Journal of Lipid Research
Volume 52, Issue 1, January 2011, Pages 175-184
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Methods
Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy[S]

https://doi.org/10.1194/jlr.D010959Get rights and content
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Plasma lipoprotein levels are predictors of risk for coronary artery disease. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in cardiovascular disease. The compositional and conformational heterogeneity of lipoproteins are major barriers to the identification of their structures, as discovered using traditional approaches. Although electron microscopy (EM) is an alternative approach, conventional negative staining (NS) produces rouleau artifacts. In a previous study of apolipoprotein (apo)E4-containing reconstituted HDL (rHDL) particles, we optimized the NS method in a way that eliminated rouleaux. Here we report that phosphotungstic acid at high buffer salt concentrations plays a key role in rouleau formation. We also validate our protocol for analyzing the major plasma lipoprotein classes HDL, LDL, IDL, and VLDL, as well as homogeneously prepared apoA-I-containing rHDL. High-contrast EM images revealed morphology and detailed structures of lipoproteins, especially apoA-I-containing rHDL, that are amenable to three-dimensional reconstruction by single-particle analysis and electron tomography.

lipoprotein structure
lipoprotein morphology
protocol

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    Abbreviations:

    apo

    apolipoprotein

    CE

    cholesteryl ester

    cryoEM

    cryo-electron microscopy

    cryo-NS

    cryo-negative-staining

    DPBS

    Dulbecco's phosphate-buffered saline

    EM

    electron microscopy

    IDL

    intermediate density lipoprotein

    NS

    negative staining

    NS-EM

    negative-staining electron microscopy

    PTA

    phosphotungstic acid

    RCT

    reverse cholesterol transport

    rHDL

    reconstituted HDL

    UC

    unesterified cholesterol

    UF

    uranyl formate

This work was supported by the Office of Science, Office of Basic Energy Sciences of the U.S. Department of Energy (contract no. DE-AC02-05CH11231) and the W. M. Keck foundations to G.R. L.Z. was supported in part by the State Scholarship of China through China Scholarship Council (file no. 2008628018).

1

L. Zhang and J. Song contributed equally to this work.

[S]

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of two figures.