Table S5.

Fit parameters from analysis of time-resolved fluorescence decays of SecA–PhoA–SecYEG complexes

	FRET pairs
Fit parameters*	SecA37–AF647–PhoA22–AF488 + ADP + SecYEG		SecA321–AF647–PhoA22–AF488 + ADP + SecYEG		SecA321C–PhoA22–AF647 + SecY292–AF488 + SecEG + ADP		SecA321C–PhoA22–AF647 + SecY292–AF488 + SecEG + ATP-γS
	Donor only	Donor–acceptor	Donor only	Donor–acceptor	Donor only	Donor–acceptor	Donor only	Donor–acceptor
α1	0.40	0.27	0.17	0.18	0.63	0.38	0.58	0.25
τ1	3.80	3.04	2.85	1.21	3.75	3.77	3.69	3.38
<τ>†	1.50	0.82	0.48	0.21	2.38	1.45	2.16	0.86
χ2	1.21	1.11	1.17	1.02	2.78‡	1.33	2.78‡	0.966
EFRET§	0.46		0.56		0.39		0.60

^*Fluorescence intensity decays (Fig. S3) were fit to a sum of exponentials using the following expression: I(t) = ${\displaystyle {\sum }_i{\alpha }_i{e}^{-t/{\tau }_i}}$. Decays were well described by a single exponential, as judged by the χ² value and a visual inspection of the residuals. Acquisition and analysis details are given in SI Materials and Methods.

^†The amplitude-weighted lifetime is proportional to the steady-state intensity and is calculated using the following expression: $\langle \tau \rangle \hspace{0.17em}=\hspace{0.17em}{\displaystyle {\sum }_i{\alpha }_i{\tau }_i}$.

^‡This sample exhibited a higher χ² value due to some precipitation of the sample during spectral acquisition.

^§E_FRET is calculated as described in SI Materials and Methods.