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J Struct Biol. 2015 Nov;192(2):196-203. doi: 10.1016/j.jsb.2015.09.006. Epub 2015 Sep 25.

The influence of frame alignment with dose compensation on the quality of single particle reconstructions.

Author information

1
Institute of Molecular Biophysics, 91 Chieftan Way, Florida State University, Tallahassee, FL 32306-4380, United States.
2
Department of Physics, 77 Chieftan Way, Florida State University, Tallahassee, FL 32306-4350, United States.
3
Department of Biochemistry & Molecular Biology, School of Medicine, Oregon Health & Science University, Portland, OR 97239-3098, United States.
4
Department of Biological Science, Florida State University, 319 Stadium Drive, Tallahassee, FL 32306, United States.
5
Institute of Molecular Biophysics, 91 Chieftan Way, Florida State University, Tallahassee, FL 32306-4380, United States; Departments of Chemistry and Biochemistry, 95 Chieftain Way, Florida State University, Tallahassee, FL 32306-4390, United States. Electronic address: sstagg@fsu.edu.

Abstract

As direct electron detection devices in cryo-electron microscopy become ubiquitous, the field is now ripe for new developments in image analysis techniques that take advantage of their increased SNR coupled with their high-throughput frame collection abilities. In approaching atomic resolution of native-like biomolecules, the accurate extraction of structural locations and orientations of side-chains from frames depends not only on the electron dose that a sample receives but also on the ability to accurately estimate the CTF. Here we use a new 2.8Å resolution structure of a recombinant gene therapy virus, AAV-DJ with Arixtra, imaged on an FEI Titan Krios with a DE-20 direct electron detector to probe new metrics including relative side-chain density and ResLog analysis for optimizing the compensation of electron beam damage and to characterize the factors that are limiting the resolution of the reconstruction. The influence of dose compensation on the accuracy of CTF estimation and particle classifiability are also presented. We show that rigorous dose compensation allows for better particle classifiability and greater recovery of structural information from negatively charged, electron-sensitive side-chains, resulting in a more accurate macromolecular model.

KEYWORDS:

Atomic resolution; Beam-induced motion; Cryo-electron microscopy; Direct electron detector; Dose compensation; Radiation damage

PMID:
26391007
PMCID:
PMC4633374
DOI:
10.1016/j.jsb.2015.09.006
[Indexed for MEDLINE]
Free PMC Article

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