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

John M. Spear, Alex J. Noble, Qing Xie, Duncan R. Sousa, Michael S. Chapman, Scott M. Stagg

Research output: Contribution to journalArticle

17 Scopus citations

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 2.8. Å 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.

Original languageEnglish (US)
Pages (from-to)196-203
Number of pages8
JournalJournal of Structural Biology
Volume192
Issue number2
DOIs
StatePublished - Nov 2015

Keywords

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

ASJC Scopus subject areas

  • Structural Biology

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