AFM images were acquired at ~32°C in tapping mode using a commercial instrument (Asylum Research Cypher) in 10 mM Hepes (pH 7.6), 100 mM KAc, and 5 mM MgAc2 or 10 mM Hepes (pH 7.6), 100 mM KAc, 5 mM MgAc2 ,and 100 μM nucleotide, as specified. Measurement-related bias of protein conformations and conformational dynamics is an ever present concern. Care was taken to control the magnitude of the tip sample force to ⪝100 pN [estimated by comparing the free amplitude (~10 nm) to the set point amplitude]. BioLever mini tips (BL-AC40TS, Olympus) with measured spring constants of ~0.06 N/m were used. Spring constants were determined using the thermal noise method. AFM images were acquired with the following parameters: scan rate, 3.6 Hz; scan size, 512 pixels × 512 pixels, 1 μm2 × 1 μm2; scan speed, 7 nm/ms. Kymographs were acquired as follows: scan rate, 5.2 Hz; scan size, 320 pixels, 300 nm; scan speed, 3 nm/ms. We analyzed at least 17 images per condition studied, at least 3 independent experiments/mica stages/sample preparations per condition, and at least 2 different tips for each independent experiment. Note that tips were recovered after each experiment and cleaned for reuse.

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