Quantifying resolution limiting factors in subtomogram averaged cryo-electron tomography using simulations

Lenard M. Voortman, Milos Vulovic, Massimiliano Maletta, Andreas Voigt, Erik M. Franken, Angelita Simonetti, Peter J. Peters, Lucas J. van Vliet, Bernd Rieger*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Cryo-electron tomography (CET) is the only available technique capable of characterizing the structure of biological macromolecules in conditions close to the native state. With the advent of subtomogram averaging, as a post-processing step to CET, resolutions in the (sub-) nanometer range have become within reach. In addition to advances in instrumentation and experiments, the reconstruction scheme has improved by inclusion of more accurate contrast transfer function (CTF) correction methods, better defocus estimation, and better alignments of the tilt-series and subtomograms. To quantify the importance of each contribution, we have split the full process from data collection to reconstruction into different steps. For the purpose of evaluation we have acquired tilt-series of ribosomes in such a way that we could precisely determine the defocus of each macromolecule. Then, we simulated tilt-series using the InSilicoTEM package and applied tomogram reconstruction and subtomogram averaging. Through large scale simulations under different conditions and parameter settings we find that tilt-series alignment is the resolution limiting factor for our experimental data. Using simulations, we find that when this alignment inaccuracy is alleviated, tilted CTF correction improves the final resolution, or equivalently, the same resolution can be achieved using less particles. Furthermore, we predict from which resolution onwards better CIF correction and defocus estimation methods are required. We obtain a final average using 3198 ribosomes with a resolution of 2.2 nm on the experimental data. Our simulations suggest that with the same number of particles a resolution of 1.2 nm could be achieved by improving the tilt-series alignment.
Original languageEnglish
Pages (from-to)103-111
JournalJournal of Structural Biology
Volume187
Issue number2
DOIs
Publication statusPublished - Aug 2014

Keywords

  • Cryo-EM
  • Tomography
  • Subtomogram averaging
  • TEM image simulation
  • Tilted CTF correction
  • Acquisition protocol
  • Ribosome

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