Re: [ccp4bb] Crystal optimization
Hi, these crystals look large enough for VMXm<https://www.diamond.ac.uk/Instruments/Mx/VMXm.html> at Diamond Light Source. We have collected excellent data out of smaller crystals. Get in touch with me (or any other members of the team) and we can discuss a possible visit. Kind regards, Jose From: CCP4 bulletin board on behalf of 白雪慧 Date: Friday, 31 May 2024 at 03:33 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystal optimization Some people who received this message don't often get email from zb20193020...@cau.edu.cn. Learn why this is important<https://aka.ms/LearnAboutSenderIdentification> Thank you very much for your suggestions. I have a question. My crystal grows microcrystals under multiple conditions, as shown in the figure. After orthogonal optimization of the precipitant and pH, the crystal growth is still very small and difficult to obtain diffraction. What is the method to optimize and increase the crystal size in this situation? To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=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/
[ccp4bb] RES: [ccp4bb] Crystal optimization
Hi, It seems to my eyes that you have multiple nucleation events happening simultaneously. I would recommend you to try crystallization under 10 or 4 ºC Best of luck Rafael Marques da Silva PhD Student – Structural Biology University of Leicester Mestre em Física Biomolecular Universidade de São Paulo Bacharel em Ciências Biológicas Universidade Federal de São Carlos phone: +55 16 99766-0021 "A sorte acompanha uma mente bem treinada" De: CCP4 bulletin board em nome de 白雪慧 Enviado: Friday, May 31, 2024 3:32:52 AM Para: CCP4BB@JISCMAIL.AC.UK Assunto: [ccp4bb] Crystal optimization Thank you very much for your suggestions. I have a question. My crystal grows microcrystals under multiple conditions, as shown in the figure. After orthogonal optimization of the precipitant and pH, the crystal growth is still very small and difficult to obtain diffraction. What is the method to optimize and increase the crystal size in this situation? To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=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/
[ccp4bb] Fwd: [ccp4bb] Crystal optimization
Apologies, my response was directly to the OP and not to the BB. I have forwarded the response to maintain the chain. -- Forwarded message - From: Nicholas Clark Date: Fri, May 31, 2024 at 7:23 AM Subject: Re: [ccp4bb] Crystal optimization To: 白雪慧 I had a protein that no matter what we did, only grew urchins very similar to what you’ve shown here. Even with extensive optimization, we were only ever able to grow urchins. Ultimately, the only way we were able to grow diffraction quality crystals and allowed us to solve the structure (with resolution ranging from 1.4- 2.2 angstroms, depending on ligand) was using MMS, as Tom suggested. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157405/ Best, Nick Nicholas D. Clark, PhD (He/Him) University at Buffalo Department of Structural Biology Jacob's School of Medicine & Biomedical Sciences 955 Main Street, RM 5130 Buffalo, NY 14203 On Thu, May 30, 2024 at 10:34 PM 白雪慧 wrote: > Thank you very much for your suggestions. I have a question. My crystal > grows microcrystals under multiple conditions, as shown in the figure. > After orthogonal optimization of the precipitant and pH, the crystal growth > is still very small and difficult to obtain diffraction. What is the method > to optimize and increase the crystal size in this situation? > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > <https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1=05%7C02%7Cndclark2%40g-mail.buffalo.edu%7Cd15f0afd4af945855efb08dc811a166f%7C96464a8af8ed40b199e25f6b50a20250%7C0%7C0%7C638527196413692164%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C0%7C%7C%7C=xWKfAAHjMInPToUjqIzNO99zljdso01mAd478t1qp%2Fk%3D=0> > -- Nicholas D. Clark, PhD (He/Him) University at Buffalo Department of Structural Biology Jacob's School of Medicine & Biomedical Sciences 955 Main Street, RM 5130 Buffalo, NY 14203 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=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/
Re: [ccp4bb] Crystal optimization
Hi, In addition, you might want to test additives. I would suggest the additive screen from Hampton Research. We made best experiences with neutral solvents like: ethylacetate (doi.org/10.1021/ic010790t and doi.org/10.1021/acs.inorgchem.6b00672), dioxane (doi.org/10.1021/acs.inorgchem.6b00672) and methanol (protein, unpublished). Best regards Bernhard To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=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/
Re: [ccp4bb] Crystal optimization
Usually, you should try to push up the protein concentration, often quite a lot (30, 50, even 100 mg/ml), and decrease precipitant (might have to be really low, eg <3% PEG is not unthinkable). To get the protein up, you may need to find a new buffer solution - there are screens for this, and DSF is one way to test. But might need a new construct or surface mutation or other protein engineering. Good luck Frank Sent from tiny silly touch screen From: 白雪慧 Sent: Friday, 31 May 2024 03:33 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystal optimization Thank you very much for your suggestions. I have a question. My crystal grows microcrystals under multiple conditions, as shown in the figure. After orthogonal optimization of the precipitant and pH, the crystal growth is still very small and difficult to obtain diffraction. What is the method to optimize and increase the crystal size in this situation? To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=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/
Re: [ccp4bb] Crystal optimization
I believe someone has already mentioned matrix seeding- taking the crystals you currently have, making microseeds and then using this in a new screen (whatever your favourite initial screens may be). This can give you different conditions that will give you better crystals as you already have seeds to nucleate from. Best of luck, tom From: CCP4 bulletin board on behalf of 白雪慧 Sent: Friday, May 31, 2024 12:32 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystal optimization You don't often get email from zb20193020...@cau.edu.cn. Learn why this is important<https://aka.ms/LearnAboutSenderIdentification> Thank you very much for your suggestions. I have a question. My crystal grows microcrystals under multiple conditions, as shown in the figure. After orthogonal optimization of the precipitant and pH, the crystal growth is still very small and difficult to obtain diffraction. What is the method to optimize and increase the crystal size in this situation? To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=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/
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