For cryo-EM sample preparation, 3 μL of concentrated sample at ∼ 8 mg/ml (100 kDa cut-off, Amicon), was applied to freshly glow-discharged (easiGlo Ted Pella, 20 mA, 40 s) grids (Quantifoil Au 1.2/1.3 200 mesh), blotted and plunge frozen in liquid ethane using a Leica GP2 plunger. Data collection was carried on a Titan Krios (Thermo Fisher Scientific) equipped with a K3 direct-electron detector (Gatan). SerialEM [50] (link) was used for automated data collection, using an image-shift based (9 holes) procedure, with a defocus range of −1.0 to −2.0 μm and a dose rate of 15 e-/px/s and a pixel size of 0.8521 A. Micrographs were collected with 50 ms exposure per frame for a total of 3 s, resulting in 60 frames total. Data processing was carried out using established protocols, as part of the software packages RELION [51] (link) (version 3.1.2) and cryoSPARC (v3.2.0). A total of 4899 micrographs were subjected to motion correction (MotionCor2 [52] (link)) and CTF estimation using CTFFIND4 [53] (link), followed by reference-free and reference-based particle picking, 2D classification and final reconstruction in cryoSPARC using non-uniform refinement [54] (link), while applying C3 symmetry, resulting in a map of 4.2 Å resolution (FSC = 0.143). All attempts to reconstruct in C1 resulted in maps of lower resolution (∼6 Å), while not showing any asymmetric features.
Gp2 plunger
Manufactured by Leica
Sourced in Austria
The GP2 plunger is a laboratory equipment component designed for precise liquid handling. It features a durable construction and a smooth, controlled dispensing action. The core function of the GP2plunger is to facilitate the accurate and repeatable transfer of liquids in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Whatman, by Cytiva (3 mentions)
Titan krios, by Thermo Fisher Scientific (1 mentions)
Au 1.2 1 3 200 mesh, by Quantifoil (1 mentions)
K3 direct electron detector, by Ametek (1 mentions)
Autogrid, by Thermo Fisher Scientific (1 mentions)
Oneview 16mp camera, by Ametek (1 mentions)
Holey carbon coated grid, by Ted Pella (1 mentions)
626 cryo holder, by Ametek (1 mentions)
Talos l120c electron microscope, by Thermo Fisher Scientific (1 mentions)
6 protocols using gp2 plunger
1
Cryo-EM Structure Determination Protocol
2
Cryo-EM Sample Preparation Protocol
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After in vitro assembly MA126CANC tubes were kept at 4°C until plunge freezing. 2/2–3C C-flat grids coated with 2 nm support carbon layer were glow discharged in the presence of amylamine. Grids were then incubated on a 5 μl drop of sample for 10 min. The rest of the sample was mixed with 10nm colloidal gold, and 2.5 μl of this solution was added to the incubated grids prior vitrification. The samples were vitrified in liquid ethane using a Leica GP2 plunger with front-side blotting (blot time of 3–4 s, humidity 90–95%, temperature 10°C) and stored in liquid nitrogen until imaging.
3
Cryo-FIB Milling of Infected Cells
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Chemically fixed infected cells grown on grids were plunge-frozen in liquid ethane cooled to −183 °C by liquid nitrogen using a Leica GP2 plunger. Before plunge freezing, 3 µl of 0.1 M PHEM buffer pH 7.4 was applied on a grid in the chamber set to 70% humidity and 24 °C. Grids were blotted for 3 seconds using No. 1 Whatman paper and clipped into Autogrids (Thermo Fisher Scientific) dedicated for cryo-focused ion beam milling and stored in liquid nitrogen.
4
Cryo-EM Sample Preparation Protocol
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To prepare samples for cryo-EM study, lacey carbon grids (Cu, 300 mesh) were glow-discharged (45s at MED RF power level) in Harrick plasma cleaner. 4 mL of sample solution was applied on to the carbon side of EM grid, which was then blotted for 2.5 s at 95% humidity and plunge-frozen into Leica GP2 plunger (Queen Mary University of London). Grids were imaged on a Jeol JEM-2100 plus (Queen Mary University of London), equipped with Gatan OneView 16MP camera at accelerating voltage of 200 kV. Images were obtained using SerialEM software at low dose mode at 50k magnification, corresponding to 2.166A ˚with the defocus range set from -2.5 to -5 mm.
5
Cryo-EM Imaging of Virus Particles
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Virus particles were fixed in 1 % glutaraldehyde (Cat# 233281000, Thermo Scientific). A 4 µL aliquot of sample was adsorbed onto holey carbon-coated grid (Lacey, Tedpella, USA), blotted with Whatman 1 filter paper and vitrified into liquid ethane at −180 °C using a Leica GP2 plunger (Leica microsystems, Austria). Frozen grids were transferred onto a Talos 200C Electron microscope (FEI, USA) using a Gatan 626 cryo-holder (GATAN, USA). Electron micrographs were recorded at an accelerating voltage of 200 kV using a low-dose system (40 e-/Å2) and keeping the sample at −175 °C. Defocus values were −2 to 3 µm. Micrographs were recorded on 4K x 4K Ceta CMOS camera.
6
Cryo-EM Sample Preparation Protocol
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Four μL sample aliquots (4 μL,
1–2 mg/mL) were adsorbed onto a holey carbon-coated grid (Lacey,
Tedpella, USA), blotted with Whatman 1 filter paper, and vitrified
into liquid ethane at −180 °C using a Leica GP2 plunger
(Leica microsystems, Austria). The frozen grids were then transferred
onto a Talos L120C Electron microscope (FEI, USA) using a Gatan multispecimen
cryo-holder Model 910 (Gatan, USA). An accelerating voltage of 120
kV using a low-dose system (40 e–/Å2) was used to record electron micrographs, while the sample was kept
at −175 °C. Defocus values were −2 to 3 μm.
A 4K × 4K Ceta CMOS camera was used to record electron micrographs.
1–2 mg/mL) were adsorbed onto a holey carbon-coated grid (Lacey,
Tedpella, USA), blotted with Whatman 1 filter paper, and vitrified
into liquid ethane at −180 °C using a Leica GP2 plunger
(Leica microsystems, Austria). The frozen grids were then transferred
onto a Talos L120C Electron microscope (FEI, USA) using a Gatan multispecimen
cryo-holder Model 910 (Gatan, USA). An accelerating voltage of 120
kV using a low-dose system (40 e–/Å2) was used to record electron micrographs, while the sample was kept
at −175 °C. Defocus values were −2 to 3 μm.
A 4K × 4K Ceta CMOS camera was used to record electron micrographs.
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