GoCTF: Geometrically optimized CTF determination for single-particle cryo-EM

Min Su

doi:10.1016/j.jsb.2018.11.012

Abstract

Preferred particle orientation represents a recurring problem in single-particle cryogenic electron microcopy (cryo-EM). A specimen-independent approach through tilting has been attempted to increase particle orientation coverage, thus minimizing anisotropic three-dimensional (3D) reconstruction. However, focus gradient is a critical issue hindering tilt applications from being a general practice in single-particle cryo-EM. The present study describes a newly developed geometrically optimized approach, goCTF, to reliably determine the global focus gradient. A novel strategy of determining contrast transfer function (CTF) parameters from a sector of the signal preserved power spectrum is applied to increase reliability. Subsequently, per-particle based local focus refinement is conducted in an iterative manner to further improve the defocus accuracy. Novel diagnosis methods using a standard deviation defocus plot and goodness of fit heatmap have also been proposed to evaluate CTF fitting quality prior to 3D refinement. In a benchmark study, goCTF processed a published single-particle cryo-EM dataset for influenza hemagglutinin trimer collected at a 40-degree specimen tilt. The resulting 3D reconstruction map was improved from 4.1 Å to 3.7 Å resolution. The goCTF program is built on the open-source code of CTFFIND4, which adopts a consistent user interface for ease of use.

Highlights

Su taken full advantage of this compustage innovation, for image acquisition at specimen tilt
The novel program goCTF was developed to determine the contrast transfer function (CTF) for singleparticle cryogenic electron microcopy (cryo-EM) customized for tilt applications
With its geometrically optimized approach, goCTF provides an ultra-reliable solution in per-particle focus refinement that is independent from the particle abundance at a given local region of interest in a micrograph

Summary

Introduction

Su taken full advantage of this compustage innovation, for image acquisition at specimen tilt. Various approaches have been explored to solve preferred particle orientation (Chowdhury et al, 2015; Frank et al, 1991; Liu et al, 2013; Lyumkis et al, 2013; Meyerson et al, 2014; Nguyen et al, 2015); these methods are only occasionally effective in increasing the range of particle orientation. These approaches are highly specimendependent and far from reproducible. In another study (Tan et al, 2017), the authors demonstrated for the first time that single-particle cryo-EM is capable of reaching near-atomic resolution with the data collected at a 40-degree specimen tilt, using influenza hemagglutinin trimer as a benchmark sample

Results

Discussion

Conclusion