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Title: Fully Mechanically Controlled Automated Electron Microscopic Tomography

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:
 [1] ;  [2] ;  [3] ;  [3] ;  [3] ;  [3] ;  [3] ;  [4] ;  [5] ;  [6] ;  [7] ;  [8] ;  [3]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Foundry; Xi'an Jiaotong Univ. (China). State Key Lab. for Manufacturing System Engineering
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Foundry; Xi'an Jiaotong Univ. (China). School of Electrical Engineering
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Foundry
  4. Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.
  5. Pfizer BioThermapeutics Pharmaceutical Sciences, Pear River, NY (United States)
  6. Pfizer BioTherapeutics Pharmaceutical Sciences, St. Louis, MO (United States)
  7. Xi'an Jiaotong Univ. (China). School of Electrical Engineering
  8. Xi'an Jiaotong Univ. (China). State Key Lab. for Manufacturing System Engineering
Publication Date:
Grant/Contract Number:
AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 6; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; electron microscopy; electronic structure of atoms and molecules; molecular imaging; single-molecule biophysics
OSTI Identifier:
1379510