Setup and Troubleshooting of Volta Phase Plate Cryo-EM Data Collection.
Fiche publication
Date publication
janvier 2021
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr KLAHOLZ Bruno
Tous les auteurs :
von Loeffelholz O, Klaholz BP
Lien Pubmed
Résumé
Cryo electron microscopy (cryo-EM) has become a method of choice in structural biology to analyze isolated complexes and cellular structures. This implies adequate imaging of the specimen and advanced image-processing methods to obtain high-resolution 3D reconstructions. The use of a Volta phase plate in cryo-EM drastically increases the image contrast while being able to record images at high acceleration voltage and close to focus, i.e., at conditions where high-resolution information is best preserved. During image processing, higher contrast images can be aligned and classified better than lower quality ones resulting in increased data quality and the need for less data. Here, we give step-by-step guidelines on how to set up high-quality VPP cryo-EM single particle data collections, as exemplified by human ribosome data acquired during a one-day data collection session. Further, we describe specific technical details in image processing that differ from conventional single particle cryo-EM data analysis.
Mots clés
Cryo electron microscopy, Human ribosome, Phase contrast, Structural biology, Volta phase plate
Référence
Methods Mol Biol. 2021 ;2305:291-299