SecA was diluted to 20 nM and incubated for 15 min in one of two solutions: (i) no substrate (apo) conditions [10 mM Hepes (pH 7.6), 100 mM KAc, and 5 mM MgAc2] or (ii) saturating nucleotide (ATP, ADP, or ADP-AlF3) conditions [10 mM Hepes (pH 7.6), 100 mM KAc, 5 mM MgAc2, and 100 μM nucleotide]. One hundred fifty microliters of this solution was then deposited onto a freshly cleaved mica surface. After 10-min incubation on mica, the sample was rinsed 10 times with 100 μl of the appropriate solution.

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