Re: [ccp4bb] Crystal Optimization
Dear All, Thanks alot for your valuable suggestion.I hope I will find out the solution now. As far as to giveup is out of question Thanks once agin Regards; Bashir On Wed, July 11, 2012 05:25, Tuhin Bhowmick wrote: Dear Muhammad, I had a similar case, and the crystals could indeed be optimized. A few things to check first, 1) How long does it take for the needles to appear? Sometimes, if the protein is degraded/ cleaved over time, a small population (possibly a fragment of the whole protein) from the heterogeneous mix can give similar crystals. Like Bryan had suggested, it is also very useful to check the mol wt. of the crystallized species through mass spect/ native page. But do make sure to give the crystals serial washes, so the test accounts for crystallizexd species, not the ones from surrounding condition. 2) If the crystals indeed contain the protein of interest, they can be used for various seeding methods. I've got results from both streak and micro seeding. Best, Tuhin. Tuhin Bhowmick Department of Physics Indian Institute of Science Bangalore: 560012 Email: tuhin.i...@gmail.com On Wed, Jul 11, 2012 at 12:34 AM, Bernhard Rupp (Hofkristallrat a.D.) hofkristall...@gmail.com wrote: Always give up. ** ** ...definitely not the kind of guy I want to sit up front in an airliner…** ** ** ** Best regards, BR - Bernhard Rupp, ATP-B737, CFII-MEI Vienna Air International Professional Aviation Services 001 (925) 209-7429 +43 (676) 571-0536 b...@vienna-air.com b...@ruppweb.org http://www.vienna-air.com/ - It is not your aptitude but your attitude that determines your altitude. (or your crystals) - ** ** ** ** ** ** From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of yybbll Sent: Tuesday, July 10, 2012 11:58 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal Optimization ** ** Hi, In my experience, it is very very very difficult to optimize this needle like crystal. Always give up. Good luck! Dear All; Could somebody give a nice suggestion how the following type crystal could be optimized, I almost tried everything. Crystal Image is attached Crystal condition: 20% w/v PEG3350 and 200mM NaCl. Thanks in advance Bashir -- Muhammad Bashir Khan ** Structural Genome Consortium (SGC). University of Toronto Toronto, Canada -- Muhammad Bashir Khan ** Department for Structural and Computational Biology Max F. Perutz Laboratories University of Vienna Campus Vienna Biocenter 5 A-1030 Vienna Austria Austria Phone: +43(1)427752224 Fax: +43(1)42779522
Re: [ccp4bb] Crystal Optimization
Give a list of all you have tried? JPK On Tue, Jul 10, 2012 at 12:22 PM, Muhammed bashir Khan muhammad.bashir.k...@univie.ac.at wrote: Dear All; Could somebody give a nice suggestion how the following type crystal could be optimized, I almost tried everything. Crystal Image is attached Crystal condition: 20% w/v PEG3350 and 200mM NaCl. Thanks in advance Bashir -- Muhammad Bashir Khan ** Structural Genome Consortium (SGC). University of Toronto Toronto, Canada -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Crystal Optimization
Have you tried multiple rounds of micro-seeding? --- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 ** Hard work often pays of in time, but Procrastination always pays off now ** - Original Message - From: Muhammed bashir Khan muhammad.bashir.k...@univie.ac.at To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, July 10, 2012 1:22:28 PM Subject: [ccp4bb] Crystal Optimization Dear All; Could somebody give a nice suggestion how the following type crystal could be optimized, I almost tried everything. Crystal Image is attached Crystal condition: 20% w/v PEG3350 and 200mM NaCl. Thanks in advance Bashir -- Muhammad Bashir Khan ** Structural Genome Consortium (SGC). University of Toronto Toronto, Canada
Re: [ccp4bb] Crystal Optimization
Dear Muhammed, In my experience, crystals like that are likely made up of contaminated or non-homogeneous protein. Have you run NATIVE PAGE and IEF gels to determine the purity of your sample? Is it the correct MW by mass spec without contaminating peaks? Good Luck! Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Greg Costakes Sent: Tuesday, July 10, 2012 2:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal Optimization Have you tried multiple rounds of micro-seeding? --- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 ** Hard work often pays of in time, but Procrastination always pays off now ** From: Muhammed bashir Khan muhammad.bashir.k...@univie.ac.at To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, July 10, 2012 1:22:28 PM Subject: [ccp4bb] Crystal Optimization Dear All; Could somebody give a nice suggestion how the following type crystal could be optimized, I almost tried everything. Crystal Image is attached Crystal condition: 20% w/v PEG3350 and 200mM NaCl. Thanks in advance Bashir -- Muhammad Bashir Khan ** Structural Genome Consortium (SGC). University of Toronto Toronto, Canada -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] Crystal Optimization
Hi, In my experience, it is very very very difficult to optimize this needle like crystal. Always give up. Good luck! Dear All; Could somebody give a nice suggestion how the following type crystal could be optimized, I almost tried everything. Crystal Image is attached Crystal condition: 20% w/v PEG3350 and 200mM NaCl. Thanks in advance Bashir -- Muhammad Bashir Khan ** Structural Genome Consortium (SGC). University of Toronto Toronto, Canada
Re: [ccp4bb] Crystal Optimization
Always give up. ...definitely not the kind of guy I want to sit up front in an airliner… Best regards, BR - Bernhard Rupp, ATP-B737, CFII-MEI Vienna Air International Professional Aviation Services 001 (925) 209-7429 +43 (676) 571-0536 b...@vienna-air.com b...@ruppweb.org http://www.vienna-air.com/ - It is not your aptitude but your attitude that determines your altitude. (or your crystals) - From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of yybbll Sent: Tuesday, July 10, 2012 11:58 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal Optimization Hi, In my experience, it is very very very difficult to optimize this needle like crystal. Always give up. Good luck! Dear All; Could somebody give a nice suggestion how the following type crystal could be optimized, I almost tried everything. Crystal Image is attached Crystal condition: 20% w/v PEG3350 and 200mM NaCl. Thanks in advance Bashir -- Muhammad Bashir Khan ** Structural Genome Consortium (SGC). University of Toronto Toronto, Canada
Re: [ccp4bb] Crystal Optimization
Dear Muhammad, I had a similar case, and the crystals could indeed be optimized. A few things to check first, 1) How long does it take for the needles to appear? Sometimes, if the protein is degraded/ cleaved over time, a small population (possibly a fragment of the whole protein) from the heterogeneous mix can give similar crystals. Like Bryan had suggested, it is also very useful to check the mol wt. of the crystallized species through mass spect/ native page. But do make sure to give the crystals serial washes, so the test accounts for crystallizexd species, not the ones from surrounding condition. 2) If the crystals indeed contain the protein of interest, they can be used for various seeding methods. I've got results from both streak and micro seeding. Best, Tuhin. Tuhin Bhowmick Department of Physics Indian Institute of Science Bangalore: 560012 Email: tuhin.i...@gmail.com On Wed, Jul 11, 2012 at 12:34 AM, Bernhard Rupp (Hofkristallrat a.D.) hofkristall...@gmail.com wrote: Always give up. ** ** ...definitely not the kind of guy I want to sit up front in an airliner…** ** ** ** Best regards, BR - Bernhard Rupp, ATP-B737, CFII-MEI Vienna Air International Professional Aviation Services 001 (925) 209-7429 +43 (676) 571-0536 b...@vienna-air.com b...@ruppweb.org http://www.vienna-air.com/ - It is not your aptitude but your attitude that determines your altitude. (or your crystals) - ** ** ** ** ** ** From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of yybbll Sent: Tuesday, July 10, 2012 11:58 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal Optimization ** ** Hi, In my experience, it is very very very difficult to optimize this needle like crystal. Always give up. Good luck! Dear All; Could somebody give a nice suggestion how the following type crystal could be optimized, I almost tried everything. Crystal Image is attached Crystal condition: 20% w/v PEG3350 and 200mM NaCl. Thanks in advance Bashir -- Muhammad Bashir Khan ** Structural Genome Consortium (SGC). University of Toronto Toronto, Canada
Re: [ccp4bb] Crystal Optimization: Summary
Dear All, Thank you for the replies to my post. I received more than 14 response. The summary is given below. The suggestions were - add another purification step. * Ni-column * remove His-tag * Ni-column * gel filtration if you have done that: add another step to test for better crystals - additive screens - room temperature data set to check if freezing hampers the crystal - seeding - crystallisation at different temperatures - removal of the His-tag (or leave it on if it is cleaved) Dehydration. Reductive methylation Add PEG400 (or set your PEG concentration ~5% higher) into the cryosolution. Cryoprotection with DMSO. Grew the crystals in the presence of 5% glycerol. Crosslink the crystals with glutaraldehyde by vapor diffusion. Adding 20% peg200 as cryoprotectant. Using the Hampton Research Crystal Screen HT as additive screen (adding 5% into the mother liquor). Slowly increase the PEG concentration to 28 - 30% Add 1-2% Glycerol or MPD or Ethylene Glycol or other cryo-agent to your protein buffer you may find similar or even identical crystallization condition in which your crystal may tolerate higher concentration of cryo-agent. To include any salts in the cryo (at least half of the concentration) that may be already in the protein solution.
[ccp4bb] Crystal Optimization
Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M BisTris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi
Re: [ccp4bb] Crystal Optimization
Dear Jobi, The usual suspects are: - add another purification step. In fact I'd do that first: * Ni-column * remove His-tag * Ni-column * gel filtration if you have done that: add another step to test for better crystals - additive screens - room temperature data set to check if freezing hampers the crystal - seeding - crystallisation at different temperatures - removal of the His-tag (or leave it on if it is cleaved) etc, etc, pp Cheers, Tim On Wed, Apr 13, 2011 at 05:44:12PM +0800, Jobichen Chacko wrote: Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M BisTris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Crystal Optimization
Dear Jobi, For a crystal which is big in size(0.2-0.3mm is pretty big) while diffract poorly, dehydration (increase concentration of the precipitant slowly) is a good choice to improve diffraction, especially for those tends to crack during cryo. Also those regular optimization approaches: Additive screen etc. Sometimes cleave the tag or change it to another end will work. Cheers, Bingfa 发件人: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] 代表 Jobichen Chacko 发送时间: 2011年4月13日 17:44 收件人: CCP4BB@JISCMAIL.AC.UK 主题: [ccp4bb] Crystal Optimization Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M BisTris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi
Re: [ccp4bb] Crystal Optimization
Dear Jobi, the paper of Heras and Martin 'Post-crystallization treatments for improving diffraction quality of protein crystals' is really helpful. Additionally, if you have lysines have you tried reductive methylation? Good luck, e On Wed, April 13, 2011 12:34, Bingfa Sun wrote: Dear Jobi, For a crystal which is big in size(0.2-0.3mm is pretty big) while diffract poorly, dehydration (increase concentration of the precipitant slowly) is a good choice to improve diffraction, especially for those tends to crack during cryo. Also those regular optimization approaches: Additive screen etc. Sometimes cleave the tag or change it to another end will work. Cheers, Bingfa 发件人: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] 代表 Jobichen Chacko 发送时间: 2011年4月13日 17:44 收件人: CCP4BB@JISCMAIL.AC.UK 主题: [ccp4bb] Crystal Optimization Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M BisTris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi -- ** Eirini Gkougkoulia Department for Structural and Computational Biology Max F. Perutz Laboratories University of Vienna Campus Vienna Biocenter 5 A-1030 Vienna Austria
Re: [ccp4bb] Crystal Optimization
Hi Jobi, I also had some crystals that were highly sensitive to glycerol. In one case, I found that DMSO at 10-15% can cryo protect it, also the crystal could grow in the presence of 10% DMSO which essentially eliminated a soaking step. In another case, I grew the crystals in the presence of 5% glycerol(it won't take DMSO this time), then the crystal could survive 30% glycerol cryo soak. Also you may try using some smaller crystals as they are easier to freeze and may still diffract decently in synchrotron. Another thing to try is to crosslink the crystals with glutaraldehyde by vapor diffusion. The crystal will partially turn to a gel and will not break when soaked with cryos. In my hand this did produce tough, diffracting crystals, but didn't solve my high mosaicity problem. Of course, RT shooting should be tested first to make sure the poor diffraction is caused by the freezing. Zhijie From: Jobichen Chacko Sent: Wednesday, April 13, 2011 5:44 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystal Optimization Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M BisTris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi
Re: [ccp4bb] Crystal Optimization
Hi Jobi- You might want to try using drop ratios ---we have had great success with this many times. My favorite additive screen is using the Hampton Research Crystal Screen HT as an additive screen. I usually start by adding 5% to the well---this has often yielded good crystals where the traditional 96 reagent additive screens and the detergent screens do not. Good Luck! Annie From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jobichen Chacko Sent: Wednesday, April 13, 2011 5:44 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystal Optimization Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M BisTris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi
Re: [ccp4bb] Crystal Optimization
Dear Jobi, See if you can slowly increase the PEG concentration to 28 - 30% and that will be a good cryo. Since BIS Tris Propane is the buffer, I think, 28% 3350 should work fine. If the crystals crack by going straight to 28% PEG 3350, 0.1M Bis Tris Propane pH:6.5, 0.2M Potassium thiocyanate solution, put the crystal in a 24% solution and slowly increase the concentration by adding the higher concentration well solution. It seems to be a good idea to keep the crystals for 2 or 3 mts in a solution before increasing the PEG concentration (well, this seems to vary with protein). Also remember to include any salts in the cryo (at least half of the concentration) that may be already in the protein solution. Kind regards, Mathews -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jobichen Chacko Sent: Wednesday, April 13, 2011 2:44 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystal Optimization Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M Bis Tris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi
