All in-air and in-fluid AFM imaging were performed with a Dimension Icon AFM (Bruker, USA) in Peak Force Mode. The tips selected for in-air AFM imaging and in-fluid imaging were ScanAsyst-Air (k = 0.4 N/m, tip radius ~2 nm; resonance frequency 70 kHz in air) and ScanAsyst-Fluid+ (k = 0.7 N/m, tip radius ~2 nm; resonance frequency 150 kHz in air) (Bruker, https://www.brukerafmprobes.com/ ), respectively. Amel and Ambn were first mixed in Tris-HCl buffer solution (25 mM, 150 mM NaCl, pH 7.4), in which the concentration of Amel was fixed at 50 μg/mL and the concentrations of Ambn were adjusted to obtain various ratios including 1:10, 1:5, 1:3, 1:2, and 1:1 by weight. In addition, 50 μg/mL Amel alone and 25 μg/mL Ambn alone solutions were prepared. These protein solutions were dropped onto a freshly cleaved muscovite mica disc (diameter 9.9 mm, Ted Pella, Inc.) and examined in fluid immediately using AFM.
A 50 μg/mL Amel solution in Tris-HCl buffer was dropped onto the mica disc for 5 min, rinsed with UPW, and then dried with argon. The same procedure was performed on 25 μg/mL Ambn, 17 μg/mL AB2, 17 μg/mL AB4, 17 μg/mL AmbnΔ5, 17 μg/mL AB2 and 50 μg/mL Amel, 17 μg/mL AB4 and 50 μg/mL Amel, and 17 μg/mL AmbnΔ5 and 50 μg/mL Amel. These samples were imaged in air using AFM. The images were analyzed using the image processing software package NanoScope Analysis version 2.0 (Bruker).
A 50 μg/mL Amel solution in Tris-HCl buffer was dropped onto the mica disc for 5 min, rinsed with UPW, and then dried with argon. The same procedure was performed on 25 μg/mL Ambn, 17 μg/mL AB2, 17 μg/mL AB4, 17 μg/mL AmbnΔ5, 17 μg/mL AB2 and 50 μg/mL Amel, 17 μg/mL AB4 and 50 μg/mL Amel, and 17 μg/mL AmbnΔ5 and 50 μg/mL Amel. These samples were imaged in air using AFM. The images were analyzed using the image processing software package NanoScope Analysis version 2.0 (Bruker).