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We could overcome the recalcitrance of holo-AMPK to crystallization in the absence of pharmacological activators by extensive construct screening and by altering three flexible solvent-exposed side chains (E471G/E474A/K476A) at the N-terminus of the ST-loop of the α-subunit (see Materials and Methods and Figure 2A).
The extensive construct modification altered ion channel function; peak currents were reduced, channel closure after removal of agonist was delayed, and agonist potency was increased [6], implying that the structure may not represent a native conformation, but rather an artificially stabilized, closed state.
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