For negative stain electron microscopy, trimers were purified by lentil lectin affinity chromatography, following which trimer fractions were collected by size exclusion chromatography using a Superdex 200 (GE Healthcare) column pre-equilibrated with buffer containing 5 mM HEPES, 150 mM NaCl (pH 7.5).
The SEC purified samples were prepared for negative staining in the following conditions – (i) NFL Wt, (ii) designed trimer NFL D1, (iii) designed trimer NFL D2, (iv) designed trimer NFL D3. All samples were prepared for Transmission Electron Microscopy imaging by conventional negative staining method. Briefly, 3.5 μl of sample (for all the constructs) was applied to glow discharged carbon coated copper grid for 30 s followed by blotted out excess buffer and sample. Negative staining was performed using 2% uranyl acetate for 25 s. All negative stained samples were visualized and imaged at room temperature using Tecnai T12 electron microscope equipped with a LaB6 filament operated at 120 kV. All images were recorded using a side-mounted Olympus VELITA (2Kx2K) CCD camera at 2.54 Å/pixel on the specimen level.
The SEC purified samples were prepared for negative staining in the following conditions – (i) NFL Wt, (ii) designed trimer NFL D1, (iii) designed trimer NFL D2, (iv) designed trimer NFL D3. All samples were prepared for Transmission Electron Microscopy imaging by conventional negative staining method. Briefly, 3.5 μl of sample (for all the constructs) was applied to glow discharged carbon coated copper grid for 30 s followed by blotted out excess buffer and sample. Negative staining was performed using 2% uranyl acetate for 25 s. All negative stained samples were visualized and imaged at room temperature using Tecnai T12 electron microscope equipped with a LaB6 filament operated at 120 kV. All images were recorded using a side-mounted Olympus VELITA (2Kx2K) CCD camera at 2.54 Å/pixel on the specimen level.