After screening in a Talos Arctica 200 kV transmission electron microscope (TEM) (Thermo Fisher Scientific, USA), the good grids were mounted into a Titan Krios 300 kV TEM (Thermo Fisher Scientific, USA) for imaging. A total of 8,745 images were collected at a nominal magnification of 64,000×, resulting in a calibrated physical pixel size at the specimen level of 2.24 Å. For images at a tilting angle at 0/30/45/60 degrees, the total dose was set to be 100 or 120/60/80/100 e-/Å2; the movies were recorded on a 0.5 s per frame base, and the exposure time of these collected datasets was set to be 28.5 or 34.5/21.5/41 or 28.5/36 or 35 s (Table S1). All movies were recorded by a Gatan K2 Summit Direct electron detector (DDD) (Gatan Company, USA) under super-resolution mode equipped with a post column GIF Quantum filter, whose slit width was set to be 20 eV. SerialEM with in-house scripts was used for data collection with the defocus value set between 1.0 μm and 4.0 μm (Mastronarde, 2005 (link); Wu et al., 2019 (link)).
300 kv tem titan krios
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 300 kV TEM Titan Krios is a transmission electron microscope (TEM) designed for high-resolution imaging of biological samples. It operates at an accelerating voltage of 300 kilovolts and incorporates advanced technologies, including a Falcon direct electron detector and Gatan imaging filter, to enable detailed structural analysis of macromolecular complexes and cellular components.
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5 protocols using 300 kv tem titan krios
1
Cryo-EM Data Collection Protocol
2
Cryo-EM Data Acquisition Workflow
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The data acquisition collection strategy of this study was basically the same with our previous reports 28 . Briefly, after screening in Talos Arctica 200 KV TEM (Thermo Fisher Scientific, USA), the good grids were mounted into Titan Krios 300 KV TEM (Thermo Fisher Scientific, USA) for imaging. 8745 images were collected at a nominal magnification of 64,000X, resulted in a calibrated physical pixel size at the specimen level of 2.24 Å. For images at tilting angle at 0/30/45/60 degrees, the total dose was set to be 100 or 120/60/80/100 e-/Å2, the movies were recorded on a 0.5 s per frame base, and the exposure time of these collected data set were set to be 28.5 or 34.5/21.5/41 or 28.5/36 or 35 s (Extended Data Table 1). All movies were recorded by a Gatan K2 Summit DDD detector (Gatan Company, USA) under super resolution mode, equipped with a post column GIF Quantum filter, whose slit width was set to be 20 eV.
SerialEM with in-house scripts was used for data collection with the defocus value set between 1.0 to 4.0 μm 44, (link)45 (link) .
SerialEM with in-house scripts was used for data collection with the defocus value set between 1.0 to 4.0 μm 44, (link)45 (link) .
3
Cryo-EM Sample Preparation for Protein Complexes
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An amount of 3 µL of protein sample (shClpP1, or shClpP1P2, or ClpC1:shClpP1P2) at a concentration of 2 mg/mL was applied to the EM grids (Quantifoil Cu R1.2/1.3, 300 mesh) that had been glow discharged for 50 s using Pelco easiGlow. The samples were plunged frozen in liquid ethane using a ThermoFisher Vitrobot with the settings of temperature 8°C, relative humidity 100%, blot force 3, and blot time 3 s. Cryo-EM data collection was performed on a ThermoFisher Scientific 300 kV TEM Titan Krios equipped with Gatan K3 direct electron detector, using the EPU software (ThermoFisher) for automated data acquisition (Table 1 ). Data were collected at a defocus range of −1.1 to −1.5 µm and at a nominal magnification of 105,000×, which resulted in a calibrated pixel size of 0.42 Å/pixel. Micrographs were recorded as movie stacks with an electron dose of 50 e−/ Å2 fractionated into a total of 50 frames.
4
Cryo-EM Sample Preparation and Imaging
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After glow-discharging for 30 seconds with Plasma cleaner, 3 μL of samples (10 mg/ml) was applied to the carbon side of Quantifoil R 0.6/1 gold 200 mesh grid grids, blotted for 4.0 seconds without blot force, and plunge-frozen in liquid ethane using Vitrobot (Thermo Fisher Scientific, USA). Cryo-EM grids were imaged on a Thermo Fisher Scientific 300 kV TEM Titan Krios using Gatan K3 direct electron detector. Raw movies were collected in super-resolution mode at a magnification of 130,000, with a super-resolution pixel size of 0.334 Å. The data was stored in 32-frame gain normalized stacks with a total dose of 55 e−/Å2. The detailed information of data collection was summarized in table S1 .
5
Structural Analysis of CHIKV nsP1
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To prepare samples of nsP1 with m7GTP, m7GMP, and FHA, purified wild type (WT) nsP1 protein were incubated with SAH-M7GTP, SAH-m7GpppAU-10RNA, and FHA for 12hrs respectively. For the sample of nsP1 with m7GpppAU-10RNA, nsP1 with H37A mutation was used to incubate with SAH-m7GpppAU-10RNA. Before preparing Cryo-EM grids, the carbon side of Quantifoil R1.2/1.3 gold 300 mesh grid was covered with one layer of graphene following a published protocol (Naydenova et al., 2019) (link). After glow-discharging for 10 s at low energy with Plasma cleaner, 3 mL of CHIKV nsP1 (concentration about 0.2 mg/mL) with different ligands or RNA was applied to the grids, blotted for 2.5 s with blot force À2, and plunge-frozen in liquid ethane using Vitrobot (Thermo Fisher Scientific). Cryo-EM grids were imaged on a Thermo Fisher Scientific 300 kV TEM Titan Krios using Gatan K2 direct electron detector or Falcon III detector. The detailed information of data collection was summarized in Table S1.
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