Biology subjects

Tan, Y. Z.

Publications and source records attributed to Tan, Y. Z..

3 recordsLinked to original sources

bioRxiv2018

Sub-2 A Ewald Curvature Corrected Single-Particle Cryo-EM

Single-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities. Here, we have exploited several technical solutions to improve resolution, including sub-Angstrom pixelation, per-particle CTF refinement, and most notably a correction for Ewald sphere curvature. The application of these methods on micrographs recorded on a base model Titan Krios enabled structure determination at [~]1.86-[A] resolution of an adeno-associated virus serotype 2 variant (AAV2), an important gene-delivery vehicle.

biophysics↗

bioRxiv2017

Spotiton: New Features and Applications

We present an update describing new features and applications of Spotiton, a novel instrument for vitrifying samples for cryoEM. We have used Spotiton to prepare several test specimens that can be reconstructed using routine single particle analysis to ~3 [A] resolution, indicating that the process has no apparent deleterious effect on the sample integrity. The system is now in routine and continuous use in our lab and has been used to successfully vitrify a wide variety of samples.

molecular biology↗

bioRxiv2017

Routine Single Particle CryoEM Sample and Grid Characterization by Tomography

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are freely floating away from the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1,000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

biophysics↗