The paper introduces the third major release of RELION, a software for automated high-resolution cryo-EM structure determination. Key improvements include CPU-based vector acceleration, reference-free autopicking using Laplacian-of-Gaussian (LoG) filtering, and execution of jobs from Python scripts for non-interactive processing during acquisition. Per-particle refinement of CTF parameters and correction of estimated beam tilt enhance resolution, especially for particles at different heights in the ice or with suboptimal coma-free alignment. Ewald sphere curvature correction improves resolution for large particles. These developments are demonstrated using publicly available datasets, showing resolution improvements of 0.2–0.7 Å compared to previous versions of RELION. The paper also highlights the introduction of Bayesian particle polishing and multi-body refinement, as well as various smaller improvements and new functionalities.The paper introduces the third major release of RELION, a software for automated high-resolution cryo-EM structure determination. Key improvements include CPU-based vector acceleration, reference-free autopicking using Laplacian-of-Gaussian (LoG) filtering, and execution of jobs from Python scripts for non-interactive processing during acquisition. Per-particle refinement of CTF parameters and correction of estimated beam tilt enhance resolution, especially for particles at different heights in the ice or with suboptimal coma-free alignment. Ewald sphere curvature correction improves resolution for large particles. These developments are demonstrated using publicly available datasets, showing resolution improvements of 0.2–0.7 Å compared to previous versions of RELION. The paper also highlights the introduction of Bayesian particle polishing and multi-body refinement, as well as various smaller improvements and new functionalities.