my protein is belong to glycosyltransferase, i got crystal size 1mm, different cryopro. were used 30% glycerol, 30% PEG 200, i got diffraction 30A, 27A, and 10A my crystal it's good enough , i though will solve structure, but no data:( ,plz can any one suggest me next step to solve the structure.. thanks in advance.
Hi Wafa,
I assume 30A, 27A and 10A are the resolutions in angstroms. Getting good looking crystal doesn't necessarily gives good diffraction. One approach I take is to minimise the disorder in the protein structure. In my experience, addition of ligands and removal of disordered residues would help. For the former, you can try co-crystallise your protein with some substrates or products. To remove disordered residues, you can do some online disorder prediction. If your N or C termini are predicted to be disordered, clone your protein without them. You can experimentally determine the disordered region by adding a small amount of protease in your sample. If your protein ended up as one major band at lower molecular weight, mass spec it to determine its sequence. Some people just add a small amount of protease when they do their crystallisation screening, and that works as well.
Best regards,
Arthur
Optimal cryoprotection is critical. Try glycerol or ethylene glycol at a final concentration of 30%, with successive 10% increments added to your initial condition. Depending on the latter you can try diffraction without cryoprotectant.
Your crystals may also never diffract at all, no matter how nice they look.
Yes, a nice crystal is not equivalent to a good enough data set. I would keep trying to get more crystals. Can I see (or can you send me) one of your images? Just to look at it and add it to my blog (only if it's ok. with you)
Smaller sized crystals could be better to be diffracted due to the compact packaging than bigger ones. Would you make the crystallization conditions change into other screening conditions like Morpheus or Wizard or something else?
Is it a membrane protein? Detergent crystals (DDM) can have similar morphology and strange looking low res scattering/diffraction.
How well do they diffract when you don't cryoprotect them? You'll probably get ice rings but at least you'll know if it's the cryoprotection step that ruins your crystals or if they were terrible from the beginning. You may also want to try a room temp. diffraction test to see if freezing is hurting your crystals.
The crystals are quite large so wash them and run them on an SDS PAGE. Mass spec the band. It's very little work and it could save you heaps of problems later on.
Once you have not determined how your crystals diffract at room temperature, you don’t know the original quality of your crystals. But independently, you can try annealing of the crystals. In my practice this approach may lead to dramatic improvement of diffraction power of the crystals. I would advice some changes in the mother liquor, for instance, it you have salt component in your crystallization solution, you can increase its concentration. An increase of at least 5-10% from the initial concentration may be a good start. If not, you can increase the molarity of the buffer. You use pretty high concentrations of cryoprotectants, and that may affect the result of cryoprotection. Some crystals do not tolerate quick changes of the mother liquor, so you may need to do small, gradual changes of the mother liquor. Best luck.
thanks to u all for ur answer and suggestion , Dr. yancho yes i have salt component in crystallization solutionton 1M(NH4)2SO4 i have screened 0.2-1M got the proper 0.8-1M , for the crystal i attached i used 0.9M salt 0..1M Bis-Tris 5.5 1-10% PEG400 (4% proper PEG conc.) , how about dehydration by increase PEG concentration after getting crystal?
I found an article in Acta D, which describes the improvement of diffraction from poorly diffracting crystals. In this protocol author explained a different approach using glycerol and ethylene glycol in the well solution 1 (glycerol or ethylene glycol)+ 9 well solution. He froze the crystals in liquid nitrogen and soak the crystal again for an hour in the cryo solution, which improved the diffraction of crystals from 12A to 2.6A. I attached the reference below. Good luck.
Spectacular improvement of X-ray diffraction through fast desiccation of protein crystals. Acta Cryst. (2004). D60, 1413±1416.
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