MagIC-cryo-EM*
* For the nucleosome, 1000~2000 nucleosomes/bead is an ideal ratio. This may vary depending on your target.
Step 1: To remove too small beads with the same buffer for the sample
1. Mix 0.1~0.5 fmol of 3HB-60nm-SAH-GFPe beads [1~5 µL of 0.1 nM], 200µL 17% sucrose gradient buffer with 0.01% Tween 20
2. Spin 16000g 20min 4ºC, collect the tube on the handmade magnetic rack
3. Remove supernatant from the tube on the handmade magnetic rack
Step 2: To remove aggregated protein
1. Transfer the sample (a fraction from a sucrose gradient containing 2~10 pmol* target complex) to a new tube.
(* For the nucleosome, around 2000 nucleosomes/bead is an ideal ratio. This may vary depending on your target.)
2. Add Tween20 to 0.01%
3. Spin at 16000g 20min 4ºC
4. Take supernatant
Step 3: Incubation
1. Resuspend the washed beads from step1 with supernatant from step2
2. Incubate (Leave 15hrs in the cold room) o/n
Step 4: Wash
* For the purified protein, this step may not be needed.
1. Spin at 16000g 20min 4ºC
2. Remove supernatant
3. Resuspend the beads with 200µL of wash buffer with 0.01% Tween
4. Spin at 16000g 20min 4ºC
5. Remove supernatant
6. Resuspend the beads with 200µL of wash buffer with 0.01% Tween
7. Spin at 16000g 20min 4ºC
8. Remove supernatant
9. Resuspend the beads with 200µL of wash buffer with 0.01% Tween
10. Spin at 16000g 20min 4ºC
11. Remove supernatant
Resuspend the beads with 80 µL of EM cryoprotectant buffer with 0.001% Tween
Step 5: grid freezing
1. Plasma clean graphene grid (or with UV/ozone cleaner)
2. Pick one grid with non-magnetic tweezers and set it in a magnetic humidity chamber
3. Apply 4µL of sample
4. Incubate 5 min
5. Transfer the tweezers to vitrobot and Freeze grid (skip sample application, 2-second blotting time at room temperature under 100% humidity
Continue
Acknowledgememt
This page was designed by Antonio Latimore in the Communications & Marketing department at the Fred Hutchinson Cancer Center and maintained by Katrina Akioka and other members in the Arimura lab at the Fred Hutchinson Cancer Center.
The Original paper of MagIC-cryo-EM, has been published (Arimura Y, Konishi HA, Funabiki H. Elife. 2025). The MagIC-cryo-EM method was developed in the Funabiki lab at the Rockefeller University under support from a National Institutes of Health grant (R35GM132111) awarded to H.F., a Japan Society for the Promotion of Science Overseas Research Fellowship awarded to H.A.K., and the Osamu Hayaishi Memorial Scholarship for Study Abroad awarded to Y.A. This research was also supported by the Stavros Niarchos Foundation (SNF) as part of its grant to the SNF Institute for Global Infectious Disease Research at The Rockefeller University.
We are grateful to Mark Ebrahim, Johanna Sotiris, and Honkit Ng for their technical advice and assistance with Cryo-EM. We also thank Genzhe Lu and Daniil Tagaev for their contributions to optimizing MagIC-cryo-EM. We extend our thanks to Seth Darst, Elizabeth Campbell, Thomas Huber, Michael Rout, Peter Fridy, Christopher Caffalette, Trevor Van Eeuwen, Hiro Furukawa, Sue Biggins, Daniel Barrero, and Mengqiu Jiang for their valuable consultations on the project.
The test data were collected at the Arimura Lab and the Electron Microscopy Facility at the Fred Hutchinson Cancer Center. We are grateful to Melody Campbell, Theo Humphreys, and Anvesh Dasari for establishing and operating the Cryo-EM equipment.