Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy

Negative stain electron microscopy (NSEM) is a simple and cost effective method to analyze a wide variety of specimens, especially proteins. In traditional NSEM, the protein sample is applied to and supported by a continuous carbon film. Unfortunately, many proteins stick to the carbon film with a l...

Full description

Bibliographic Details
Main Authors: Katayoun Mansouri, Robert J. Edwards
Format: Article
Language:English
Published: Elsevier 2019-01-01
Series:MethodsX
Online Access:http://www.sciencedirect.com/science/article/pii/S2215016119300433
id doaj-3240870d4cc24063855c2a3e0a57992a
record_format Article
spelling doaj-3240870d4cc24063855c2a3e0a57992a2020-11-25T01:30:23ZengElsevierMethodsX2215-01612019-01-016582586Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopyKatayoun Mansouri0Robert J. Edwards1Duke Human Vaccine Institute, Duke University, Durham, NC, United StatesCorresponding author.; Duke Human Vaccine Institute, Duke University, Durham, NC, United StatesNegative stain electron microscopy (NSEM) is a simple and cost effective method to analyze a wide variety of specimens, especially proteins. In traditional NSEM, the protein sample is applied to and supported by a continuous carbon film. Unfortunately, many proteins stick to the carbon film with a limited number of orientations. Because the restricted orientation limits the available views of the molecule, information about the three-dimensional structure of the molecule is likewise limited. The method presented here overcomes this limitation by using a carbon holey film combined with 1-octadecanol as a spreading agent. We demonstrate this method with solubilized envelope (Env) proteins from HIV, which typically show a restricted orientation on continuous carbon film, and show the following: • 1-octadecanol added to negative stain aids the formation of a continuous sample-stain layer spanning the holes of a holey carbon film. • Samples negatively stained over holes show less restricted orientation, resulting in better single particle reconstructions. Method name: Negative stain over holes, Keywords: 1-Octadecanol, Uranyl formate, Carbon film, Preferred orientation, Single particle reconstructionshttp://www.sciencedirect.com/science/article/pii/S2215016119300433
collection DOAJ
language English
format Article
sources DOAJ
author Katayoun Mansouri
Robert J. Edwards
spellingShingle Katayoun Mansouri
Robert J. Edwards
Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy
MethodsX
author_facet Katayoun Mansouri
Robert J. Edwards
author_sort Katayoun Mansouri
title Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy
title_short Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy
title_full Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy
title_fullStr Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy
title_full_unstemmed Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy
title_sort suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy
publisher Elsevier
series MethodsX
issn 2215-0161
publishDate 2019-01-01
description Negative stain electron microscopy (NSEM) is a simple and cost effective method to analyze a wide variety of specimens, especially proteins. In traditional NSEM, the protein sample is applied to and supported by a continuous carbon film. Unfortunately, many proteins stick to the carbon film with a limited number of orientations. Because the restricted orientation limits the available views of the molecule, information about the three-dimensional structure of the molecule is likewise limited. The method presented here overcomes this limitation by using a carbon holey film combined with 1-octadecanol as a spreading agent. We demonstrate this method with solubilized envelope (Env) proteins from HIV, which typically show a restricted orientation on continuous carbon film, and show the following: • 1-octadecanol added to negative stain aids the formation of a continuous sample-stain layer spanning the holes of a holey carbon film. • Samples negatively stained over holes show less restricted orientation, resulting in better single particle reconstructions. Method name: Negative stain over holes, Keywords: 1-Octadecanol, Uranyl formate, Carbon film, Preferred orientation, Single particle reconstructions
url http://www.sciencedirect.com/science/article/pii/S2215016119300433
work_keys_str_mv AT katayounmansouri suspendingsamplesovercarbonholeyfilmsincreasesheterogeneityofmolecularorientationsinnegativestainelectronmicroscopy
AT robertjedwards suspendingsamplesovercarbonholeyfilmsincreasesheterogeneityofmolecularorientationsinnegativestainelectronmicroscopy
_version_ 1725091733563244544