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Optimized Negative-Staining Protocol for Lipid–Protein Interactions Investigated by Electron Microscopy

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Book cover Lipid-Protein Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2003))

Abstract

A large number of proteins are capable of inserting themselves into lipids, and interacting with membranes, such as transmembrane proteins and apolipoproteins. Insights into the lipid-protein interactions are important in understanding biological processes, and the structure of proteins at the lipid binding stage can help identify their roles and critical functions. Previously, such structural determination was challenging to obtain because the traditional methods, such as X-ray crystallography, are unable to capture the conformational and compositional heterogeneity of protein–lipid complexes. Electron microscopy (EM) is an alternative approach to determining protein structures and visualizing lipid–protein interactions directly, and negative-staining (OpNS), a subset of EM techniques, is a rapid, frequently used qualitative approach. The concern, however, is that current NS protocols often generate artifacts with lipid-related proteins, such as rouleaux formation from lipoproteins. To overcome this artifact formation, Ren and his colleagues have refined early NS protocols, and developed an optimized NS protocol that validated by comparing images of lipoproteins from cryo-electron microscopy (cryo-EM). This optimized NS protocol produces “near native-state” particle images and high contrast images of the protein in its native lipid-binding state, which can be used to create higher-quality three-dimensional (3D) reconstruction by single-particle analysis and electron tomography (e.g. IPET). This optimized protocol is thus a promising hands-on approach for examining the structure of proteins at their lipid-binding status.

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Acknowledgments

We thank Drs. Ron Krauss, Paul Alivisatos, Haijun Sun, Xiayang Qiu, Lei Zhang, and Mark Garewal, for providing the samples, preparing the EM samples, or preparing the draft. This work was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health (R01HL115153, 2R01HL115153-06, and R01GM104427,). Work at the Molecular Foundry was supported by the Office of Science, Office of Basic Energy Sciences of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

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Authors and Affiliations

  1. Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Jianfang Liu, Hao Wu, Changyu Huang, Dongsheng Lei, Meng Zhang & Gang Ren

  2. Department of Computer Science, College of Information Science and Technology, Beijing Normal University, Beijing, China

    Hao Wu

  3. State Key Laboratory for Biocontrol, School of Life Sciences, The Sun Yat-Sen University, Guangzhou, Guangdong, China

    Wei Xie

  4. Center for Cellular and Structural Biology, The Sun Yat-Sen University, Guangzhou, Guangdong, China

    Wei Xie

  5. Department of Biomedical Sciences, Mercer University School of Medicine, Savannah, GA, USA

    Jinping Li

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  1. Jianfang Liu
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  2. Hao Wu
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  3. Changyu Huang
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  4. Dongsheng Lei
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  5. Meng Zhang
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  6. Wei Xie
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  7. Jinping Li
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  8. Gang Ren
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Correspondence to Gang Ren .

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  1. Department of Biophysics, Institute of Biology, FB10 and Center for Interdisciplinary Nanostructure Science and Technology (CINSaT), University of Kassel, Kassel, Germany

    Jörg H. Kleinschmidt

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Liu, J. et al. (2019). Optimized Negative-Staining Protocol for Lipid–Protein Interactions Investigated by Electron Microscopy. In: Kleinschmidt, J. (eds) Lipid-Protein Interactions. Methods in Molecular Biology, vol 2003. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9512-7_8

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  • DOI: https://doi.org/10.1007/978-1-4939-9512-7_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9511-0

  • Online ISBN: 978-1-4939-9512-7

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