Cf300 cu ul

Glow-discharged continuous carbon grids (Cat# CF300-CU-UL, Electron Microscopy Sciences, Hatfiled, PA) were used for negative staining. The sample was diluted 10× in the elution buffer (20 mM HEPES, pH7.6, 150 mM NaCl, and 0.5 mM TCEP) and 3 µl was applied on the carbon side of the grid. After incubation for 1 min, extra protein was washed away by touching a piece of filter paper followed by two washes in 10 µl distilled water and one wash in 2% uranium acetate (UA). The sample was then stained using 2% UA for 1 min and excess liquid was wicked out using a filter paper. The stained sample was dried in air and evaluated on a JEOL-1400 Electron Microscope (JEOL USA inc, Peabody, USA).

For negative stain imaging, purified β-Klotho or β-Klotho/Fab 39F7 complex was diluted to 10 μg/ml in 25 mm Hepes, pH 7.5, and 150 mm NaCl. Five molar ratio of FGF21 was added to 10 μg/ml β-Klotho to make FGF21/β-Klotho complex. Continuous carbon grids (CF300-Cu-UL, Electron Microscopy Sciences) were plasma cleaned for 30 s. Then, 4 μl of sample was applied and wicked away before staining with 2% uranyl acetate (36 (link)). Images were manually acquired on an FEI Talos F200C operated at 200 kV, at a nominal magnification of 57,000× on a Ceta camera with a pixel size of 2.58 Å/pixel. All image processing was carried out with the EMAN2 package (37 (link)). Particles were picked with e2boxer and CTF correction was carried out on a per particle basis with e2ctf. The CTF corrected particles were then subjected to reference-free 2D classification using e2refine2d.

Square mesh thin carbon-coated copper EM grids (CF300-CU-UL, Electron Microscopy Sciences) were prepared for negative stain EM images. Uranyl formate (UF) was prepared as a 1% w/v solution in deionized water and protected from light. The solution was filtered at 0.02 μm before use. Samples were diluted in 10 mM HEPES pH 7.5, 150 mM NaCl to 0.125 mg mL⁻¹ (protein and lipids each). An aliquot of 4 μL of sample was added to a glow discharged grid, positioned over ice. The sample was then blotted for 1 or 2 min with Whatman filter paper. The blotted grid was then touched to a 20 μL well of UF for 30 s, then blotted with filter paper, followed by an additional UF touching/blotting step for 1 min. Drying of the grid was initiated with a slow stream of N₂ gas for approximately 30 s, then the grids were left overnight to dry completely on filter paper in a covered petri dish before storing. Negative stain EM images were collected on a FEI Technai TF200 microscope operated at 200 kV. Images were collected at 1700 and ×6500 magnification using a Gatan US1000 2k × 2k CCD camera.