Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography

Autor: Hsuan-Fu Liu, Bradley P. Klemm, Roel M. Schaaper, Xiaochen Du, Mario J. Borgnia, Ye Zhou, Rick Huang, Andrew P. Sikkema, Jonathan Bouvette, Juliana da Fonseca Rezende e Mello, Alberto Bartesaghi
Jazyk: angličtina
Rok vydání: 2021
Předmět:
0301 basic medicine
Electron Microscope Tomography
Materials science
Macromolecular Substances
Science
General Physics and Astronomy
Electron
Biochemistry
General Biochemistry
Genetics and Molecular Biology
Article
law.invention
Inorganic Chemistry
03 medical and health sciences
0302 clinical medicine
Optics
Data acquisition
Imaging
Three-Dimensional
law
Atomic resolution
Image Processing
Computer-Assisted
General Materials Science
Physical and Theoretical Chemistry
Particle Size
Multidisciplinary
Tomographic reconstruction
business.industry
Resolution (electron density)
Cryoelectron Microscopy
Reproducibility of Results
General Chemistry
Condensed Matter Physics
Weighting
030104 developmental biology
Cryo-electron tomography
Particle
Cryoelectron tomography
Electron microscope
business
Structural biology
Tomography
X-Ray Computed
030217 neurology & neurosurgery
Beam (structure)
Algorithms
Zdroj: Nature Communications
Nature Communications, Vol 12, Iss 1, Pp 1-11 (2021)
ISSN: 2041-1723
Popis: Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Here, we introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by up to an order of magnitude and improve map resolution compared to existing approaches. We achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. We validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300 kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved.
Tomographic reconstructions of cryopreserved specimens enable in-situ structural studies. Here, the authors present the beam image-shift electron cryo-tomography (BISECT) approach that accelerates data collection speed and improves the map resolution compared to earlier approaches and present the in vitro structure of a 300 kDa protein complex that was solved at 3.6 Å resolution as a test case.
Databáze: OpenAIRE
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