Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions

Atherton, Joe and Moores, Carolyn A. (2021) Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions. Acta Crystallographica Section D Biological Crystallography D77 , pp. 411-423. ISSN 0907-4449.

Abstract

Kinesin-binding protein (KBP) is an important selective inhibitor of specific kinesin family members and its genetic disruption causes Goldberg-Shprintzen syndrome (GOSHS). We recently used cryo-electron microscopy to reveal the structure of KBP alone (72 kDa) and in complex with the motor domain of the mitotic kinesin-12 KIF15 (110 kDa). KBP is an α-solenoid, tetratricopeptide repeat protein that interacts with the microtubule-binding region of the kinesin motor domain, and blocks microtubule attachment. Numerous challenges arose relating to the behaviour of KBP and KBP-kinesin complexes during cryo-EM sample preparation. This included partial denaturation of KBP by air-water interfaces, protein aggregation resulting from carbon interaction and preferential orientation. Sample preparation with a graphene oxide substrate enabled eventual structure determination. Here we detail our experiences with preparing these samples, bringing attention to some of the challenges and opportunities likely arising from protein-surface interactions.

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