Movie
Controller
Structure viewers
About Yorodumi Papers
+Search query
-Structure paper
| Title | Untangling the effects of flexibility and the AWI in cryoEM sample preparation: A case study using KtrA. |
|---|---|
| Journal, issue, pages | J Struct Biol, Vol. 217, Issue 2, Page 108206, Year 2025 |
| Publish date | May 3, 2025 |
 Authors | Isobel Jackson Hirst / Wesley Tien Chiang / Nien-Jen Hu / Charlotte A Scarff / Rebecca F Thompson / Michele C Darrow / Stephen P Muench /   |
| PubMed Abstract | Single particle cryo-electron microscopy (cryoEM) is a powerful tool for elucidating the structures of biological macromolecules without requiring crystallisation or fixation. However, certain ...Single particle cryo-electron microscopy (cryoEM) is a powerful tool for elucidating the structures of biological macromolecules without requiring crystallisation or fixation. However, certain barriers to obtaining high-resolution structures persist, particularly during grid preparation when samples are in a thin liquid film. At this stage, extensive exposure to the air-water interface (AWI) can lead to subunit dissociation, denaturation, and preferred orientation of particles. Another obstacle to high-resolution cryoEM is molecular flexibility, which introduces heterogeneity in the dataset, weakening the signal during image processing. This study explores the effects of AWI interactions and molecular flexibility on the cryoEM density maps of KtrA, the soluble regulatory subunit of the potassium transporter KtrAB from Bacillus subtilis. From grids prepared using a standard blotting technique, we observed a lack of density in the C-lobe domains and preferred orientation. Modifications such as reducing AWI exposure through faster vitrification times (6 s vs ≤100 ms) notably improved C-lobe density. Moreover, the addition of cyclic di-AMP, which binds to the C-lobes, combined with a 100 ms plunge time, further enhanced C-lobe density and eliminated preferred orientation. These findings demonstrate that both AWI interactions and flexibility had to be addressed to obtain density for the C-lobe domains of KtrA. This study underscores the ongoing complexities in achieving high-resolution cryoEM for many samples. |
 External links | J Struct Biol / PubMed:40324569 |
| Methods | EM (single particle) |
| Resolution | 3.8 - 7.1 Å |
| Structure data |  EMDB-51919: Grid prepared using the vitrobot of KtrA.ADP from B. subtilis, small dataset  EMDB-51921: KtrA.ADP with a 100ms plunge time on the chameleon  EMDB-51922: KtrA.ADP with a 300ms plunge time on the chameleon  EMDB-51923: KtrA.ADP with 2500ms plunge time on the chameleon  EMDB-51924: KtrA.ADP with cyclic di-AMP prepared on the vitrobot  EMDB-51925: KtrA.ADP, chameleon with 180ms plunge time and DDM added  EMDB-51926: KtrA.ADP from chameleon with 2500ms plunge time and DDM added as a surfactant  EMDB-51927: KtrA.ADP with cyclic di-AMP from chameleon with 100ms plunge time.  EMDB-51928: KtrA.ADP prepared on the vitrobot, full particle stack |
| Source |
|
Bascillus subtilis (bacteria)