We use SerialEM for the data collection. Tips for the data collection include the following:

Step 1: LMM map collection

A higher resolution map can be helpful in avoiding the square with aggregated beads. The Low Mag Montage (LMM) maps can be collected in the same way as normal cryo-EM grids, and aggregated MagIC cryo-EM beads appear as black clouding.

Step 2: MMM collection

The Middle Mag Montage (MMM) maps must be collected at a high resolution sufficient to distinguish MagIC cryo-EM beads and ice particles. We typically collect MMM maps at x1,250 magnification and 0.5~1 sec exposure for each image. Here is example of the MMM map with ideal resolution.

Step 1: To remove too small beads with the same buffer for the sample

1. Mix 10 µL of 3HB-60nm-SAH-GFPe beads [1 fmol], 100 µL 17% sucrose gradient buffer with 0.01% Tween20
2. Spin 16000g 20 min 4ºC, collect the tube on the handmade magnetic rack
3. Remove supernatant from the tube on the handmade magnetic rack

Step 3: movie collection

The MagIC-cryo-EM data has to be collected on each bead. Using MMM maps, the beads are manually selected. Using ‘Dummy’ SerialEM is helpful in choosing the beads while collecting the data on the microscope. Examples of the bead images include the following:

MagIC-cryo-EM analysis with sample data (to be updated in summer!!)

to be updated 

Troubleshooting and updates (we will continue to update this page)

Identified issues and solutions will be listed here

Issue 1: Biotinylation of the spacer proteins is inefficient. 

Solution 1:

The protocol described in the paper, established at Rockefeller University, did not work at Fred Hutch because the MilliQ at the Hutch is under oxidizing conditions. Using a low concentration of TECP resolved the issue (see the step-by-step protocol above).

Issue 2: The MagIC-cryo-EM beads do not capture the target protein.

Solution 2:

It is possible that the beads are not assembled as intended. We highly recommend performing SDS-PAGE at each step of the bead preparation process, including for the streptavidin beads, prior to protein assembly. In particular, non-specific binding of the target-capturing module can reduce the efficiency of the pull-down.

Acknowledgemet

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. 

View the MagIC-cryo-EM Frequently Asked Questions