Samples were analyzed using a NanoWizard ULTRA Speed AFM (JPK Instruments) mounted on an inverted optical microscope (Nikon Eclipse TE2000-U or Zeiss AxioObserver.A1), or equipped with a JPK TopViewOptics. Samples were imaged in buffer at ambient temperature in amplitude-modulation or phase-modulation AC mode. Fast-scanning high-resonant ultra-short cantilevers (USC-F0.3-k0.3, NanoWorld) with a nominal resonance frequency of 300 kHz in air, spring constant of 0.3 N/m, reflective chromium/gold-coated silicon chip, and high-density carbon tips with a radius of curvature of 10 nm were used. Prior to deposition on substrate, RNA and HBDs molecules were incubated in filtered nuclear-like buffer (NLB; 5 mM NaCl, 140 mM K+, 0.5 mM Mg2+, 10−4 mM Ca2+, pH = 7.2) for 30 min at 37°C. For cryo-EM, HOTAIR-bearing grids were plunge-frozen in liquid ethane cooled by liquid nitrogen, using a Leica EM-GP plunger (4 sec blotting time, 80% humidity), and imaged at liquid nitrogen temperature on an FEI Tecnai TF20 electron microscope operated at 200 kV with a Gatan side entry 626 cryo-holder. Images were recorded on a K2 Summit direct detector (Gatan) mounted at the end of a GIF Quantum energy filter (Gatan). Images were collected in counting mode, at a calibrated magnification of 16,218 yielding a pixel size of 3.083 Å. Additional detailed methods can be found in the Supplemental Notes .
Em gp plunger
Manufactured by Leica camera
The EM GP plunger is a precision tool designed for use in electron microscopy sample preparation. It is engineered to gently and consistently apply pressure to specimens, facilitating the process of vitrification for cryo-electron microscopy. The EM GP plunger's core function is to ensure uniform, high-quality sample preparation without extraneous interpretation or extrapolation.
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Lab products found in correlation
Axio observer a1, by Zeiss (3 mentions)
Quantum energy filter, by Ametek (1 mentions)
Usc f0 k0, by NanoWorld (1 mentions)
Eclipse te2000 u, by Zeiss (1 mentions)
Tecnai tf20 electron microscope, by Ametek (1 mentions)
Nanowizard ultra speed afm, by Bruker (1 mentions)
Topviewoptics, by Bruker (1 mentions)
626 cryo holder, by Ametek (1 mentions)
F20 microscope, by Thermo Fisher Scientific (1 mentions)
Titan krios electron microscope, by Thermo Fisher Scientific (1 mentions)
Krios tem, by Thermo Fisher Scientific (1 mentions)
5 protocols using em gp plunger
1
Atomic Force Microscopy of RNA-Protein Complexes
2
Cryo-EM Sample Preparation for High-Resolution Imaging
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3.5 μl of FAS-I solution at 10 mg/ml was applied to glow-discharged C-flat 2/2, 200 mesh holey carbon grids and plunge frozen in liquid ethane cooled by liquid nitrogen, using a Leica EM-GP plunger (3 s blotting time, 80% humidity). Grids were screened on an FEI F20 microscope equipped with a K2 Summit direct detector (Gatan). Cryo-EM data were collected on a Titan Krios electron microscope (FEI) operated at 300 kV. Coma-free alignment was performed with AutoCTF (FEI) and beam size was 645 nm. Movies were recorded on a K2 Summit direct detector mounted at the end of a GIF Quantum energy filter (Gatan) using a slit of 20 eV. 5248 Movies were collected in super-resolution counting mode at a nominal magnification of 130,000× corresponding to a physical pixel size of 1.054 Å. The dose rate was set to 4.49 electrons per physical pixel per second and the total exposure time was 20 s, resulting in an accumulated dose of 80.1 electrons per Å2. Each Movie was fractionated into 40 frames of 0.5 s. Nominal defocus range was −0.5 to −2.5 μm. All dose-fractionated images were recorded using an automated low dose procedure implemented in SerialEM26 (link). Stage navigation was used to navigate to hole centers and image shift used to target 5 distinct imaging locations within each hole.
3
Binding of Adamantyl Compounds to Virions
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Purified virus at a titre of approximately 1010 pfu/mL was incubated at RT for 10 min (preparation 1), with 17 uM 1-adamantylmethyl 5-aminoisoxazole-3-carboxylate (preparation 2) or with 34 uM 1-adamantylmethyl 5-amino-4-(2-phenylethyl)isoxazole-3-carboxylate (preparation 3) before inactivation to investigate the binding of these compounds to the virion [23 (link)]. Both compounds were kindly provided by Dmitry Osolodkin. The samples were inactivated by irradiation with 0.1 J/cm2 UV-C using a UVP Crosslinker (Analytik, Jena, Germany). The samples were vitrified on glow-discharged electron microscopy grids using a Leica EM GP plunger at 80% humidity and 1.5 s blotting time using front blotting. Lacey carbon graphene oxide-coated grids (300 micrometer grid, Electron Microscopy Sciences, Hatfield, PA, USA) were used for preparations 1 and 2, and UltrAuFoil gold grids (2/2, 300 micrometer grid, Electron Microscopy Sciences) for preparation 3.
4
Vitrification of Cells on EM Grids
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WI-38 cells and HDMECs were grown on EM grids as described previously (44 (link)). Briefly, cells were plated on gold QUANTIFOIL grids and grown to 30 to 70% confluence, typically over 2 to 3 days. The cells were subsequently vitrified in liquid ethane using a Leica EM GP plunger and stored in liquid nitrogen until use.
5
Structural Imaging of BKV VLP-scFv Complex
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BKV ST1 VLPs were incubated with the scFv fragment of 41F17 (360 scFv molecules per VLP, total protein concentration of 1 mg/mL) at RT C for 1 h. The sample was then concentrated 10-fold. 4.0 mL of the concentrated VLP-scFv complex was applied onto the grid (R1.2/1.3, Cu 300 mesh, Quantifoil Micro Tools GmbH, Grosslo ¨bichau, Germany) coated with an additional thin amorphous carbon layer. Grids were vitrified using a Leica EM GP plunger. Images were acquired with a Cs-corrected FEI Titan Krios TEM operated at 300 kV equipped with a Quantum-LS Gatan Image Filter (GIF) and recorded on a K2-Summit direct electron detector (Gatan GmbH). Images were collected automatically (with EPU, Thermo Fisher) in electron-counting mode (nominal post-GIF magnification of 3 105,000 and calibrated pixel size of 1.12 A ˚). Exposures of 7 s were dose-fractionated into 40 frames. The total exposure dose was $40 e-/A ˚2 . Defocus values varied from À0.8 to À2.5 mm.
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