Difference between revisions of "2010Zhang OpNS"

Latest revision as of 14:28, 30 June 2020

Citation

Rames, Matthew, Yu, Yadong, and Ren, Gang. Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy. United States: N. p., 2014. Web. doi:10.3791/51087

Zhang L, Song J, Newhouse Y, Zhang S, Weisgraber KH, Ren G. An optimized negative-staining protocol of electron microscopy for apoE4 POPC lipoprotein. J Lipid Res. 2010;51(5):1228-1236. doi:10.1194/jlr.D002493

Abstract

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high-resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography. Moreover, OpNS can be a high-throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

Keywords

protein structure, lipoprotein structure, electron microscopy, negative-staining, optimized negative-staining protocol, individual-particle electron tomography

Links

https://www.jove.com/video/51087/optimized-negative-staining-high-throughput-protocol-for-examining

https://pubmed.ncbi.nlm.nih.gov/19965615/

https://www.ncbi.nlm.nih.gov/pubmed/29129068

http://www.ncbi.nlm.nih.gov/pubmed/20978167

http://www.nature.com/articles/ncomms11083

Related software

https://pubmed.ncbi.nlm.nih.gov/25145703/

https://pubmed.ncbi.nlm.nih.gov/20978167/

https://www.springer.com/gp/book/9781627032742

https://pubmed.ncbi.nlm.nih.gov/23032862/

Difference between revisions of "2010Zhang OpNS"

Latest revision as of 14:28, 30 June 2020

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@@ Line 1: / Line 1: @@
 == Citation ==
+Rames, Matthew, Yu, Yadong, and Ren, Gang. Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy. United States: N. p., 2014. Web. doi:10.3791/51087
 Zhang L, Song J, Newhouse Y, Zhang S, Weisgraber KH, Ren G. An optimized negative-staining protocol of electron microscopy for apoE4 POPC lipoprotein. J Lipid Res. 2010;51(5):1228-1236. doi:10.1194/jlr.D002493
 == Abstract ==
-Apolipoprotein E (apoE), one of the major protein components of lipoproteins in the peripheral and central nervous systems, regulates cholesterol metabolism through its interaction with members of the low density lipoprotein receptor family. One key to understanding apoE function is determining the structure of lipid-bound forms of apoE. Negative-staining (NS) electron microscopy (EM) is an easy and rapid approach for studying the structure and morphology of lipid-bound forms of apoE. However, an artifact of using the conventional NS protocol is that the apoE phospholipid particles form rouleaux. In this study, we used cryo-electron microscopy (cryo-EM) to examine apoE4 palmitoyl-oleoylphosphatidylcholine (POPC) particles in a frozen-hydrated native state. By comparing the particle sizes and shapes produced by different NS protocols to those produced by cryo-EM, we propose an optimized protocol to examine apoE4 POPC particles. Statistical analysis demonstrated that the particle sizes differ by less than 5% between the optimized protocol and the cryo-EM method, with similar shapes. The high contrast and fine detail of particle images produced using this optimized protocol lend themselves to the structural study of lipid-bound forms of apoE.
+Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high-resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography. Moreover, OpNS can be a high-throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.
 == Keywords ==
@@ Line 9: / Line 11: @@
 == Links ==
+https://www.jove.com/video/51087/optimized-negative-staining-high-throughput-protocol-for-examining
 https://pubmed.ncbi.nlm.nih.gov/19965615/
 https://www.ncbi.nlm.nih.gov/pubmed/29129068
 http://www.ncbi.nlm.nih.gov/pubmed/20978167
 http://www.nature.com/articles/ncomms11083
@@ Line 18: / Line 25: @@
 == Related methods ==
 https://pubmed.ncbi.nlm.nih.gov/20978167/
 https://pubmed.ncbi.nlm.nih.gov/25145703/
 https://pubmed.ncbi.nlm.nih.gov/20978167/
 https://www.springer.com/gp/book/9781627032742
 https://pubmed.ncbi.nlm.nih.gov/23032862/
 == Comments ==