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TitleThrough-grid wicking enables high-speed cryoEM specimen preparation.
Journal, issue, pagesActa Crystallogr D Struct Biol, Vol. 76, Issue Pt 11, Page 1092-1103, Year 2020
Publish dateNov 1, 2020
AuthorsYong Zi Tan / John L Rubinstein /
PubMed AbstractBlotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air-water interface. Here, ...Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air-water interface. Here, it is shown that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass-fiber filter held against the opposite side, often called the `back', of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. Ultrasonic specimen application and through-grid wicking were combined in a high-speed specimen-preparation device that was named `Back-it-up' or BIU. The high liquid-absorption capacity of the glass fiber compared with self-wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The speed of the device increases the number of views for a specimen that suffers from preferred orientations.
External linksActa Crystallogr D Struct Biol / PubMed:33135680
MethodsEM (single particle)
Resolution2.0 - 2.9 Å
Structure data

21951

6wx6

EMDB-21951, PDB-6wx6:
Cryo-EM Structure of Human Apoferritin Light Chain Vitrified Using Back-it-up
Method: EM (single particle) / Resolution: 2.0 Å

21954

6wxb

EMDB-21954, PDB-6wxb:
Cryo-EM Structure of Influenza Hemagglutinin (HA) Trimer Vitrified Using Back-it-up
Method: EM (single particle) / Resolution: 2.9 Å

Chemicals

ChemComp-CA:
Unknown entry

ChemComp-HOH:
WATER

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

ChemComp-BMA:
beta-D-mannopyranose

Source
  • homo sapiens (human)
  • H3N2 subtype (virus)
  • influenza a virus (strain a/hong kong/1/1968 h3n2)
KeywordsMETAL BINDING PROTEIN / Human / apoferritin / ferritin / light chain / back-it-up / through-grid wicking / VIRAL PROTEIN / Hemagglutinin / trimer / Influenza

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