A case for glycerol as an acceptable additive for single-particle cryoEM samples.
aldolase
apoferritin
cryoEM
glycerol
sample buffer
Journal
Acta crystallographica. Section D, Structural biology
ISSN: 2059-7983
Titre abrégé: Acta Crystallogr D Struct Biol
Pays: United States
ID NLM: 101676043
Informations de publication
Date de publication:
01 Jan 2022
01 Jan 2022
Historique:
received:
14
09
2021
accepted:
13
11
2021
entrez:
4
1
2022
pubmed:
5
1
2022
medline:
25
3
2022
Statut:
ppublish
Résumé
Buffer-composition and sample-preparation guidelines for cryo-electron microscopy are geared towards maximizing imaging contrast and reducing electron-beam-induced motion. These pursuits often involve the minimization or the complete removal of additives that are commonly used to facilitate proper protein folding and minimize aggregation. Among these admonished additives is glycerol, a widely used osmolyte that aids protein stability. In this work, it is shown that the inclusion of glycerol does not preclude high-resolution structure determination by cryoEM, as demonstrated by an ∼2.3 Å resolution reconstruction of mouse apoferritin (∼500 kDa) and an ∼3.3 Å resolution reconstruction of rabbit muscle aldolase (∼160 kDa) in the presence of 20%(v/v) glycerol. While it was found that generating thin ice that is amenable to high-resolution imaging requires long blot times, the addition of glycerol did not result in increased beam-induced motion or an inability to pick particles. Overall, these findings indicate that glycerol should not be discounted as a cryoEM sample-buffer additive, particularly for large, fragile complexes that are prone to disassembly or aggregation upon its removal.
Identifiants
pubmed: 34981768
pii: S2059798321012110
doi: 10.1107/S2059798321012110
pmc: PMC8725161
doi:
Substances chimiques
Buffers
0
Apoferritins
9013-31-4
Fructose-Bisphosphate Aldolase
EC 4.1.2.13
Glycerol
PDC6A3C0OX
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
124-135Subventions
Organisme : NIA NIH HHS
ID : R21 AG067594
Pays : United States
Organisme : NIH HHS
ID : S10 OD021634
Pays : United States
Organisme : NIH HHS
ID : R01 GM14305
Pays : United States
Organisme : NIH HHS
ID : R21 AG067594
Pays : United States
Informations de copyright
open access.
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