BACKGROUND: Negative-staining (NS), a rapid, simple and conventional technique of electron microscopy (EM), has been commonly used to initially study the morphology and structure of proteins for half a century. Certain NS protocols however can cause artifacts, especially for structurally flexible or lipid-related proteins, such as lipoproteins. Lipoproteins were often observed in the form of rouleau as lipoprotein particles appeared to be stacked together by conventional NS protocols. The flexible components of lipoproteins, i.e. lipids and amphipathic apolipoproteins, resulted in the lipoprotein structure being sensitive to the NS sample preparation parameters, such as operational procedures, salt concentrations, and the staining reagents. SCOPE OF REVIEW: The most popular NS protocols that have been used to examine lipoprotein morphology and structure were reviewed. MAJOR CONCLUSIONS: The comparisons show that an optimized NS (OpNS) protocol can eliminate the rouleau artifacts of lipoproteins, and that the lipoproteins are similar in size and shape as statistically measured from two EM methods, OpNS and cryo-electron microscopy (cryo-EM). OpNS is a high-throughput, high-contrast and high-resolution (near 1nm, but rarely better than 1nm) method which has been used to discover the mechanics of a small protein, 53kDa cholesterol ester transfer protein (CETP), and the structure of an individual particle of a single protein by individual-particle electron tomography (IPET), i.e. a 14Å-resolution IgG antibody three-dimensional map. GENERAL SIGNIFICANCE: It is suggested that OpNS can be used as a general protocol to study the structure of proteins, especially highly dynamic proteins with equilibrium-fluctuating structures. Published by Elsevier B.V.
BACKGROUND: Negative-staining (NS), a rapid, simple and conventional technique of electron microscopy (EM), has been commonly used to initially study the morphology and structure of proteins for half a century. Certain NS protocols however can cause artifacts, especially for structurally flexible or lipid-related proteins, such as lipoproteins. Lipoproteins were often observed in the form of rouleau as lipoprotein particles appeared to be stacked together by conventional NS protocols. The flexible components of lipoproteins, i.e. lipids and amphipathic apolipoproteins, resulted in the lipoprotein structure being sensitive to the NS sample preparation parameters, such as operational procedures, salt concentrations, and the staining reagents. SCOPE OF REVIEW: The most popular NS protocols that have been used to examine lipoprotein morphology and structure were reviewed. MAJOR CONCLUSIONS: The comparisons show that an optimized NS (OpNS) protocol can eliminate the rouleau artifacts of lipoproteins, and that the lipoproteins are similar in size and shape as statistically measured from two EM methods, OpNS and cryo-electron microscopy (cryo-EM). OpNS is a high-throughput, high-contrast and high-resolution (near 1nm, but rarely better than 1nm) method which has been used to discover the mechanics of a small protein, 53kDa cholesterol ester transfer protein (CETP), and the structure of an individual particle of a single protein by individual-particle electron tomography (IPET), i.e. a 14Å-resolution IgG antibody three-dimensional map. GENERAL SIGNIFICANCE: It is suggested that OpNS can be used as a general protocol to study the structure of proteins, especially highly dynamic proteins with equilibrium-fluctuating structures. Published by Elsevier B.V.
Authors: Lei Zhang; Feng Yan; Shengli Zhang; Dongsheng Lei; M Arthur Charles; Giorgio Cavigiolio; Michael Oda; Ronald M Krauss; Karl H Weisgraber; Kerry-Anne Rye; Henry J Pownall; Xiayang Qiu; Gang Ren Journal: Nat Chem Biol Date: 2012-02-19 Impact factor: 15.040
Authors: R van Antwerpen; G C Chen; C R Pullinger; J P Kane; M LaBelle; R M Krauss; C Luna-Chavez; T M Forte; J C Gilkey Journal: J Lipid Res Date: 1997-04 Impact factor: 5.922
Authors: A Inazu; M L Brown; C B Hesler; L B Agellon; J Koizumi; K Takata; Y Maruhama; H Mabuchi; A R Tall Journal: N Engl J Med Date: 1990-11-01 Impact factor: 91.245
Authors: Gang Ren; Gabby Rudenko; Steven J Ludtke; Johann Deisenhofer; Wah Chiu; Henry J Pownall Journal: Proc Natl Acad Sci U S A Date: 2009-12-28 Impact factor: 11.205
Authors: Jianfang Liu; Anurag Misra; M V V V Sekhar Reddy; Mark Andrew White; Gang Ren; Gabby Rudenko Journal: J Mol Biol Date: 2018-09-05 Impact factor: 5.469