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-Structure paper
Title | Through-grid wicking enables high-speed cryoEM specimen preparation. |
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Journal, issue, pages | Acta Crystallogr D Struct Biol, Vol. 76, Issue Pt 11, Page 1092-1103, Year 2020 |
Publish date | Nov 1, 2020 |
Authors | Yong Zi Tan / John L Rubinstein / |
PubMed Abstract | 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, ...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 links | Acta Crystallogr D Struct Biol / PubMed:33135680 |
Methods | EM (single particle) |
Resolution | 2.0 - 2.9 Å |
Structure data | EMDB-21951, PDB-6wx6: EMDB-21954, PDB-6wxb: |
Chemicals | ChemComp-CA: ChemComp-HOH: ChemComp-NAG: ChemComp-BMA: |
Source |
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Keywords | METAL BINDING PROTEIN / Human / apoferritin / ferritin / light chain / back-it-up / through-grid wicking / VIRAL PROTEIN / Hemagglutinin / trimer / Influenza |