Oct 30, 2018

Eliminating effects of particle adsorption to the air/water interface in single-particle cryo-electron microscopy: Bacterial RNA polymerase and CHAPSO

BioRxiv : the Preprint Server for Biology
James ChenSeth Darst

Abstract

Preferred particle orientation presents a major challenge for many single particle cryo-electron microscopy (cryo-EM) samples. Orientation bias limits the angular information used to generate three-dimensional maps and thus affects the reliability and interpretability of the structural models. The primary cause of preferred orientation is presumed to be due to adsorption of the particles at the air/water interface during cryo-EM grid preparation. To ameliorate this problem, detergents are often added to cryo-EM samples to alter the properties of the air/water interface. We have found that many bacterial transcription complexes suffer severe orientation bias when examined by cryo-EM. The addition of non-ionic detergents, such as NP-40, does not remove the orientation bias but the Zwitter-ionic detergent CHAPSO significantly broadens the particle orientation distributions, yielding isotropically uniform maps. We used cryo-electron tomography to examine the particle distribution within the ice layer of cryo-EM grid preparations of Escherichia coli 6S RNA/RNA polymerase holoenzyme particles. In the absence of CHAPSO, essentially all of the particles are located at the ice surfaces. CHAPSO at the critical micelle concentration elimi...Continue Reading

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Mentioned in this Paper

Electron Microscope Tomography
Study
Three-dimensional
DNA-Directed RNA Polymerase
RNA Polymerase Assembly Pathway
Complex (molecular entity)
Adsorption
Detergents
Electron Microscopy
Angular

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