Ac160

Titin samples were imaged with a high-resolution atomic force microscope (Cypher, AsylumResearch, Santa Barbara, CA). Dehydrated samples were imaged in tapping mode with a stiff cantilever (Olympus AC160 or AC55TS with nominal tip radii of 9 and 7 nm, respectively) at a line-scan rate of 3–6 Hz and pixel resolution of 0.5 - 2 nm. Hydrated samples were scanned in tapping mode with a soft, high-resonance-frequency cantilever (Olympus BL-RC150VB, BioLever B, nominal tip radius 25 nm) a a line-scan rate of 2–3 Hz and a pixel resolution of 2–4 nm. Images were corrected for flatness of field and color contrast (offset and range) by using built-in algorithms of the AFM driver software. Topographical distance (e.g., filament width) measurements were corrected for tip convolution as published earlier [29] (link). Accordingly, titin molecule width (W) was calculated as where W_m is measured width at half of the maximum, r is tip radius and h is topographical height in the filament axis. The width of topographical gaps (G) was obtained as where G_m is the measured gap width.

An aggregated sample drop (5 µl) was placed on freshly cleaved mica and incubated at RT for 5 min. The excess contacting a filter paper to the edge of the drop. The mica was washed 3-4 times with water and subsequently dried with a nitrogen stream. The sample was imaged on an AFM (JPK Nano wizard 4 AFM, Germany) using AC240 or AC160 cantilevers in tapping mode. The images were processed with JPK data processing software. The mechanical properties of the peptides were measured using “peak force QNM” on a Multimode AFM (Bruker). AC160 (Olympus) or RTESP probes (Bruker) were used with calibrated spring constants between 40 and 50 N/m. The tip area function was calibrated on a sample of HOPG with a modulus of 18 GPa. Deformation depths were kept to about 1 nm. Finally, the built-in software computed the elastic modulus by fitting the force vs. deformation curves to a DMT model.