Because the MilliQ at the Fred Hutch is under oxidizing conditions, the protocol described in the afore mentioned paper, established at Rockefeller University, did not work at Fred Hutch. However, using a low concentration of TECP resolved the issue.
Day 1
1. Express the spacer protein in Rosetta (DE3)
2. Resuspend E.coli pellet with sonication buffer (1x PBS, 400 mM Additional NaCl, 5 % glycerol, 5 mM 2-me, 1 mM PMSF, 30 mM imidazole, 1x protease inhibitor)
3. Sonication
4. Remove insoluble fraction with centrifugation at 30000rpm, 30min, 4ºC
5. Filter supernatant with 0.22µm syringe filter
6. Load sample to His-Trap HP 5ml column using a peristaltic pump
7. Wash column with 50 ml wash buffer 1 (1 x PBS, 800 mM Additional NaCl, 5 % Glycerol, 30 mM imidazole, 2 mM TCEP, and 1 mM PMSF)
8. Wash column with 50 ml wash buffer 2 (1 x PBS, 5 % Glycerol, 30 mM imidazole, 2 mM TCEP)
9. Connect on AKTA
Pump A: 1 x PBS, 5 % Glycerol, 30 mM imidazole, 2 mM TCEP
Pump B: 1 x PBS, 5 % Glycerol, 500 mM imidazole, 2 mM TCEP
10. Elute with B10% (5CV) => B40% (5CV) => B100% (7CV)
11. Run SDS-PAGE (Peak in B40% may contain spacer protein)
12. Collect fractions containing the spacer protein
13. Add SENP1 protease
14. Dialyze to 500 ml Capto HiResQ wash buffer (20mM KH2PO4 / K2HPO4 (pH8), 5% glycerol, 2 mM TCEP), 4ºC, o/n
Day 2
15. Apply the sample to His-Trap HP 5ml column and collect the flow-through
16. Apply the flow-through to Capto HiResQ
17. Elute with linear gradient
Pump A: 20 mM KH2PO4-K2HPO4 (pH 8), 5 % Glycerol, 2 mM TCEP
Pump B: 1x PBS, 1 M Additional NaCl, 5 % glycerol, 2 mM TCEP
18. SDS-PAGE (Peak in B10-20% may contain spacer protein)
19. Collect fractions containing the spacer protein
20. Apply the sample to Superdex S200 16/600 (x1 PBS + 5% gltcerol + 1 mM TCEP)
21. SDS-PAGE (Peak in 50-60ml elution volume may contain 60 nm SAH spacer protein)
22. Collect fractions containing the spacer protein
23. Concentrate the protein using Amicon 10K to 1ml
24. Estimate the concentration using nanodrop (The extinction coefficient of 60 nm SAH is: 11460)
25. Mix Meleimide-biotin with proteins (50-fold molar excess Meleimide-biotin)
26. On ice, o/n
Day 3
27. Dialyze sample to Quench buffer (1x PBS, 5 % Glycerol, 2 mM 2-me)
28. Maintain in Cold room overnight
Day 4
29. Apply sample to Superdex S200 16/600 (1x PBS, 5% glycerol) *inadequate modification on 60nm SAH disrupts the single alpha helix and changes the Superdex profile from the day 2’s result
30. Concentrate the protein using Amicon 10K to 1ml
31. Estimate the concentration using a nanodrop
32. Add equal volume of storage buffer (1x PBS, 90% glycerol)
33. Store at -20ºC
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.