Recently, I purified and crystallized a novel protein . The crystal of my protein from initial screening is big but unfortunately no diffraction in the X-ray crystallography.In the meantime, I apply different methods to improve the quality of crystals but with no improvement. Kindly your suggestions will be highly appreciated .Thanks in advance
Hi Muslim,
are you sure you crystals are protein? Sometimes salts or PEG or other components can form crystals. To test if your crystal is your protein of interest you can run it on a SDS-PAGE gel. Just take the crystal out of your drop and wash ~5times in a few microliter reservoir solution then transfer to SDS loading buffer and run like you would a normal protein sample.
If you are sure that your crystal is protein it could be that the crystal is not well ordered. If your big crystal grows very fast it may be an option to slow down crystal growth by reducing temperature or varying protein to precipitate ratio. I would also try an additive screen.Hope that helps. Good luck.
Dear Prof Bernhard Lechtenberg,
First of all thank you very much for your response and fruitful discussions.....The crystal I got is the purified protein and not the salt crystals but actually the crystals is big in size but weak and thin I am screening and optimizing the precipitant, PH and screening the additives then If I find some thing more fruitful then I will discuss with you......
Hi Muslim,
I had that several time before that a rather huge crystal did not diffract any X-rays at all. In one case I could optimize the cryo so that we could see 3-5 reflections close to the beam stop. I interpreted those negative results as very high degree of long-range disorder in the crystals, resulting in essentially a zero signal to noise ratio during the diffraction experiment.
There are several methods to improve the long range order in a crystal (cross linking the crystals, drying out the crystals, etc.), however, in such a severe case there is little hope of archiving anything useful by trying to improving the long-range order. There is clearly something wrong with the way your sample crystallizes. I would try dehydrating the crystals first, because it is cheap and you have crystals, but you might need to recrystallize the sample in a (slightly? totally?) different condition.
good luck,
S.
Hi Muslim
having a 'weak crystal' to me suggests that you have very high solvent content and that the protein molecules are probably very loosely packed inside the crystal. Thus as Stefan Gajewski suggests, I would definitely try dehydrating the crystal. Also be as careful as possible while handling the crystals since high solvent content crystals are often very fragile. Another thing I would try if you have the setup to do it is to collect a few diffraction images at room temperature. This might tell you whether the crystal itself is not well ordered, or whether cryo protection does harm your crystal (assuming you are testing diffraction under cryogenic temperatures at the moment). If the latter is the case, you need to optimize your cryo conditions. Again be as gentle as possible, try step-wise increase of cyro solution or slowly add more cryo buffer to your crystallization drop.
Hi Musilm
In our experience this is quite normal to have almost no diffraction with some protein crystals. Specially true for big-huge, or complexes or flexible samples. You could try to find other crystallization conditions until find one with better diffraction. For some very difficult cases we used partial digestion (Contrearas-Martel et al. 2006) and the SERp approach (Wong & Dessen 2014) (http://services.mbi.ucla.edu/SER/). In this last case the improvements were remarkable.
Good luck
Carlos
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