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Finally, TraK is required for or strongly stimulates the transfer of cognate (pKM101, TraI relaxase) and noncognate (RSF1010, MobA relaxase) substrates. We propose that TraK functions not only to nucleate pKM101 relaxosome assembly, but also to activate the TrapKM101 T4SS via interactions with the ATPase energy center positioned at the channel entrance. © 2020 John Wiley Sons Ltd.External quenching method based on flow microreactors allowed generating and using short-lived fluoro-substituted methyllithiums such as fluoromethyllithi