Functional NiTi grids for in situ straining in the TEM

Schürmann, Ulrich; Chluba, Christoph; Wolff, Niklas; Smazna, Daria; Miranda, Rodrigo Lima de; Junker, Philipp; Adelung, Rainer; Quandt, Eckhard; Kienle, Lorenz

doi:10.1016/j.ultramic.2017.06.003

Cited by 2 publications

(2 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is now evident that this also significantly affects the orientation distribution of macromolecules in the thin films formed in the holes. We note the following two caveats in this study: (i) all of the grids used were from Quantifoil with amorphous carbon as the support film, and it is unclear whether similar behaviour will be observed with C-flat grids from Protochips that are manufactured differently or with other grids such as UltraFoil or NiTi alloy (Fan et al, 2021;Quispe et al, 2007;Russo & Passmore, 2016;Schu ¨rmann et al, 2017), and (ii) only one sample (catalase) was tested and further extensive study with more samples is required to generalize the effect of the holey grid geometry.…”

Section: Discussionmentioning

confidence: 98%

Factors affecting macromolecule orientations in thin films formed in cryo-EM

Yadav,

Vinothkumar

2024

Acta Cryst Sect D Struct Biol

View full text Add to dashboard Cite

The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge-freeze method first described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the air–water interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, the effect of physical and chemical factors on the orientations of macromolecules was investigated through an analysis of selected well studied macromolecules, and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of the interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and to provide a framework to design small-molecule additives to enhance sample stability and behaviour.

show abstract

Section: Discussionmentioning

confidence: 98%

Factors affecting macromolecule orientations in thin films formed in cryo-EM

Yadav,

Vinothkumar

2024

Acta Cryst Sect D Struct Biol

View full text Add to dashboard Cite

show abstract

“…It is now evident that this also significantly affects the orientation distribution of macromolecules in the thin films formed in the holes. We note two caveats in this study: a) all the grids used were from Quantifoil with amorphous carbon as the support film, and it is unclear if similar behaviour will be observed with C-flat grids from Protochips that are manufactured differently or other grids such as UltraFoil or NiTi alloy (Fan et al, 2021;Quispe et al, 2007;Russo & Passmore, 2016;Schürmann et al, 2017). b) only one sample (catalase) was tested and further extensive study with more samples is required to generalise the effect of the holey grid geometry.…”

Section: Discussionmentioning

confidence: 99%

Factors affecting macromolecule orientations in thin films formed in cryo-EM

Yadav,

Vinothkumar

2023

Preprint

View full text Add to dashboard Cite

The formation of vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge freeze method described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the air-water interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, through an analysis of selected well-studied macromolecules, the effect of physical and chemical factors on the orientations of macromolecules was investigated, and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and provide a framework to design small-molecule additives to enhance sample stability and behaviour.

show abstract

Functional NiTi grids for in situ straining in the TEM

Cited by 2 publications

References 17 publications

Factors affecting macromolecule orientations in thin films formed in cryo-EM

Factors affecting macromolecule orientations in thin films formed in cryo-EM

Factors affecting macromolecule orientations in thin films formed in cryo-EM

Contact Info

Product

Resources

About