Crosslinking of transient interacting complex?

Hi,

I want to make a cryo-EM sample from a complex of 2 proteins that have a total size of around 130 KDa. The problem is that these proteins form a transient complex, and I have been told that such transient interactions generate multiple different pictures in Cryo-EM and a lot of people recommend to crosslink the complex.

My question is, Will crosslinking modify/reduce/increase rigidity? Will it affect protein complex conformation? Will the crosslinker bind to such a high degree (many molecules bound) that later it will interfere with elucidating the real protein structure?

Will the length of the crosslinker also be a factor to consider? Is it better for example to use short-length crosslinkers rather than long such as DTSSP etc?

Thank you

I

During imaging the particles are basically destroyed - the structure is recovered in the average because each one is damaged differently. The same concept applies to crosslinked particles - the crosslinkers will be in mostly different places from particle to particle.

The degree of crosslinking is up to you. At some point, any of the properties you mention may be affected, but unless the crosslinking is very aggressive it will most likely simply prevent exchange between different states and “lock in” the distribution you had before.

Defined length crosslinkers like BS3 may be better than e.g. gluteraldehyde which polymerizes (unless you use the GraFix method to separate aggregates). Some people I know like to add BS3 to the droplet on the grid right before freezing. You should always try your sample as is first too.

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Thank you so much for this explanation, I will try the BS3 crosslinker with my protein complexes, seems that this one is quite popular for Cryo-EM experiments.

If I have the time for the Cryo-EM measurement, I will try to test both with and without a crosslinker; it seems quite trivial, but since I have no experience with this, it seems to me that is one extra factor to be considered during the sample preparation.

Hopefully I will get some data,

Thank you :slight_smile:

Don’t forget to test crosslinking by SDS-PAGE or native PAGE too! (Cheaper than a microscope session).