[ccp4bb] Crystallization condition for trimeric protein
Hi, I am working with a protein which can assume different oligomerization forms, starting from monomers to trimers and even penta-decamers. We conformed this by Native PAGE and HPLC studies. The protein's theoretical monomeric molecular weight is 14.6 KDa (pI - 5.9) and it has some 140 amino acids with high Glutamic acid (24), Lysine (10) and Arginine (13) content. I have tried to crystallize it but not getting any hit as far now. Previous study showed that this protein gets some stability by Calcium ion. With the calcium chloride conditions, I am getting spherical shaped structures, but not sure what are they; calcium chloride crystals or protein crystals. Can protein crystals be spherical in shape, specially when the protein behaves like an oligomer? Also please let me know what is the minimum protein concentration required to obtain crystal for such small protein (if there is any empirical rule/idea). Any suggestion will be highly appreciated. Thanks and regards, Debasish Kumar Ghosh CSIR- Junior Research Fellow (PhD Scholar) C/o: Dr. Akash Ranjan Computational and Functional Genomics Group Centre for DNA Fingerprinting and Diagnostics Hyderabad, INDIA Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com Telephone: 0091-9088787619 (M), 0091-40-24749396 (Lab) Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html
Re: [ccp4bb] Crystallization condition for trimeric protein
As far as I know, there is no specific rule with the concentration for the crystallization. There are some pre-crystallization tests available, mainly from the hampton research which may help you to determine the appropriate concentration for the crystallization. Coming to the spherical structures, it may be quasicrystals. Just try by increasing the concentration when you screen again. On Thu, Sep 12, 2013 at 4:42 PM, Debasish Kumar Ghosh wrote: > Hi, > > I am working with a protein which can assume different oligomerization > forms, starting from monomers to trimers and even penta-decamers. We > conformed this by Native PAGE and HPLC studies. The protein's theoretical > monomeric molecular weight is 14.6 KDa (pI - 5.9) and it has some 140 amino > acids with high Glutamic acid (24), Lysine (10) and Arginine (13) content. > I have tried to crystallize it but not getting any hit as far now. > Previous study showed that this protein gets some stability by Calcium > ion. With the calcium chloride conditions, I am getting spherical shaped > structures, but not sure what are they; calcium chloride crystals or > protein crystals. Can protein crystals be spherical in shape, specially > when the protein behaves like an oligomer? > Also please let me know what is the minimum protein concentration required > to obtain crystal for such small protein (if there is any empirical > rule/idea). > Any suggestion will be highly appreciated. > > Thanks and regards, > Debasish Kumar Ghosh > > CSIR- Junior Research Fellow (PhD Scholar) > C/o: Dr. Akash Ranjan > Computational and Functional Genomics Group > Centre for DNA Fingerprinting and Diagnostics > Hyderabad, INDIA > > Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com > Telephone: 0091-9088787619 (M), 0091-40-24749396 (Lab) > Lab URL: > http://www.cdfd.org.in/labpages/computational_functional_genomics.html > -- Vikram Babu Kasaragod Graduate Student Hermann Schindelin Group Rudolf Virchow Center University of Wuerzburg Josef-Schneider-Straße 2, Haus D15 D - 97080 Würzburg. email: vikram.kasara...@uni-wuerzburg.de website: www.rudolf-virchow-zentrum.de Phone : +49 931 31-89938 Fax: +49 931 31-83255
Re: [ccp4bb] Crystallization condition for trimeric protein
Perhaps you should try finding buffer conditions and protein concentration that pushes the self-association equilibrium to one particular oligomeric state. Sent from Jack's iPad On Sep 12, 2013, at 9:53 AM, "Debasish Kumar Ghosh" wrote: > Hi, > > I am working with a protein which can assume different oligomerization forms, > starting from monomers to trimers and even penta-decamers. We conformed this > by Native PAGE and HPLC studies. The protein's theoretical monomeric > molecular weight is 14.6 KDa (pI - 5.9) and it has some 140 amino acids with > high Glutamic acid (24), Lysine (10) and Arginine (13) content. I have tried > to crystallize it but not getting any hit as far now. > Previous study showed that this protein gets some stability by Calcium ion. > With the calcium chloride conditions, I am getting spherical shaped > structures, but not sure what are they; calcium chloride crystals or protein > crystals. Can protein crystals be spherical in shape, specially when the > protein behaves like an oligomer? > Also please let me know what is the minimum protein concentration required to > obtain crystal for such small protein (if there is any empirical rule/idea). > Any suggestion will be highly appreciated. > > Thanks and regards, > Debasish Kumar Ghosh > > CSIR- Junior Research Fellow (PhD Scholar) > C/o: Dr. Akash Ranjan > Computational and Functional Genomics Group > Centre for DNA Fingerprinting and Diagnostics > Hyderabad, INDIA > > Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com > Telephone: 0091-9088787619 (M), 0091-40-24749396 (Lab) > Lab URL: > http://www.cdfd.org.in/labpages/computational_functional_genomics.html
Re: [ccp4bb] Crystallization condition for trimeric protein
HI debashish , These crystal might be spherulites. You can try reducing protein concentration in this situation and Microseeding will be the best idea for this. You should crush these crystals by using seed bead or needles and do either seeding with full screen or dilution or streak seeding with low concentration of protein. I had positive experience with these type of spherulites. good luck with your seeding vandna On Thu, Sep 12, 2013 at 9:42 AM, Debasish Kumar Ghosh wrote: > Hi, > > I am working with a protein which can assume different oligomerization > forms, starting from monomers to trimers and even penta-decamers. We > conformed this by Native PAGE and HPLC studies. The protein's theoretical > monomeric molecular weight is 14.6 KDa (pI - 5.9) and it has some 140 amino > acids with high Glutamic acid (24), Lysine (10) and Arginine (13) content. > I have tried to crystallize it but not getting any hit as far now. > Previous study showed that this protein gets some stability by Calcium > ion. With the calcium chloride conditions, I am getting spherical shaped > structures, but not sure what are they; calcium chloride crystals or > protein crystals. Can protein crystals be spherical in shape, specially > when the protein behaves like an oligomer? > Also please let me know what is the minimum protein concentration required > to obtain crystal for such small protein (if there is any empirical > rule/idea). > Any suggestion will be highly appreciated. > > Thanks and regards, > Debasish Kumar Ghosh > > CSIR- Junior Research Fellow (PhD Scholar) > C/o: Dr. Akash Ranjan > Computational and Functional Genomics Group > Centre for DNA Fingerprinting and Diagnostics > Hyderabad, INDIA > > Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com > Telephone: 0091-9088787619 (M), 0091-40-24749396 (Lab) > Lab URL: > http://www.cdfd.org.in/labpages/computational_functional_genomics.html > -- Vandna Kukshal Postdoctral Research Associate Dept. Biochemistry and Molecular Biophysics Washington University School of Medicine 660 S. Euclid, Campus Box 8231 St. Louis, MO 63110
