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Cryo‐EM of kinesin‐binding protein: challenges and opportunities from protein‐surface interactions
Author(s) -
Atherton Joseph,
Moores Carolyn A.
Publication year - 2021
Publication title -
acta crystallographica section d
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.374
H-Index - 138
ISSN - 2059-7983
DOI - 10.1107/s2059798321001935
Subject(s) - kinesin , microtubule , tetratricopeptide , motor protein , biophysics , microbiology and biotechnology , plasma protein binding , mitosis , biology , chemistry , genetics , gene
Kinesin‐binding protein (KBP) is an important selective inhibitor of specific kinesin family members and its genetic disruption causes Goldberg–Shprintzen syndrome. Cryo‐electron microscopy (cryo‐EM) has recently been used 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 behavior of KBP and KBP–kinesin complexes during cryo‐EM sample preparation. These included the 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 the eventual structure determination. Here, experiences with preparing these samples are detailed, bringing attention to some of the challenges and opportunities that are likely to arise from protein‐surface interactions.