The article presents a comprehensive image processing pipeline for electron cryo-tomography (cryo-ET) in RELION-5, a free and open-source software. The pipeline addresses the challenges of processing noisy, three-dimensional (3D) reconstructions of macromolecular complexes embedded in complex environments. Key features of the pipeline include: 1. **Metadata Definition**: The pipeline defines metadata items for tilt series, tomograms, and extracted particles, promoting interoperability with other software tools. 2. **Import and Preparation**: It handles the import of unprocessed movies and acquisition metadata, generating metadata files in the STAR format. 3. **Motion Correction**: Uses UCSF MotionCor2 or RELION's own implementation for movie frame alignment. 4. **CTF Estimation**: Wraps the CTFFIND4 algorithm to estimate contrast transfer function (CTF) parameters, enhancing the robustness of CTF estimation at high tilt angles. 5. **Tilt Series Alignment**: Implements wrappers for IMOD and AreTomo to align tilt series, improving the accuracy of 3D reconstructions. 6. **Tomogram Reconstruction**: Conducts real-space weighted back-projection to reconstruct 3D tomograms, suitable for particle picking and subtomogram averaging. 7. **Denoising**: Utilizes the cryo-CARE program to denoise tomograms, reducing noise and improving resolution. 8. **Particle Picking**: Provides tools for interactive 3D annotation and particle picking, allowing users to select particles from spheres or filaments. 9. **Subtomogram Averaging**: Offers methods for 3D initial reference, 3D classification, 3D auto-refine, and 3D multi-body, enabling the extraction of pseudo-subtomograms or 2D particle stacks. 10. **Bayesian Polishing and CTF Refinement**: Enhances the accuracy of 3D reconstructions through Bayesian polishing and CTF refinement. The pipeline has been successfully applied to two cryo-ET datasets, achieving high-resolution reconstructions and atomic models. The authors emphasize the importance of continuous updates and standardization in the field of cryo-ET image processing, suggesting that the explicit metadata definitions in RELION-5 can contribute to broader efforts in standardization and interoperability.The article presents a comprehensive image processing pipeline for electron cryo-tomography (cryo-ET) in RELION-5, a free and open-source software. The pipeline addresses the challenges of processing noisy, three-dimensional (3D) reconstructions of macromolecular complexes embedded in complex environments. Key features of the pipeline include: 1. **Metadata Definition**: The pipeline defines metadata items for tilt series, tomograms, and extracted particles, promoting interoperability with other software tools. 2. **Import and Preparation**: It handles the import of unprocessed movies and acquisition metadata, generating metadata files in the STAR format. 3. **Motion Correction**: Uses UCSF MotionCor2 or RELION's own implementation for movie frame alignment. 4. **CTF Estimation**: Wraps the CTFFIND4 algorithm to estimate contrast transfer function (CTF) parameters, enhancing the robustness of CTF estimation at high tilt angles. 5. **Tilt Series Alignment**: Implements wrappers for IMOD and AreTomo to align tilt series, improving the accuracy of 3D reconstructions. 6. **Tomogram Reconstruction**: Conducts real-space weighted back-projection to reconstruct 3D tomograms, suitable for particle picking and subtomogram averaging. 7. **Denoising**: Utilizes the cryo-CARE program to denoise tomograms, reducing noise and improving resolution. 8. **Particle Picking**: Provides tools for interactive 3D annotation and particle picking, allowing users to select particles from spheres or filaments. 9. **Subtomogram Averaging**: Offers methods for 3D initial reference, 3D classification, 3D auto-refine, and 3D multi-body, enabling the extraction of pseudo-subtomograms or 2D particle stacks. 10. **Bayesian Polishing and CTF Refinement**: Enhances the accuracy of 3D reconstructions through Bayesian polishing and CTF refinement. The pipeline has been successfully applied to two cryo-ET datasets, achieving high-resolution reconstructions and atomic models. The authors emphasize the importance of continuous updates and standardization in the field of cryo-ET image processing, suggesting that the explicit metadata definitions in RELION-5 can contribute to broader efforts in standardization and interoperability.