Electrospray-assisted cryo-EM sample preparation to mitigate interfacial effects.
Nat Methods
; 21(6): 1023-1032, 2024 Jun.
Article
en En
| MEDLINE
| ID: mdl-38664529
ABSTRACT
Addressing interfacial effects during specimen preparation in cryogenic electron microscopy remains challenging. Here we introduce ESI-cryoPrep, a specimen preparation method based on electrospray ionization in native mass spectrometry, designed to alleviate issues associated with protein denaturation or preferred orientation induced by macromolecule adsorption at interfaces. Through fine-tuning spraying parameters, we optimized protein integrity preservation and achieved the desired ice thickness for analyzing target macromolecules. With ESI-cryoPrep, we prepared high-quality cryo-specimens of five proteins and obtained three-dimensional reconstructions at near-atomic resolution. Our findings demonstrate that ESI-cryoPrep effectively confines macromolecules within the middle of the thin layer of amorphous ice, facilitating the preparation of blotting-free vitreous samples. The protective mechanism, characterized by the uneven distribution of charged biomolecules of varying sizes within charged droplets, prevents the adsorption of target biomolecules at air-water or graphene-water interfaces, thereby avoiding structural damage to the protein particles or the introduction of dominant orientation issues.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Manejo de Especímenes
/
Microscopía por Crioelectrón
/
Espectrometría de Masa por Ionización de Electrospray
Límite:
Humans
Idioma:
En
Revista:
Nat Methods
/
Nat. methods
/
Nature methods
Asunto de la revista:
TECNICAS E PROCEDIMENTOS DE LABORATORIO
Año:
2024
Tipo del documento:
Article
País de afiliación:
China
Pais de publicación:
Estados Unidos