2016Liu Aquasition

Citation

Fully Mechanically Controlled Automated Electron Microscopic Tomography , Jinxin Liu, Hongchang Li, Lei Zhang, Matthew Rames, Meng Zhang, Yadong Yu, Bo Peng, César Díaz Celis, April Xu, Qin Zou, Xu Yang, Xuefeng Chen, Gang Ren, Scientific Reports, (2016), 6:29231, doi: 10.1038/srep29231

Abstract

Knowledge of three-dimensional (3D) structures of each individual particles of asymmetric and flexible proteins is essential in understanding those proteins’ functions; but their structures are difficult to determine. Electron tomography (ET) provides a tool for imaging a single and unique biological object from a series of tilted angles, but it is challenging to image a single protein for three-dimensional (3D) reconstruction due to the imperfect mechanical control capability of the specimen goniometer under both a medium to high magnification (approximately 50,000–160,000×) and an optimized beam coherence condition. Here, we report a fully mechanical control method for automating ET data acquisition without using beam tilt/shift processes. This method could reduce the accumulation of beam tilt/shift that used to compensate the error from the mechanical control, but downgraded the beam coherence. Our method was developed by minimizing the error of the target object center during the tilting process through a closed-loop proportional-integral (PI) control algorithm. The validations by both negative staining (NS) and cryo-electron microscopy (cryo-EM) suggest that this method has a comparable capability to other ET methods in tracking target proteins while maintaining optimized beam coherence conditions for imaging.

Keywords

cryo-electron tomography (cryo-ET); Data acquasition; automation; individual-particle electron tomography (IPET); single-protein 3D structure; single-molecule 3D reconstruction.

Publication link

https://www.nature.com/articles/srep29231