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J Struct Biol. 2014 Aug;187(2):103-111. doi: 10.1016/j.jsb.2014.06.007. Epub 2014 Jul 3.

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

Author information

1
Quantitative Imaging Group, Department of Imaging Physics, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
2
Quantitative Imaging Group, Department of Imaging Physics, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands; Institute of Nanoscopy, Faculty of Health, Medicine and Life Sciences, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands.
3
NKI Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital, Plesmanlaan 121 B6, 1066 CX Amsterdam, The Netherlands.
4
FEI Company, Achtseweg Noord 5, 5651 GG Eindhoven, The Netherlands.
5
Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 67084 Strasbourg Cedex, France.
6
Institute of Nanoscopy, Faculty of Health, Medicine and Life Sciences, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands; NKI Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital, Plesmanlaan 121 B6, 1066 CX Amsterdam, The Netherlands.
7
Quantitative Imaging Group, Department of Imaging Physics, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands. Electronic address: b.rieger@tudelft.nl.

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 CTF correction and defocus estimation methods are required. We obtain a final average using 3198 ribosomes with a resolution of 2.2nm on the experimental data. Our simulations suggest that with the same number of particles a resolution of 1.2nm could be achieved by improving the tilt-series alignment.

KEYWORDS:

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

PMID:
24998892
DOI:
10.1016/j.jsb.2014.06.007
[Indexed for MEDLINE]

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