Re: [ccp4bb] Improvement in crystal quality
Dear Nishant, are you sure your crystals are protein ? The condition contains 1 M of different salts and PEG as well - I wouldn't exclude that you are having salt crystals. You can easy check this with Izit dye or simply by trying to destroy the crystal with a microtool. If it is salt you can not crush ist. Best wishes, Ulrike
Re: [ccp4bb] Improvement in crystal quality
Niks Seeding often works very well with needles. But start with seeding in RANDOM SCREENS, i.e. rMMS References - Allan D’Arcy, Frederic Villarda, May Marsh. 'An automated microseed matrix-screening method for protein crystallization'. Acta Crystallographica section D63 (2007) 550–554. On-line at http://scripts.iucr.org/cgi-bin/paper?S0907444907007652 **Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E. Chayen and Peter F.M. Baldock. 'Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization'. Cryst. Growth Des., 2011, 11 (8), pp 3432–3441. On-line at http://pubs.acs.org/doi/abs/10.1021/cg2001442 *Chatty article that I wrote for SerCat newsletter in 2009*: http://www.douglas.co.uk/SER-CAT09_1.html *A bit of theory*: http://www.douglas.co.uk/mms.htm *Procedure*: http://www.douglas.co.uk/MMS_proc.htm A. G. Villaseñor, A. Wong, A. Shao, A. Garg, A. Kuglstatter and S. F. Harris. 'Acoustic matrix microseeding: improving protein crystal growth with minimal chemical bias.' Acta Crystallographica Section D66 (2010) 568-576. On-line at http://scripts.iucr.org/cgi-bin/paper?S0907444910005512 Galina Obmolova,* Thomas J. Malia, Alexey Teplyakov, Raymond Sweet and Gary L. Gilliland. 'Promoting crystallization of antibody–antigen complexes via microseed matrix screening.' Acta Crystallographica Section D66 (2010) 927–933. Open-access at http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf On 24 July 2012 14:40, Niks nik...@gmail.com wrote: Dear All, I am trying to crystallize a recombinant dehydrogenase protein. Got five hits in PEG ION Screen from Hamptons (20% PEG 3350 with 0.2M Sodium Acetate, 0.2M Potassium acetate, 0.2M ammonium acetate, 0.2M sodium formate and 0.2M Potassium Chloride) after two days. Crystals looks like needles most of time, sometime broader needles (Pictures attached). UV crystal scanner says those are protein crystals, but when we tried to pick up one and shoot at room temperature, diffraction patterns looks like similar like of powder diffraction (picture attached). I have tried 50 of the 96 additives(whichever I can arrange of) mentioned in the additive screen from Hamptons . I have tried detergent screen from Hamptons (this time original screen solutions). I have tried incubating the plate at 28degrees as well as 10degrees, Though waiting for 10degree results but one drop showed needles again after normal two days of growth period. I tried to slow down the supersaturation by adding 100ul of 1:1 ratio of silicon oil and paraffin oil over the 1ml of well solution. This time no crystals but some precipitation. If anyone spare any word of wisdom to improve these crystal quality, I will be very grateful. If seeding is the only obvious thing to try, any reference for the seeding procedure will be highly appreciated. Thanks very much Nishant Varshney PhD student, National Chemical Laboratory,Pune,India -- The most difficult phase of life is not when No one understands you;It is when you don't understand yourself -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Improvement in crystal quality
4Mb attachments, posted to a mailing list NOT COOL. On 24/07/2012 14:40, Niks wrote: Dear All, I am trying to crystallize a recombinant dehydrogenase protein. Got five hits in PEG ION Screen from Hamptons (20% PEG 3350 with 0.2M Sodium Acetate, 0.2M Potassium acetate, 0.2M ammonium acetate, 0.2M sodium formate and 0.2M Potassium Chloride) after two days. Crystals looks like needles most of time, sometime broader needles (Pictures attached). UV crystal scanner says those are protein crystals, but when we tried to pick up one and shoot at room temperature, diffraction patterns looks like similar like of powder diffraction (picture attached). I have tried 50 of the 96 additives(whichever I can arrange of) mentioned in the additive screen from Hamptons . I have tried detergent screen from Hamptons (this time original screen solutions). I have tried incubating the plate at 28degrees as well as 10degrees, Though waiting for 10degree results but one drop showed needles again after normal two days of growth period. I tried to slow down the supersaturation by adding 100ul of 1:1 ratio of silicon oil and paraffin oil over the 1ml of well solution. This time no crystals but some precipitation. If anyone spare any word of wisdom to improve these crystal quality, I will be very grateful. If seeding is the only obvious thing to try, any reference for the seeding procedure will be highly appreciated. Thanks very much Nishant Varshney PhD student, National Chemical Laboratory,Pune,India -- The most difficult phase of life is not when No one understands you;It is when you don't understand yourself
Re: [ccp4bb] Improvement in crystal quality
The diffraction you see is probably not the best diffraction you could obtain from these crystals. I have found long thin needles are very susceptible to manipulation. I would highly recommend seeding (I like the Hampton Seed Bead personally, http://hamptonresearch.com/product_detail.aspx?cid=18sid=44pid=42 ). You need to try to change the shape of these crystals to beef them up. Other Recommendations: Vary protein concentration Vary pH (if there is not a buffer in the well, determine pH of well and add buffer to stabilize pH and screen around - I've had success changing needle clusters with a few 0.1 pH unit changes) Dioxane to limit crystal nucleation, try to only add it to the well, not mother liquor present in drop, 1 - 5 % Dioxane Different MW PEGs There are almost limitless possibilities of things to try with protein crystallography. Good luck, Kelly Daughtry *** Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 *** On Tue, Jul 24, 2012 at 9:40 AM, Niks nik...@gmail.com wrote: Dear All, I am trying to crystallize a recombinant dehydrogenase protein. Got five hits in PEG ION Screen from Hamptons (20% PEG 3350 with 0.2M Sodium Acetate, 0.2M Potassium acetate, 0.2M ammonium acetate, 0.2M sodium formate and 0.2M Potassium Chloride) after two days. Crystals looks like needles most of time, sometime broader needles (Pictures attached). UV crystal scanner says those are protein crystals, but when we tried to pick up one and shoot at room temperature, diffraction patterns looks like similar like of powder diffraction (picture attached). I have tried 50 of the 96 additives(whichever I can arrange of) mentioned in the additive screen from Hamptons . I have tried detergent screen from Hamptons (this time original screen solutions). I have tried incubating the plate at 28degrees as well as 10degrees, Though waiting for 10degree results but one drop showed needles again after normal two days of growth period. I tried to slow down the supersaturation by adding 100ul of 1:1 ratio of silicon oil and paraffin oil over the 1ml of well solution. This time no crystals but some precipitation. If anyone spare any word of wisdom to improve these crystal quality, I will be very grateful. If seeding is the only obvious thing to try, any reference for the seeding procedure will be highly appreciated. Thanks very much Nishant Varshney PhD student, National Chemical Laboratory,Pune,India -- The most difficult phase of life is not when No one understands you;It is when you don't understand yourself
Re: [ccp4bb] Improvement in crystal quality
Dear Nishant, To me, the diffraction looks like powder diffraction from a salt. In your case, I would look at the protein buffer to see if there are components present (phosphate, detergent etc.) which might be prone to form crystals on their own and see if you can find a minimal buffer where the protein is still happy. With this buffer, I would do another full screening to see if you get hits with more protein-like diffraction. Best, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Niks Sent: Tuesday, July 24, 2012 4:02 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Improvement in crystal quality Dear All, I am trying to crystallize a recombinant dehydrogenase protein. Got five hits in PEG ION Screen from Hamptons (20% PEG 3350 with 0.2M Sodium Acetate, 0.2M Potassium acetate, 0.2M ammonium acetate, 0.2M sodium formate and 0.2M Potassium Chloride) after two days. Crystals looks like needles most of time, sometime broader needles (Pictures attached). UV crystal scanner says those are protein crystals, but when we tried to pick up one and shoot at room temperature, diffraction patterns looks like similar like of powder diffraction (picture attached). I have tried 50 of the 96 additives(whichever I can arrange of) mentioned in the additive screen from Hamptons . I have tried detergent screen from Hamptons (this time original screen solutions). I have tried incubating the plate at 28degrees as well as 10degrees, Though waiting for 10degree results but one drop showed needles again after normal two days of growth period. I tried to slow down the supersaturation by adding 100ul of 1:1 ratio of silicon oil and paraffin oil over the 1ml of well solution. This time no crystals but some precipitation. If anyone spare any word of wisdom to improve these crystal quality, I will be very grateful. If seeding is the only obvious thing to try, any reference for the seeding procedure will be highly appreciated. Thanks very much Nishant Varshney PhD student, National Chemical Laboratory,Pune,India -- The most difficult phase of life is not when No one understands you;It is when you don't understand yourself -- The most difficult phase of life is not when No one understands you;It is when you don't understand yourself
