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

Joseph Atherton,Carolyn A Moores

doi:10.1107/s2059798321001935

Abstract

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.

Highlights

Since the recent revolution in hardware and software (Kuhlbrandt, 2014), cryo-electron microscopy has become a popular and effective method of macromolecular structure determination
kinesin-binding protein (KBP) becomes partially denatured at the air–water interface, a process prevented by adherence to a graphene oxide substrate
We have detailed our experiences in preparing KBP and KBP–kinesin motor domain (MD) complexes for cryo-electron microscopy (cryo-EM), in particular relating to sample behavior on different EM grid types

Summary

Introduction

Since the recent revolution in hardware and software (Kuhlbrandt, 2014), cryo-electron microscopy (cryo-EM) has become a popular and effective method of macromolecular structure determination. There has been a growing awareness that macromolecules interact with various surfaces on EM grids during sample preparation and that this can cause protein unfolding and/or conformational artifacts (Glaeser & Han, 2017). In this technical report, we detail our experiences when preparing samples of kinesin-binding protein (KBP; 72 kDa) alone or in complex with two different kinesin motor domains ($40 kDa). We highlight technical issues with the use of GO, and with combining Volta phase plate (VPP) cryo-EM with tilted data collection

Methods

Results

Conclusion