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Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography
Published on: March 12, 2017
Validation of the orthogonal tilt reconstruction method with a biological test sample
Preethi Chandramouli1, Rogelio Hernandez-Lopez, Hong-Wei Wang
1Department of Molecular and Cellular Biology, Harvard University, USA.
Journal of Structural Biology
|May 4, 2011
Summary
Orthogonal tilt reconstruction (OTR) solves the initial model problem in cryo-electron microscopy (cryo-EM) for novel macromolecules. This method bypasses artifacts, enabling automated structure determination.
Area of Science:
- Structural biology
- Biophysics
- Biochemistry
Background:
- Cryo-electron microscopy (cryo-EM) determines high-resolution macromolecular structures.
- The initial model problem, obtaining orientations ab initio, is a bottleneck for novel macromolecules, especially those lacking symmetry.
Purpose of the Study:
- Validate the orthogonal tilt reconstruction (OTR) method for generating initial models in cryo-EM.
- Demonstrate OTR's superiority over existing methods like random conical tilt (RCT) and angular reconstitution (AR).
Main Methods:
- Applied OTR to a biological test sample (yeast exosome-Rrp44 complex) with a known structure.
- Compared OTR-generated initial models with those from RCT, focusing on "missing cone" artifacts and information distribution.
Main Results:
- OTR successfully generated initial models without "missing cone" artifacts.
- OTR-derived models exhibited an isotropic distribution of information, aligning with the known structure.
- Proposed quantitative criteria for selecting reliable OTR reconstructions for further refinement.
Conclusions:
- OTR effectively addresses the initial model problem in cryo-EM.
- OTR's artifact-free output facilitates automation and analysis of heterogeneous samples.
- The proposed criteria aid in selecting high-quality initial models for high-resolution cryo-EM structure determination.
