CryoEM Methods: Beam-Induced Motion Attenuation and Selective Particle Capture with Carbon Nanotubes

Modern-day cryo-electron microscopy (cryoEM) has radically changed the scientific landscape, allowing for the atomic mapping of biological molecules without the need for crystallization or the utilization of large-scale facilities such as synchrotrons or free-electron lasers. While accounted for, beam-induced motion remains one of the biggest causes of information loss in cryoEM and has been the subject of ongoing research that has led to the development of various transmission electron grid technologies. This work investigates the use of single-walled carbon nanotubes (SWCNTs) as a support network for the attenuation of beam-induced motion and selective particle capture of cryoEM specimens. The inclusion of SWCNTs within the vitreous meniscus of cryoEM specimens was found to attenuate beam-induced motion due to mechanical reinforcement and also inhibited preferential orientation. Particle motions from experiments with both nanoparticles and proteins were found to have less motion when using SWCNT networks compared to control trials without. Euler angle distributions of particles within SWCNT networks were also found to have less preferential orientation than those of the control according to distance correlations. The reduction in beam-induced motion resulted in greater B-factor weightings of the first movie frames indicating that more information from the first several frames were utilized to build the map. The same SWCNT networks were functionalized with a pyrene–Nitrilotriacetic acid (NTA) moiety such that histidine-tagged proteins would undergo selectively binding to the SWCNT network. Pyrene–NTA moieties were synthesized using two separate methods with the later structures being verified by nuclear magnetic resonance spectroscopy. Binding of the pyrene-NTA moiety to a SWCNT homologue, graphene, was verified with X-ray photoelectron spectroscopy. Selective binding was verified with fluorescence microscopy of histidine tagged green fluorescing protein on functionalized SWCNT grids.