MagIC-cryo-EM Frequently Asked Questions
1. Target capturing modules
Q: Can I replace the target-capturing modules with other proteins?
A: Yes! As long as the target-capturing modules are not aggregable, you can use any proteins as the target-capturing module. The target-capturing module may need to be passed through the size-exclusion column. Even if the target-capturing module is purified by size exclusion, if the target-capturing module is aggregable, beads will form clusters (See image below).
2. Inner layer by triple helical bundle (3HB)
Q: Is the 3HB layer essential?
A: Yes and NO.
“Yes” for 300 kV microscope. The 3HB layer is essential to avoid the noise signal from the beads. Having an inner layer of 3HB and mono-SPYtag-streptavidin significantly increases the density of the SAH spacers, allowing the targets to stretch out from the bead.
“No” for 200 kV microscope. Since the noise signals generated by 200 kV microscope do not spread too far, 3HB may not be essential.
3. Spacer length
Q: Does the 60 nm SAH spacer work for all proteins?
A: To maintain the appropriate spacing between captured complexes, longer spacer proteins may be needed. The single alpha helix can be extended to the desired length using MluI and AscI sites that were inserted at the top and bottom of the DNA segment encoding 30 nm SAH, respectively.
4. Graphene grid
Q: Is graphene necessary for this experiment?
A: it is not essential. However, using graphene significantly increases the number of beads in holes.
5. Orientation
Q: Does the protein capturing by MagIC-cryo-EM limit the particle orientation?
A: In theory, the particle orientation bias should be reduced in MagIC-cryo-EM, as particles are submerged, and the bias is thought to arise from particle accumulation at the air-water interface. However, while the preferred orientation appears to be mitigated, the issue is not completely resolved.
A possible explanation for the remaining preferred orientation bias in MagIC-cryo-EM data is that many particles are localized on graphene-water interfaces.
For more questions, please contact me through yarimura@fredhutch.org
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