title: Fully Mechanically Controlled Automated Electron Microscopic Tomography.
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.
authors.affiliation: The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.|State Key Laboratory for Manufacturing System Engineering, Xi'an Jiaotong University, Xi'an, 710049, P.R. China.
authors.forename: Jinxin
authors.initials: J
authors.lastname: Liu
authors.affiliation: The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.|School of Electrical Engineering, Xi'an Jiaotong University, Xi'an, 710049, P.R. China.
authors.forename: Hongchang
authors.initials: H
authors.lastname: Li
authors.affiliation: The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Rames
authors.affiliation: The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Meng
authors.initials: M
authors.lastname: Zhang
authors.affiliation: The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Yadong
authors.initials: Y
authors.lastname: Yu
authors.affiliation: The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Peng
authors.affiliation: Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, USA.
authors.forename: César Díaz
authors.initials: CD
authors.lastname: Celis
authors.affiliation: Pfizer BioTherapeutics Pharmaceutical Sciences, 401 N Middletown Rd, Pear River, NY 10956, USA.
authors.forename: April
authors.initials: A
authors.lastname: Xu
authors.affiliation: Pfizer BioTherapeutics Pharmaceutical Sciences, 700 Chesterfield Parkway West, St Louis, MO 63017, USA.
authors.forename: Qin
authors.initials: Q
authors.lastname: Zou
authors.affiliation: School of Electrical Engineering, Xi'an Jiaotong University, Xi'an, 710049, P.R. China.
authors.forename: Xu
authors.initials: X
authors.lastname: Yang
authors.affiliation: State Key Laboratory for Manufacturing System Engineering, Xi'an Jiaotong University, Xi'an, 710049, P.R. China.
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Chen
authors.affiliation: The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Ren
mesh_terms: D000465:Algorithms; D000818:Animals; D001331:Automation; D035843:Biomedical Research; D020285:Cryoelectron Microscopy; D055032:Electron Microscope Tomography / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional / Q000379:methods*; D016624:Negative Staining / Q000379:methods*