GP2 Automatic Plunge Freezer at 23° C and >85% humidity and plunge-frozen in liquid ethane cooled by liquid nitrogen.

Zubcevic L., Borschel W.F., Hsu A.L., Borgnia M.J, & Lee S.Y. (2019). Regulatory switch at the cytoplasmic interface controls TRPV channel gating. eLife, 8, e47746.

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3

Cryo-EM Specimen Preparation for TRPV3

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Cryo-EM grid preparation was performed similarly for each TRPV3 specimen. Briefly, 3 μl of purified TRPV3 (0.35–0.5 mg ml⁻¹) was dispensed on a freshly plasma cleaned (Gatan Solarus) UltrAuFoil R1.2/1.3 300-mesh grid (Electron Microscopy Services) and manually blotted^{50 (link)} using a custom-built manual plunger housed in a 4 °C cold room (>85% relative humidity). Samples were blotted for 4–5 s with Whatman No. 1 filter paper immediately before plunge-freezing in liquid ethane cooled by liquid nitrogen.

Zubcevic L., Herzik MA J.r., Wu M., Borschel W.F., Hirschi M., Song A.S., Lander G.C, & Lee S.Y. (2018). Conformational ensemble of the human TRPV3 ion channel. Nature Communications, 9, 4773.

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4

Negative Staining and Cryo-EM of TRPML3

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For negatively-stained TRPML3, 4 μL of amphipol-solubilized protein (0.04 mg/mL) was applied to a freshly glow-discharged 400 Cu-Rh Maxtaform grid (Electron Microscropy Services) that had been coated with a thin layer of carbon. After incubation on grid for ~90s, excess solution was wicked away using Whatman #1 filter paper and 5 μL of ~1% (w/v) uranyl formate solution was immediately applied directly to the grid. After ~10s, excess stain was wicked away and the grid surface was 3x repeated washed with 25 μL 1% (w/v) UF solution to yield thorough embedding of the sample. Following the final wash the grid was blotted to dryness and allowed to air-dry in a fume hood.
For cryo-EM, 3 uL of purified amphipol-solubilized TRPML3 (0.5 mg/ml) was applied to a freshly plasma cleaned (75% argon/25% oxygen atmosphere, 15 Watts for 6 seconds in a Gatan Solarus) UltrAuFoil® R1.2/1.3 300-mesh grid (Electron Microscopy Services) and manually blotted using filter paper (Whatman No.1) for ~4 seconds (≥95% relative humidity, 4°C) immediately prior to plunge freezing in liquid ethane cooled by liquid nitrogen.

Hirschi M., Herzik MA J.r., Wie J., Suo Y., Borschel W.F., Ren D., Lander G.C, & Lee S.Y. (2017). Cryo-EM structure of the lysosomal Ca2+-permeable channel TRPML3. Nature, 550(7676), 411-414.

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5

TRPV2 Protein Cryo-EM Sample Preparation

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TRPV2_RTx-APOL and TRPV2_RTx-ND were frozen using the same protocol. Before freezing, the concentrated protein sample was supplemented with 300 μM RTx and incubated ~30 min at 4°C. 3 μl sample was dispensed on a freshly glow discharged (30 s) UltrAuFoil R1.2/1.3 300-mesh grid (Electron Microscopy Sciences), blotted for 3 s with Whatman No. one filter paper using the Leica EM GP2 Automatic Plunge Freezer at 23°C and >85% humidity and plunge-frozen in liquid ethane cooled by liquid nitrogen.

Zubcevic L., Hsu A.L., Borgnia M.J, & Lee S.Y. (2019). Symmetry transitions during gating of the TRPV2 ion channel in lipid membranes. eLife, 8, e45779.

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6

Cryo-EM Preparation of Rabbit Muscle Aldolase

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Pure aldolase isolated from rabbit muscle was purchased as a lyophilized powder (Sigma Aldrich) and solubilized in 20 mM HEPES (pH 7.5), 50 mM NaCl at 1.6 mg/ml. Sample as dispensed on freshly plasma cleaned UltrAuFoil R1.2/1.3 300-mesh grids (Electron Microscopy Services) and applied to grid in the chamber of a Vitrobot (Thermo Fisher) at ~95% relative humidity, 4°C. The sample was blotted for 4 seconds with Whatman No. #1 filter paper immediately prior to plunge freezing in liquid ethane cooled by liquid nitrogen.

Li Y., Cash J.N., Tesmer J.J, & Cianfrocco M.A. (2020). High-throughput cryo-EM enabled by user-free preprocessing routines. Structure (London, England : 1993), 28(7), 858-869.e3.

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7

Cryo-EM sample preparation for aldolase and proteasome

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Pure aldolase isolated from rabbit muscle was purchased as a lyophilized powder (Sigma Aldrich) and solubilized in 20 mM HEPES (pH 7.5), 50 mM NaCl at ~3 mg/ml. Aldolase was further purified by size-exclusion chromatography using a Sepharose 6 10/300 (GE Healthcare) column equilibrated in solubilization buffer. Peak fractions were pooled and concentrated to 1.6 mg/ml immediately prior to cryo-electron microscopy (cryo-EM) grid preparation.
Archaeal 20S proteasome (T. acidophilum) was kindly donated by Drs. Zanlin Yu and Yifan Cheng at The University of California, San Francisco and used as-is without further modification.
For cryo-EM, 3 μL of purified aldolase (1.6 mg/ml) or 20S proteasome (0.5 mg/ml) were dispensed on freshly plasma cleaned UltrAuFoil® R1.2/1.3 300-mesh grids (Electron Microscopy Services) and manually blotted^{25 (link)} using a custom-built manual plunger in a cold room (≥95% relative humidity, 4°C). Sample was blotted for ~4 seconds with Whatman No.1 filter paper immediately prior to plunge freezing in liquid ethane cooled by liquid nitrogen. In order to provide enough signal for proper CTF estimation we strived to achieve a particle concentration that maximized the number of particles contained within the holes without resulting in overlapping particles and/or aggregation (see Supplemental Note 3).

Herzik MA J.r., Wu M, & Lander G.C. (2017). Achieving better than 3 Å resolution by single particle cryo-EM at 200 keV. Nature methods, 14(11), 1075-1078.

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8

Cryo-EM sample preparation for aldolase and proteasome

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Pure aldolase isolated from rabbit muscle was purchased as a lyophilized powder (Sigma Aldrich) and solubilized in 20 mM HEPES (pH 7.5), 50 mM NaCl at ~3 mg/ml. Aldolase was further purified by size-exclusion chromatography using a Sepharose 6 10/300 (GE Healthcare) column equilibrated in solubilization buffer. Peak fractions were pooled and concentrated to 1.6 mg/ml immediately prior to cryo-electron microscopy (cryo-EM) grid preparation.
Archaeal 20S proteasome (T. acidophilum) was kindly donated by Drs. Zanlin Yu and Yifan Cheng at The University of California, San Francisco and used as-is without further modification.
For cryo-EM, 3 μL of purified aldolase (1.6 mg/ml) or 20S proteasome (0.5 mg/ml) were dispensed on freshly plasma cleaned UltrAuFoil® R1.2/1.3 300-mesh grids (Electron Microscopy Services) and manually blotted^{25 (link)} using a custom-built manual plunger in a cold room (≥95% relative humidity, 4°C). Sample was blotted for ~4 seconds with Whatman No.1 filter paper immediately prior to plunge freezing in liquid ethane cooled by liquid nitrogen. In order to provide enough signal for proper CTF estimation we strived to achieve a particle concentration that maximized the number of particles contained within the holes without resulting in overlapping particles and/or aggregation (see Supplemental Note 3).

Herzik MA J.r., Wu M, & Lander G.C. (2017). Achieving better than 3 Å resolution by single particle cryo-EM at 200 keV. Nature methods, 14(11), 1075-1078.

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9

Cryo-EM Sample Preparation for Csy-AcrIF9 Complexes

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Csy–Acr complexes were previously found to exhibit preferred orientation when suspended in open holes during preparation for cryo-EM imaging^{10 (link)}. Therefore, purified Csy–AcrIF9 was mixed with 0.05% v v⁻¹ Lauryl Maltose Neopentyl Glycol prior to freezing. UltrAuFoil R1.2/1.3 300-mesh grids (Electron Microscopy Sciences) were plasma cleaned immediately prior to sample application in a Solarus plasma cleaner (Gatan, Inc.) with a 75% nitrogen, 25% oxygen atmosphere at 15 W for 7 s. Cryo-EM grids were prepared by application of 4 µL Csy–AcrF9 at a concentration of 2.5 mg mL⁻¹. Grids were manually blotted with Whatman No. 1 filter paper for 5 s followed by plunge freezing in liquid ethane at 4 °C in 95% humidity.

Hirschi M., Lu W.T., Santiago-Frangos A., Wilkinson R., Golden S.M., Davidson A.R., Lander G.C, & Wiedenheft B. (2020). AcrIF9 tethers non-sequence specific dsDNA to the CRISPR RNA-guided surveillance complex. Nature Communications, 11, 2730.

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10

Cryo-EM Sample Preparation Protocol

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A volume of 3 µL of purified ADH (~2.5 mg mL⁻¹), metHb (~12 mg mL⁻¹), or inhibited PKA (~5 mg mL⁻¹) were dispensed on UltrAuFoil R1.2/1.3 300-mesh grids (Electron Microscopy Services) that had been freshly plasma cleaned using a Solarus plasma cleaner (Gatan, Inc.) with a 75% argon/25% oxygen atmosphere at 15 Watts for 6 s. Grids were blotted manually^{29 (link)} using a custom-built manual plunger in a cold room (≥95% relative humidity, 4 °C)^{12 (link)}. Samples were blotted for 4–5 s with Whatman No. 1 filter paper immediately before plunge freezing in liquid ethane cooled by liquid nitrogen. We aimed to achieve a particle concentration that maximized the number of particles contained within the holes without resulting in overlapping particles and/or aggregation in order to maintain a consistent ice thickness across the center of holes and provide sufficient signal for accurate CTF estimation, as observed previously^{12 (link)}.

Herzik MA J.r., Wu M, & Lander G.C. (2019). High-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM. Nature Communications, 10, 1032.

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Ultraufoil r1.2 1 3 300 mesh grid

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11 protocols using ultraufoil r1.2 1 3 300 mesh grid

Negative Staining and Cryo-EM of TRPML3

TRPV3 K169A Cryo-EM Grid Preparation

Cryo-EM Specimen Preparation for TRPV3

Negative Staining and Cryo-EM of TRPML3

TRPV2 Protein Cryo-EM Sample Preparation

Cryo-EM Preparation of Rabbit Muscle Aldolase

Cryo-EM sample preparation for aldolase and proteasome

Cryo-EM sample preparation for aldolase and proteasome

Cryo-EM Sample Preparation for Csy-AcrIF9 Complexes

Cryo-EM Sample Preparation Protocol

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