Cryogenic electron microscopy (Cryo-EM) has been established as one of the key players in structural biology. It can reconstruct a 3D model of a sample at a near-atomic resolution. With the increasing number of facilities, faster microscopes, and new imaging techniques, there is a growing demand for algorithms and programs able to process the so-called movie data produced by the microscopes in real time while preserving a high resolution and maximal information. In this article, we conduct a comparative analysis of the quality and performance of the most commonly used software for movie alignment. More precisely, we compare the most recent versions of FlexAlign (Xmipp v3.23.03), MotionCor2 (v1.6.4), Relion MotionCor (v4.0-beta), Warp (v1.0.9), and CryoSPARC (v4.0.3). We tested the quality of the alignment using generated phantom data, as well as real datasets, comparing the alignment precision, power spectra density, and performance scaling of each program.

Institute of Computer Science Masaryk University Botanická 68a 60200 Brno Czech Republic

Spanish National Centre for Biotechnology Spanish National Research Council Calle Darwin 3 28049 Madrid Spain

Zobrazit více v PubMed

Glaeser R. Chapter Two—Specimen Behavior in the Electron Beam. In: Crowther R., editor. Methods in Enzymology. Volume 579. Academic Press; Cambridge, MA, USA: 2016. pp. 19–50. PubMed DOI

Hattne J., Martynowycz M.W., Penczek P.A., Gonen T. MicroED with the Falcon III direct electron detector. IUCrJ. 2019;6:921–926. doi: 10.1107/S2052252519010583. PubMed DOI PMC

Fréchin L., Holvec S., Von Loeffelholz O., Hazemann I., Klaholz B. High-resolution cryo-EM performance comparison of two latest-generation cryo electron microscopes on the human ribosome. J. Struct. Biol. 2023;215:107905. doi: 10.1016/j.jsb.2022.107905. PubMed DOI

Marko A. CryoEM Takes Center Stage: How Compute, Storage, and Networking Needs Are Growing with CryoEM Research. 2019. [(accessed on 1 February 2023)]. Available online: https://www.microway.com/hpc-tech-tips/cryoem-takes-center-stage-how-compute-storage-networking-needs-growing.

Zhou X., Li Y., Zhang C., Zheng W., Zhang G., Zhang Y. Progressive assembly of multi-domain protein structures from cryo-EM density maps. Nat. Comput. Sci. 2022;4:265–275. doi: 10.1038/s43588-022-00232-1. PubMed DOI PMC

Frenz B., Walls A.C., Egelman E.H., Veesler D., DiMaio F. RosettaES: A sampling strategy enabling automated interpretation of difficult cryo-EM maps. Nat. Methods. 2017;8:797–800. doi: 10.1038/nmeth.4340. PubMed DOI PMC

Punjani A., Rubinstein J.L., Fleet D.J., Brubaker M.A. cryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods. 2017;14:290–296. doi: 10.1038/nmeth.4169. PubMed DOI

Střelák D., Filipovič J., Jiménez-Moreno A., Carazo J., Sánchez Sorzano C. FlexAlign: An Accurate and Fast Algorithm for Movie Alignment in Cryo-Electron Microscopy. Electronics. 2020;9:1040. doi: 10.3390/electronics9061040. DOI

Zivanov J., Nakane T., Forsberg B.O., Kimanius D., Hagen W.J., Lindahl E., Scheres S.H. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife. 2018;7:e42166. doi: 10.7554/eLife.42166. PubMed DOI PMC

Zheng S.Q., Palovcak E., Armache J.P., Verba K.A., Cheng Y., Agard D.A. MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods. 2017;14:331–332. doi: 10.1038/nmeth.4193. PubMed DOI PMC

Tegunov D., Cramer P. Real-time cryo-electron microscopy data preprocessing with Warp. Nat. Methods. 2019;16:1146–1152. doi: 10.1038/s41592-019-0580-y. PubMed DOI PMC

De la Rosa-Trevín J.M., Otón J., Marabini R., Zaldívar A., Vargas J., Carazo J.M., Sorzano C.O.S. Xmipp 3.0: An improved software suite for image processing in electron microscopy. Elsevier. 2013;184:321–328. doi: 10.1016/j.jsb.2013.09.015. PubMed DOI

Strelak D., Jiménez-Moreno A., Vilas J., Ramírez-Aportela E., Sánchez-García R., Maluenda D., Vargas J., Herreros D., Fernández-Giménez E., de Isidro-Gómez F., et al. Advances in Xmipp for Cryo–Electron Microscopy: From Xmipp to Scipion. Molecules. 2021;26:20. doi: 10.3390/molecules26206224. PubMed DOI PMC

Krishna K.K., Shalev-Benami M., Robertson M., Hu H., Banister S., Hollingsworth S., Latorraca N., Kato H., Hilger D., Maeda S., et al. Cryo Electron Microscopy of Cannabinoid Receptor 1-G Protein Complex. Cell. 2019;176:448–458.e12. doi: 10.1016/j.cell.2018.11.040. PubMed DOI PMC

Suga M., Ozawa S., Yoshida-Motomura K., Akita F., Miyazaki N., Takahashi Y. Structure of the green algal photosystem I supercomplex with a decameric light-harvesting complex I. Nat. Plants. 2019;5:626–636. doi: 10.1038/s41477-019-0438-4. PubMed DOI

Nureki O., Kasuya G., Nakane T., Yokoyama T., Jia Y., Inoue M., Watanabe K., Nakamura R., Nishizawa T., Kusakizako T., et al. Cryo-EM structures of the human volume-regulated anion channel LRRC8. Nat. Struct. Mol. Biol. 2018;25:797–804. doi: 10.1038/s41594-018-0109-6. PubMed DOI

Conesa P., Fonseca Y.C., de la Morena J.J., Sharov G., de la Rosa-Trevín J.M., Cuervo A., Mena A.G., de Francisco B.R., del Hoyo D., Herreros D., et al. Scipion3: A workflow engine for cryo-electron microscopy image processing and structural biology. Biol. Imaging. 2023;3:e13. doi: 10.1017/S2633903X23000132. PubMed DOI PMC

Střelák D., Marchan D. Performance and Quality Comparison of Movie Alignment Software for Cryo-EM. Zenodo. 2023. [(accessed on 1 August 2023)]. Available online: https://zenodo.org/record/8186837. PubMed PMC

Střelák D., Marchan D. Benchmarking Phantom Movies for Performance and Quality Comparison of Movie Alignment software for Cryo-EM. Empiar. 2023. pending for acceptance . PubMed PMC

Performance and Quality Comparison of Movie Alignment Software for Cryogenic Electron Microscopy