Abstract
Accurate estimation of the contrast transfer function (CTF) is critical for a near-atomic resolution cryo electron microscopy (cryoEM) reconstruction. Here, a GPU-accelerated computer program, Gctf, for accurate and robust, real-time CTF determination is presented. The main target of Gctf is to maximize the cross-correlation of a simulated CTF with the logarithmic amplitude spectra (LAS) of observed micrographs after background subtraction. Novel approaches in Gctf improve both speed and accuracy. In addition to GPU acceleration (e.g. 10–50×), a fast ‘1-dimensional search plus 2-dimensional refinement (1S2R)’ procedure further speeds up Gctf. Based on the global CTF determination, the local defocus for each particle and for single frames of movies is accurately refined, which improves CTF parameters of all particles for subsequent image processing. Novel diagnosis method using equiphase averaging (EPA) and self-consistency verification procedures have also been implemented in the program for practical use, especially for aims of near-atomic reconstruction. Gctf is an independent program and the outputs can be easily imported into other cryoEM software such as Relion (Scheres, 2012) and Frealign (Grigorieff, 2007). The results from several representative datasets are shown and discussed in this paper.
Highlights
Recent progress has allowed cryo-electron microscopy to determine structures of biomacromolecules to near-atomic resolution(Nogales and Scheres, 2015)
In contrast to a simple projection of a 3-dimensional object, the cryo-electron microscopy (cryoEM) image of vitrified specimen is modulated by contrast transfer function (CTF) in Fourier space
2.1 Definition of Contrast transfer function Image formation in a weak-phase approximation is modulated by the CTF which can be defined as Eq (1)
Summary
Recent progress has allowed cryo-electron microscopy (cryoEM) to determine structures of biomacromolecules to near-atomic resolution(Nogales and Scheres, 2015) This is due to developments in multiple fields, but especially better detectors and image processing methods(Bai et al, 2015). The oscillation of the CTF becomes more severe at higher frequency or under a higher defocus For this reason, image restoration is quite challenging, especially for the high frequency information, which makes accurate CTF determination an important factor for near-atomic 3D reconstructions. GPU acceleration as well as an optimized programming strategy makes Gctf very fast It can process thousands of micrographs within minutes using a single GPU card. Beside the determination and refinement of defocus in Gctf, automatic self-consistency verification and micrographs quality evaluation is available for better automation of cryoEM data processing
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