You have mentioned that you have 20-30% crosslinked product (I suppose in the crude reaction mix before any further purification). Does this percentage change (where your 300kDa complex is predominant, and 200kDa protein is much less) change after any of the downstream purification schemes?

Though not impossible, computational 'purification' may be difficult due to small difference in size (300kDa vs 200kDa). A number of parameters will hold crucial clues to this problem. 

It is therefore recommended to exhaustively try to enrich your target complex (300kDa).

Good luck!

You should run iex at different conditions using cation and anion exchange media. You can also try hic. Be sure to use chromatographic media with large pore size.

Hey Sunil Singh,

    You have an interesting problem and project and, as stated by Smarajit, there are a number of issues pressing the need for separation techniques.

    Using a "raw" preparation of your cross-linked material would likely produce, given the correct imaging conditions, images of your complex. That is true. And, you do not know if the uncomplexed 200 Kda molecule has a tertiary structure which, in the absence of ligand, looks something like your 300 Kda complex. Hence, you will spend inordinate amounts of time only to find that you cannot bring your structure to a given single structure. That brings me to the next suggestion, Marin van Heel.

    Some years ago, Marin produces, along with his colleagues, a manuscript dealing with the simultaneous reconstruction of multiple models. His work using IMAGIC 4D provides this capability and, if I am not mistaken, uses FREALIGN approaches in addition to his other reference-based reconstruction tools. I would suggest you attempt to contact Marin or the folks at Image Sciences in Germany regarding the capabilities of IMAGIC 4D.

    Lastly, in a project I had started just prior to the end of my science career due to funding constraints, I was considering an X-ray/Cryo-EM problem where knowing the overall Structure Factor (X-ray) over all Fourier space would be useful. The question for all of our projects has to do with sample purity and concentration. A question came to mind: Where, in our analytical chemistry toolbox, do we ever obtain increases in concentration? The answer, in a band of complex separated on a gel. The concentration of a pure complex within a band on a Gel Electrophoresis band approaches that necessary to obtain the continuous Structure Factor (X-ray) or Shape Factor (Cryo-EM) necessary to resolve an ultimate end result. I was able to obtain a protein at sufficiently high a concentration and was able to determine the contribution of the Gel to background X-ray Scatter...negligible. 

    For your project, have you ever looked at your complex in a Non-Reducing Gel Electorphoresis Gel? You can easily resolve 200 Kda and 300 Kda. Cutting the band out and removing the gel under controlled "Nano" conditions would allow you to obtain the 1 mg/ml concentrations necessary to obtain the Cryo-EM images. More, you can look at your unseparated complex, the 200 Kda  complex post reaction. Given these kinds of controls, you should be able to determine the conditions necessary to obtain the desired end result.

    All I can say to you is, don't give up, there is always a way to skin a cat, you need only figure out which cat and how you'd like it skinned!

    Enjoy. I do hope my gel method is possibly of benefit to you. I was certain it would have been for my macromolecule were I to have had the time to get that project further down the road.

David

 Sunil Singh, I am sorry for having forgotten yet another approach. Does you  complex have a His-tag or other such tag? I had been doing some Cryo-EM work with preparing a 2D His-tag binding array containing a Nickel reaction center. Forming a 2D "tape" structure on the surface of a tiny Langmuir-Blodgett Trough allowed me the ability to drop a Holey Carbon Grid with very thin Carbon across the holes...it did work without the carbon as these were 1 micron holes. I was able to prove that the Ni-DithioBiuret complex did hold my His-tagged complex to the surface of the Holey Carbon Grid. This is yet one more method I'd developed which did not get published for reasons I would rather not discuss. If you do a search for my name and DTB, you will find at least one Biophysical Society Poster I'd done using this approach... For clarity, the work and subsequent Poster came as a result of a long series of experiments ultimately ending with this Poster done by a Summer Student working with me.

Dear All,

Thank you for your suggestions.

Dear David,

Thank you for sharing your expertise with me.

Regards

Sunil Singh

Hi Sunil Singh,

I think you need to  increase the yield of your XL complex. Do you pre-purify your complex before adding the cosslinker? I wonder if the free protein A in not corsslinked because it was never in the complex. If this is the case, you van try to form the complex adding an excess of B and C, then purifying the complex by gel filtration. You can corosslink the already purified complex and run again a gel filtration to purify from unspecific crosslinked species.

Also, there are several groups using graFix to purify and stabilise complexes for EM:

Methods Enzymol. 2010;481:109-26. doi: 10.1016/S0076-6879(10)81005-5.

good Luck!