Talos f200c

T4 bacteriophages that were treated with plasma or plasma-activated water or were untreated were dropped onto carbon-coated grids and kept for 10 min at 22°C. Then, the excess liquid was discarded, and the grids were covered with 1% uranyl acetate for 30 s at 22°C. These stained samples were examined using an FEI Talos F200C transmission electron microscope operating at 200 kV at ×28,000 magnification.

The polydispersity index (PDI) and zeta potential of MOF@GSK-J1 and HA@MOF@GSK-J1 NPs were determined using a Zetasizer Nano ZS90 instrument (Malvern Instruments, Malvern, United Kingdom) at 25°C. The morphologies of MOF@GSK-J1 and HA@MOF@GSK-J1 NPs were imaged via transmission electron microscopy (TEM, Talos F200C, FEI, United States) with an acceleration voltage of 200 kV. The phase and crystal structures of MOF and MOF@HA NPs without GSK-J1 were examined by X-ray diffraction (XRD) patterns using a Rigaku X-ray diffractometer with Cu-Kα radiation (Rigaku, Japan). The FTIR spectra were recorded by Fourier transform-infrared spectroscopy (Perkin Elmer, United States).

Chemically fixed scallop eyes were washed with 0.1 M cacodylate buffer for 2 h and post-fixed with 1% osmium tetroxide and 2% uranyl acetate according to the method published in ref. ¹⁷ . Ultra-thin sections were prepared with an ultra-microtome (RMC, Arizona, USA) and imaged with HRSEM Gemini 300 SEM (Zeiss) STEM detector, and Thermo Fisher Scientific (former FEI) Talos F200C transmission electron microscope operating at 200 kV. The images were taken with Ceta 16 M CMOS camera. The electron diffraction patterns were obtained with a Thermo Fisher Scientific (FEI) Tecnai T12 G² TWIN TEM operating at 120 kV. Images and electron diffraction (ED) patterns were recorded using a Gatan 794 MultiScan CCD camera. ED analysis was done using Gatan Digital Micrograph software with the DIFPack module. Images and ED patterns were recorded with consideration of potential beam damage to the sample, thus appropriate illumination conditions (spot size) were used to avoid it.

Purified NiV F and Fabs were mixed at a molar ratio of 1:1.2 (NiV-F:Fab) in 2 mM Tris pH 8, 200 mM NaCl, 0.02% (w/v) sodium azide and 0.1% (w/v) amphipol A8-35. The final concentration of NiV F in each sample ranged from 0.5-0.7 mg/mL, or approximately 3-5 μM. The samples were incubated for 30 minutes at room temperature to form complexes, then centrifuged at 20,000 x g. Approximately 3-4 μL of supernatant was deposited onto plasma-cleaned UltrAuFoil R 1.2/1.3, 300 mesh grids (Electron Microscopy Sciences). Excess liquid was blotted away for 3-4 seconds, and the grids were plunge frozen in liquid ethane using a Vitrobot Mark IV (FEI) operating at 22 °C and 100% humidity. Frozen grids were stored in liquid nitrogen until further use.
Grids were screened for quality using a Talos F200C (FEI) transmission electron microscope (TEM). Grids that passed quality control were loaded onto a separate microscope for high resolution imaging. All high-resolution structures were imaged on a Titan Krios TEM (ThermoFisher Scientific) operating at 300 kV equipped with a K3 camera (Gatan), except for prefusion NiV F complexed with Fab 1H1, which was imaged on a Glacios TEM equipped with a Falcon 4 detector (ThermoFisher Scientific). Movies were collected using SerialEM. Calibrated pixel sizes were 1.075 Å (1H8 and 4H3), 0.66 Å (2D3 and 2B12), 0.94 Å (prefusion NiV+1H1), and 0.81 Å (1A9).

A final polishing step to confirm isolation of 12-mer CaMKII holoenzymes was performed using a Superose 6 10/100 size-exclusion column resulting in an elution fraction of 20 μg/mL in final buffer 20 mM HEPES, pH 7.5, and 200 mM NaCl. The sample (3 μL) was then incubated on a glow-discharged, carbon-coated 400 mesh copper grid (CF400-Cu, Electron Microscopy Sciences) for 1 min, followed by washing with three drops of a freshly-prepared 1% uranyl formate solution, then wicked dry. Grids were imaged on an FEI Talos F200C equipped with a 4k x 4k BM-Ceta CCD camera operating at 200 kV in low-dose mode (10 e^-/A²sec) at 73,000 X magnification and -5 μm defocus.