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Heterogeneous cryo-EM projection image classification is an effective way to discover conformational heterogeneity of biological macromolecules in different functional states. However, due to the low signal-to-noise ratio of the projection images, the classification of heterogeneous cryo-EM projection images is a very challenging task. In this paper, two novel distance measures between projection images integrating the reliability of common lines, pixel intensity and class averages are designed, and then a two-stage spectral clustering algorithm based on the two distance measures is proposed for heterogeneous cryo-EM projection image classification. In the first stage, the novel distance measure integrating common lines and pixel intensities of projection images is used to obtain preliminary classification results through spectral clustering. In the second stage, another novel distance measure integrating the first novel distance measure and class averages generated from each group of projection images is used to obtain the final classification results through spectral clustering. The proposed two-stage spectral clustering algorithm is applied on a simulated and a real cryo-EM dataset for heterogeneous reconstruction. Results show that the two novel distance measures can be used to improve the classification performance of spectral clustering, and using the proposed two-stage spectral clustering algorithm can achieve higher classification and reconstruction accuracy than using RELION and XMIPP.<\/jats:p>","DOI":"10.1093\/bib\/bbac032","type":"journal-article","created":{"date-parts":[[2022,1,26]],"date-time":"2022-01-26T12:10:29Z","timestamp":1643199029000},"source":"Crossref","is-referenced-by-count":4,"title":["Heterogeneous cryo-EM projection image classification using a two-stage spectral clustering based on novel distance measures"],"prefix":"10.1093","volume":"23","author":[{"given":"Xiangwen","family":"Wang","sequence":"first","affiliation":[{"name":"School of Information Science and Engineering, Lanzhou University, 730000, Lanzhou, China"},{"name":"College of Computer Science and Engineering, Northwest Normal University, 730070, Lanzhou, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yonggang","family":"Lu","sequence":"additional","affiliation":[{"name":"School of Information Science and Engineering, Lanzhou University, 730000, Lanzhou, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xianghong","family":"Lin","sequence":"additional","affiliation":[{"name":"College of Computer Science and Engineering, Northwest Normal University, 730070, Lanzhou, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2022,3,7]]},"reference":[{"issue":"7794","key":"2022051813071971300_ref1","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1038\/d41586-020-00341-9","article-title":"Revolutionary cryo-EM is taking over structural biology","volume":"578","author":"Callaway","year":"2020","journal-title":"Nature"},{"issue":"7675","key":"2022051813071971300_ref2","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1038\/nature.2017.22738","article-title":"Cryo-electron microscopy wins chemistry Nobel","volume":"550","author":"Cressey","year":"2017","journal-title":"Nature"},{"issue":"10","key":"2022051813071971300_ref3","doi-asserted-by":"crossref","first-page":"837","DOI":"10.1016\/j.tibs.2019.04.008","article-title":"Cryo-electron microscopy methodology: current aspects and future directions","volume":"44","author":"Danev","year":"2019","journal-title":"Trends Biochem Sci"},{"issue":"7832","key":"2022051813071971300_ref4","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1038\/s41586-020-2829-0","article-title":"Single-particle cryo-EM at atomic resolution","volume":"587","author":"Nakane","year":"2020","journal-title":"Nature"},{"issue":"7832","key":"2022051813071971300_ref5","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1038\/s41586-020-2833-4","article-title":"Atomic-resolution protein structure determination by cryo-EM","volume":"587","author":"Yip","year":"2020","journal-title":"Nature"},{"key":"2022051813071971300_ref6","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1038\/d41586-020-02924-y","article-title":"Cryo-electron microscopy reaches atomic resolution","volume":"587","author":"Herzik Jr","year":"2020","journal-title":"Nature"},{"issue":"4","key":"2022051813071971300_ref7","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1016\/j.tibs.2021.01.001","article-title":"Seeing atoms by single-particle Cryo-EM","volume":"46","author":"Bai","year":"2021","journal-title":"Trends Biochem Sci"},{"key":"2022051813071971300_ref8","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/j.abb.2015.05.003","article-title":"Three-dimensional reconstruction methods in single particle analysis from transmission electron microscopy data","volume":"581","author":"Carazo","year":"2015","journal-title":"Arch Biochem Biophys"},{"key":"2022051813071971300_ref9","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1016\/j.sbi.2018.11.004","article-title":"Advances in image processing for single-particle analysis by electron cryomicroscopy and challenges ahead","volume":"52","author":"Vilas","year":"2018","journal-title":"Curr Opin Struct Biol"},{"issue":"1","key":"2022051813071971300_ref10","doi-asserted-by":"crossref","DOI":"10.1016\/j.jsb.2020.107695","article-title":"Algorithmic robustness to preferred orientations in single particle analysis by CryoEM","volume":"213","author":"Sorzano","year":"2021","journal-title":"J Struct Biol"},{"key":"2022051813071971300_ref11","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1016\/j.csbj.2016.10.002","article-title":"Cryo-electron microscopy analysis of structurally heterogeneous macromolecular complexes","volume":"14","author":"Joni\u0107","year":"2016","journal-title":"Comput Struct Biotechnol J"},{"issue":"18","key":"2022051813071971300_ref12","doi-asserted-by":"crossref","first-page":"4262","DOI":"10.3390\/molecules25184262","article-title":"Xinpei Li, and Khanh Dao Duc. 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