WikiSysop: Created page with "== Citation == Esfahani, Behrouz Ghazi / Randolph, Peter S. / Peng, Ruizhi / Stroupe, Elizabeth / Grant, Tim / Stagg, Scott M. SPOT-RASTR—a cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface. 2024. PNAS Nexus, Vol. 3, p. 184 == Abstract == In cryogenic electron microscopy (cryo-EM), specimen preparation remains a bottleneck despite recent advancements. Classical plunge freezing methods often result i..."

2024-08-21T07:05:53Z

Created page with "== Citation == Esfahani, Behrouz Ghazi / Randolph, Peter S. / Peng, Ruizhi / Stroupe, Elizabeth / Grant, Tim / Stagg, Scott M. SPOT-RASTR—a cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface. 2024. PNAS Nexus, Vol. 3, p. 184 == Abstract == In cryogenic electron microscopy (cryo-EM), specimen preparation remains a bottleneck despite recent advancements. Classical plunge freezing methods often result i..."

New page

== Citation ==

Esfahani, Behrouz Ghazi / Randolph, Peter S. / Peng, Ruizhi / Stroupe, Elizabeth / Grant, Tim / Stagg, Scott M. SPOT-RASTR—a cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface. 2024. PNAS Nexus, Vol. 3, p. 184

== Abstract ==

In cryogenic electron microscopy (cryo-EM), specimen preparation remains a bottleneck despite recent advancements. Classical plunge
freezing methods often result in issues like aggregation and preferred orientations at the air/water interface. Many alternative methods
have been proposed, but there remains a lack a universal solution, and multiple techniques are often required for challenging samples.
Here, we demonstrate the use of lipid nanotubes with nickel NTA headgroups as a platform for cryo-EM sample preparation. His-tagged
specimens of interest are added to the tubules, and they can be frozen by conventional plunge freezing. We show that the nanotubes
protect samples from the air/water interface and promote a wider range of orientations. The reconstruction of average subtracted
tubular regions (RASTR) method allows for the removal of the nanotubule signal from the cryo-EM images resulting in isolated images
of specimens of interest. Testing with β-galactosidase validates the method’s ability to capture particles at lower concentrations,
overcome preferred orientations, and achieve near-atomic resolution reconstructions. Since the nanotubules can be identified and
targeted automatically at low magnification, the method enables fully automated data collection. Furthermore, the particles on the
tubes can be automatically identified and centered using 2D classification enabling particle picking without requiring prior
information. Altogether, our approach that we call specimen preparation on a tube RASTR holds promise for overcoming air–water
interface and preferred orientation challenges and offers the potential for fully automated cryo-EM data collection and structure
determination.

== Keywords ==

== Links ==

https://academic.oup.com/pnasnexus/article/3/8/pgae284/7727607

== Related software ==

== Related methods ==

== Comments ==

2024Esfahani SPOTRASTR - Revision history