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

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 d...

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Autores principales: Atherton, Joseph, Moores, Carolyn A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: International Union of Crystallography 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025885/
https://www.ncbi.nlm.nih.gov/pubmed/33825702
http://dx.doi.org/10.1107/S2059798321001935
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author Atherton, Joseph
Moores, Carolyn A.
author_facet Atherton, Joseph
Moores, Carolyn A.
author_sort Atherton, Joseph
collection PubMed
description 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.
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spelling pubmed-80258852021-04-30 Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions Atherton, Joseph Moores, Carolyn A. Acta Crystallogr D Struct Biol Ccp-EM 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. International Union of Crystallography 2021-03-30 /pmc/articles/PMC8025885/ /pubmed/33825702 http://dx.doi.org/10.1107/S2059798321001935 Text en © Atherton & Moores 2021 http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.http://creativecommons.org/licenses/by/4.0/
spellingShingle Ccp-EM
Atherton, Joseph
Moores, Carolyn A.
Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions
title Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions
title_full Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions
title_fullStr Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions
title_full_unstemmed Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions
title_short Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions
title_sort cryo-em of kinesin-binding protein: challenges and opportunities from protein-surface interactions
topic Ccp-EM
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025885/
https://www.ncbi.nlm.nih.gov/pubmed/33825702
http://dx.doi.org/10.1107/S2059798321001935
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