Gold fiducial markers (10 or 5 nm) (BBI Solutions) in buffer A were added to the tubulation reaction (1:10 fiducials:reaction volume ratio). Four microliters of this mixture was backside-blotted for 6 s at a relative humidity of 98% and a temperature of 19°C on a glow-discharged holey carbon grid (CF-2/1-3C; Protochips) before plunge-freezing in liquid ethane (Leica EM GP2 automatic plunger). Dose-symmetrical tilt series acquisition (55 (link)) was performed on an FEI Titan Krios electron microscope operated at 300 kV using a Gatan Quantum energy filter with a slit width of 20 eV and a K2 or K3 direct detector operated in counting mode. The total exposure of ~130 e−/Å2 was equally distributed between 41 tilts. Ten frame movies were acquired for each tilt. The details of data collection are given in table S1. The selection of acquisition areas was guided by suitability for high-resolution tomographic data collection (i.e., vitreous ice quality, lack of crystalline ice contaminations, and intactness of the carbon support) and was not based on the morphology of tubules.
Em gp2 automatic plunger
Manufactured by Leica
Sourced in Germany
The EM GP2 automatic plunger is a piece of laboratory equipment designed for the preparation of samples for electron microscopy. It is used to automatically apply a controlled amount of cryoprotectant or other liquids to a sample, ensuring consistent and efficient sample preparation.
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5 protocols using em gp2 automatic plunger
1
Cryo-EM Acquisition of Tubulation Samples
2
Cryo-EM Analysis of AP2 Liposome Binding
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AP2 was recruited to liposomes alone or in the presence of the FCHO2 linker by mixing 1.5 μM AP2 or 1.5 μM AP2 and 7.5 μM FCHO2 linker, with 100-nm extruded liposomes (0.2 mg/ml) containing 10% brain PtdIns(4,5)P2 and 10% DOPS in a POPC/POPE (3:2) mixture in HKT buffer. Reactions were performed in parallel and incubated for 30 min at 21°C. After incubation, the reactions were supplemented with 1:10 of 10-nm gold fiducial markers in HKT buffer. A total of 3.5 μl of this mixture was applied on a glow-discharged holey carbon grid (CF-2/1-3C, Protochips) and back-side blotted for 4 s at relative humidity of 98% and 18°C, followed by plunge-freezing in liquid ethane (Leica EM GP2 automatic plunger).
Data acquisition was performed as in (14 (link)). Dose-symmetrical tomographic tilt series (84 (link)) were collected in a FEI Titan Krios electron microscope operated at 300 kV with a Gatan Quantum energy filter with a slit width of 20 eV and a K3 direct detector operated in counting mode using tilt series controller in Serial EM software. Tilt series contained 41 tilted images (−60° to +60° with 3° increment) with 10-frame movies acquired for each tilt and were imaged with a total exposure of ~130 e−/A2 equally distributed between tilts. The details of data collection are given in table S1.
Data acquisition was performed as in (14 (link)). Dose-symmetrical tomographic tilt series (84 (link)) were collected in a FEI Titan Krios electron microscope operated at 300 kV with a Gatan Quantum energy filter with a slit width of 20 eV and a K3 direct detector operated in counting mode using tilt series controller in Serial EM software. Tilt series contained 41 tilted images (−60° to +60° with 3° increment) with 10-frame movies acquired for each tilt and were imaged with a total exposure of ~130 e−/A2 equally distributed between tilts. The details of data collection are given in table S1.
3
Cryo-EM Isolation of Malaria Merozoites
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For merozoite preparation, cultures were scaled up to 60 mL at 5% hematocrit. Late-stage schizonts were purified from highly synchronous cultures by magnetic activated cell sorting (MACS). Isolated schizonts were treated with 10 μM E64 for 6-8 hours, followed by passage through 1.2 μm syringe filters (preincubated in 1% BSA + PBS for 20 minutes) to mechanically isolate merozoites39 (link). Isolated merozoites were spun down at 2200 g for 15 minutes and resuspended in 20 μL complete media. 10 nm colloidal gold fiducials (Ted Pella, Redding, USA) were added to the suspension (for alignment during tomogram reconstruction from tilt series). 4 μL of suspended merozoites were applied onto Quantifoil 200 mesh copper R2/2 holey carbon electron microscopy grids, excess liquid blotted away, and plunge frozen into a liquid ethane/propane mixture (pre-cooled with liquid nitrogen) using an EM GP2 automatic plunger (Leica Microsystems, Wetzlar, Germany)40 (link). The blotting chamber was set to 95% relative humidity at 24°C and blotting was done from the sample side of the grid using Whatman filter paper #1. Plunge-frozen grids were subsequently loaded into autogrid c-clip rings (Thermo Fisher). The autogrid box containing frozen grids were stored in liquid nitrogen and maintained at ≤−170°C throughout storage, transfer, and cryo-ET imaging.
4
Cryo-EM Isolation of Malaria Merozoites
5
Cryo-EM Merozoite Isolation Protocol
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For merozoite preparation, cultures were scaled up to 60 mL at 5% hematocrit. Late-stage schizonts were purified from highly synchronous cultures by magnetic activated cell sorting (MACS). Isolated schizonts were treated with 10 𝜇M E64 for 6-8 hours, followed by passage through 1.2 𝜇m syringe filters (preincubated in 1% BSA + PBS for 20 minutes) to mechanically isolate merozoites 44 . Isolated merozoites were spun down at 2200 g for 15 minutes and resuspended in 20 𝜇L complete media. 10 nm colloidal gold fiducials (Ted Pella, Redding, USA) were added to the suspension (for alignment during tomogram reconstruction from tilt series).
4 𝜇L of suspended merozoites were applied onto Quantifoil 200 mesh copper R2/2 holey carbon EM grids, excess liquid blotted away, and plunge frozen into a liquid ethane/propane mixture (pre-cooled with liquid nitrogen) using an EM GP2 automatic plunger (Leica Microsystems, Wetzlar, Germany) 45 . The blotting chamber was set to 95% relative humidity at 24°C and blotting was done from the sample side of the grid using Whatman filter paper #1. Plunge-frozen grids were subsequently loaded into autogrid c-clip rings (Thermo Fisher). The autogrid box containing frozen grids were stored in liquid nitrogen and maintained at ≤-170°C throughout storage, transfer, and cryo-ET imaging.
4 𝜇L of suspended merozoites were applied onto Quantifoil 200 mesh copper R2/2 holey carbon EM grids, excess liquid blotted away, and plunge frozen into a liquid ethane/propane mixture (pre-cooled with liquid nitrogen) using an EM GP2 automatic plunger (Leica Microsystems, Wetzlar, Germany) 45 . The blotting chamber was set to 95% relative humidity at 24°C and blotting was done from the sample side of the grid using Whatman filter paper #1. Plunge-frozen grids were subsequently loaded into autogrid c-clip rings (Thermo Fisher). The autogrid box containing frozen grids were stored in liquid nitrogen and maintained at ≤-170°C throughout storage, transfer, and cryo-ET imaging.
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