Hi Saurabh, I have also encountered a similar problem. Sometimes, though your protein looks pure in size exclusion chromatography, but actually is not very pure and may contain traces of partially unfolded form or some transient oligomeric forms. In such a case, your size exclusion profile would be very broad and if such impurities remain within, it would affect the packing of crystal resulting in poor diffraction.
What I have done to get rid of it is, I tried mild heating at 42 degrees C for 5 min, spin down the protein at very high speed, take out the supernatant and straight away go for crystallization. If by any chance your protein has some thermal stability, you could also, try mild heating before crystallization. This removes the trace contaminants and makes your sample more homogenous. Alternatively, if your protein sequence has long disorder regions internally, then you could try in-situ proteolysis also. Using a protease at a very low concentration in your crystallization drop would chop off the disordered loops in your protein, which would favor a better packing. Also, you can try slightly altering the length of your construct, wherein if the ends are disordered or highly charged, you can trim those regions from your construct to get a more stable and homogenous sample. Another thing I noticed is your two conditions have PEG in them. For those conditions sometimes a low concentration (20-25%) of PEG 400 works best as a cryoprotectant. You could try using that as well. I wish you all the best with your trials. Best Ashish Kumar On Sun, Mar 21, 2021 at 11:04 AM Saurabh Upadhyay <saurabh807...@gmail.com> wrote: > Dear All, > > I am trying to crystalize a protein, which occurs in monomeric (60kDa) and > tetrameric (240kDa) form. *The protein during crystallization was in > MES buffer pH-6 *and the method of crystallization was "*Sitting drop*" > in "*Proplex*" condition. Some of the conditions in which crystals were > obtained are: > > *1) 0.1M HEPES pH-7; 10% (w/v) PEG 4000* > *2) 0.1M Tris, pH-8; 8% (w/v) PEG 8000* > *3) 0.1M Tris, pH-8; 25% (v/v) PEG 400* > > For reference, I have attached the images of the crystals observed below. > > *However, the diffraction pattern obtained and the resolution of crystals > were very poor. Even some mosaicity has been observed. * > > Kindly suggest some methods or modifications, to improve the resolution > and diffraction pattern of the crystals obtained. > > Thanking You, > Sincerely, > Saurabh Upadhyay, > Ph.D. Scholar > c/o Dr. Ashok kumar Patel, > Kusuma School of Biological Sciences, > Indian Institute of Technology, Hauz Khas, > New Delhi-110016, India > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
