中国物理B ›› 2024, Vol. 33 ›› Issue (8): 88702-088702.doi: 10.1088/1674-1056/ad4cdb

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Subtraction of liposome signals in cryo-EM structural determination of protein-liposome complexes

Shouqing Li(李首卿)1,2, Ming Li(李明)1,2,†, Yumei Wang(王玉梅)1,8,‡, and Xueming Li(李雪明)3,4,5,6,7,§   

  1. 1 Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China;
    2 University of Chinese Academy of Sciences, Beijing 100049, China;
    3 Key Laboratory for Protein Sciences of Ministry of Education, School of Life Sciences, Tsinghua University, Beijing 100084, China;
    4 State Key Laboratory of Membrane Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China;
    5 Tsinghua——Peking Joint Center for Life Sciences, Beijing 100084, China;
    6 Beijing Frontier Research Center for Biological Structure, Beijing 100084, China;
    7 School of Life Sciences, Tsinghua University, Beijing 100084, China;
    8 Beijing Branch of Songshan Lake Materials Laboratory, Beijing 100190, China
  • 收稿日期:2024-04-08 修回日期:2024-04-28 出版日期:2024-08-15 发布日期:2024-07-29
  • 通讯作者: Ming Li, Yumei Wang, Xueming Li E-mail:mingli@iphy.ac.cn;wangym@iphy.ac.cn;lixueming@tsinghua.edu.cn
  • 基金资助:
    Project supported by the National Natural Science Foundation of China (Grant Nos. 32241023 and 92254306) and the Fund from the Tsinghua-Peking Joint Center for Life Sciences, and Beijing Frontier Research Center for Biological Structure.

Subtraction of liposome signals in cryo-EM structural determination of protein-liposome complexes

Shouqing Li(李首卿)1,2, Ming Li(李明)1,2,†, Yumei Wang(王玉梅)1,8,‡, and Xueming Li(李雪明)3,4,5,6,7,§   

  1. 1 Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China;
    2 University of Chinese Academy of Sciences, Beijing 100049, China;
    3 Key Laboratory for Protein Sciences of Ministry of Education, School of Life Sciences, Tsinghua University, Beijing 100084, China;
    4 State Key Laboratory of Membrane Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China;
    5 Tsinghua——Peking Joint Center for Life Sciences, Beijing 100084, China;
    6 Beijing Frontier Research Center for Biological Structure, Beijing 100084, China;
    7 School of Life Sciences, Tsinghua University, Beijing 100084, China;
    8 Beijing Branch of Songshan Lake Materials Laboratory, Beijing 100190, China
  • Received:2024-04-08 Revised:2024-04-28 Online:2024-08-15 Published:2024-07-29
  • Contact: Ming Li, Yumei Wang, Xueming Li E-mail:mingli@iphy.ac.cn;wangym@iphy.ac.cn;lixueming@tsinghua.edu.cn
  • Supported by:
    Project supported by the National Natural Science Foundation of China (Grant Nos. 32241023 and 92254306) and the Fund from the Tsinghua-Peking Joint Center for Life Sciences, and Beijing Frontier Research Center for Biological Structure.

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摘要： Reconstituting membrane proteins in liposomes and determining their structure is a common method for determining membrane protein structures using single-particle cryo-electron microscopy (cryo-EM). However, the strong signal of liposomes under cryo-EM imaging conditions often interferes with the structural determination of the embedded membrane proteins. Here, we propose a liposome signal subtraction method based on single-particle two-dimensional (2D) classification average images, aimed at enhancing the reconstruction resolution of membrane proteins. We analyzed the signal distribution characteristics of liposomes and proteins within the 2D classification average images of protein-liposome complexes in the frequency domain. Based on this analysis, we designed a method to subtract the liposome signals from the original particle images. After the subtraction, the accuracy of single-particle three-dimensional (3D) alignment was improved, enhancing the resolution of the final 3D reconstruction. We demonstrated this method using a PIEZO1-proteoliposome dataset by improving the resolution of the PIEZO1 protein.

关键词: cryo-EM, protein-liposome complexes, liposome signal subtraction, 2D classification averaging

Abstract: Reconstituting membrane proteins in liposomes and determining their structure is a common method for determining membrane protein structures using single-particle cryo-electron microscopy (cryo-EM). However, the strong signal of liposomes under cryo-EM imaging conditions often interferes with the structural determination of the embedded membrane proteins. Here, we propose a liposome signal subtraction method based on single-particle two-dimensional (2D) classification average images, aimed at enhancing the reconstruction resolution of membrane proteins. We analyzed the signal distribution characteristics of liposomes and proteins within the 2D classification average images of protein-liposome complexes in the frequency domain. Based on this analysis, we designed a method to subtract the liposome signals from the original particle images. After the subtraction, the accuracy of single-particle three-dimensional (3D) alignment was improved, enhancing the resolution of the final 3D reconstruction. We demonstrated this method using a PIEZO1-proteoliposome dataset by improving the resolution of the PIEZO1 protein.

Key words: cryo-EM, protein-liposome complexes, liposome signal subtraction, 2D classification averaging

中图分类号:  (Spectroscopic and microscopic techniques in biophysics and medical physics)

  • 87.64.-t
87.80.-y (Biophysical techniques (research methods)) 87.64.Ee (Electron microscopy) 87.85.jf (Bio-based materials)