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The Ni-NTA resin was packed into a glass column and washed with 25 column volumes of 20 mM HEPES pH 7.4, 100 mM NaCl, 2 mM MgCl 2 , 0.01% (w/v) LMNG and 0.0006% (w/v) CHS, 1 M peptide, followed by elution using the same buffer supplemented with 250 mM imidazole and 3 mM CaCl 2

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H296 ECL2 adopts a distinct rotamer in Cagri-bound CTR relative to Cagri-bound-AMY 1 R to accommodate the Glu linker that extends proximal to the top segment of TM5

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