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Updated: Feb 1, 2026

A Robust Single-Particle Cryo-Electron Microscopy cryo-EM Processing Workflow with cryoSPARC, RELION, and Scipion
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goCTF: Geometrically optimized CTF determination for single-particle cryo-EM.

Min Su1

  • 1University of Michigan Life Sciences Institute, Ann Arbor 48109, USA.

Journal of Structural Biology
|November 30, 2018
PubMed
Summary

A new method, goCTF, reliably determines focus gradients in single-particle cryo-electron microscopy (cryo-EM), improving 3D reconstruction resolution. This addresses a key challenge hindering tilt-based cryo-EM imaging techniques.

Keywords:
Contrast transfer functionCryo-electron microscopyFocus gradientLocal focus refinement

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Area of Science:

  • Structural Biology
  • Biophysics
  • Microscopy

Background:

  • Preferred particle orientation is a significant challenge in single-particle cryo-electron microscopy (cryo-EM).
  • Specimen tilting can improve orientation coverage but is limited by focus gradients.
  • Accurate contrast transfer function (CTF) determination is crucial for high-resolution cryo-EM reconstruction.

Purpose of the Study:

  • To develop a robust method for determining global focus gradients in cryo-EM data.
  • To enhance the accuracy of defocus estimation for improved 3D reconstructions.
  • To overcome limitations hindering the widespread adoption of tilt-based cryo-EM imaging.

Main Methods:

  • A geometrically optimized approach (goCTF) was developed to reliably determine global focus gradients.
  • A novel strategy using signal-preserved power spectrum sectors was employed for CTF parameter determination.
  • Per-particle local focus refinement and novel diagnostic plots (defocus standard deviation, goodness of fit heatmap) were utilized.

Main Results:

  • goCTF successfully determined global focus gradients, enabling more accurate CTF correction.
  • Iterative local focus refinement significantly improved defocus accuracy.
  • Application to a tilted influenza hemagglutinin dataset improved 3D reconstruction resolution from 4.1 Å to 3.7 Å.

Conclusions:

  • goCTF effectively addresses the focus gradient problem in cryo-EM, particularly for tilted datasets.
  • The developed methods enhance the reliability and accuracy of CTF determination and focus refinement.
  • goCTF offers a user-friendly, open-source solution built upon existing tools (CTFFIND4) for improved cryo-EM data processing.