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. 2019 Jul 16;28(3):723–734.e6. doi: 10.1016/j.celrep.2019.06.052

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© 2019 The Author(s)

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Conformational States of ATP Sites I and II of ABCE1 Revealed by ALEX-Based smFRET

(A and C) 2D-ALEX histogram of site I (A) and site II (C) variants labeled with Cy3B and Atto647N reveals three conformational states (open, intermediate, and closed). 2D-ALEX histogram of free ABCE1 (top panel); ABCE1 with AMP-PNP, 70S, and aRF1/aPelota (middle panel); and AMP-PNP and 30S (bottom panel). Interaction partners (3 μM 70S and aRF1/aPelota or 1 μM 30S ribosome, 2 mM AMP-PNP) affect these states. For clarity, only the double-labeled donor-acceptor species are shown. Cartoons depict the percentage of each state in the conformational equilibrium, with different transparencies as indicated.

(B and D) Burst-size histogram of site I (B) and II (D) variants of free ABCE1 and the pre-splitting state and post-splitting state of ABCE1 reveals the diffusion properties of the complexes and allows determination of a relative diffusion coefficient D by fitting the histogram to Equation 1 (Method Details). Errors indicate 95% confidence interval.