[ccp4bb] Adding ligand to crystallization drop
Hi, I've just got a quick question about getting ligands bound to proteins in crystals. I've managed to co-crystallize my proteins with the various ligands, and I'm aware that soaking will work as well but I want to speed up the process. Would this work? If I had a bunch of crystals that have grown in their drop, can I pipette the ligand (say 0.6ul to get a desired conc.) onto the drop and just let the crystals soak that way. Any dilution that would have occurred when then readjust as the drop will be sealed again. Perhaps the crystals would get a little stressed when adding the high concentrated ligand and this method would not work…? Thanks, Chris -- Christopher Browning, Ph. D Vertex Pharmaceuticals (Europe) Ltd 86-88 Jubilee Avenue Milton Park Abingdon Oxfordshire OX14 4RW United Kingdom Tel +44 (0) 1235 438327 christopher_b_brown...@vrtx.com www.vrtx.comhttp://www.vrtx.com/ This email message and any attachments are confidential and intended for use by the addressee(s) only. If you are not the intended recipient, please notify me immediately by replying to this message, and destroy all copies of this message and any attachments. Thank you. Vertex Pharmaceuticals (Europe) Ltd. Registered in England and Wales, company no. 2907620 Registered Office: 86 - 88 Jubilee Avenue, Milton Park, Abingdon, Oxfordshire, OX14 4RW, UK
Re: [ccp4bb] Adding ligand to crystallization drop
Hi Again, Thanks for the suggestions. I just tried it at 2 ligand concentrations and the crystals seem not to mind. The drop does go a little cloudy but I can't say if this is protein precipitation, or the ligand coming out of solution. My feeling its a bit of protein because of the high DMSO conc. being added. I'll co-crystallize just to have a backup! Cheers C -- Christopher Browning, Ph. D Vertex Pharmaceuticals (Europe) Ltd 86-88 Jubilee Avenue Milton Park Abingdon Oxfordshire OX14 4RW United Kingdom Tel +44 (0) 1235 438327 christopher_b_brown...@vrtx.com www.vrtx.comhttp://www.vrtx.com/ From: Noinaj, Nicholas [E] (NIH/NIDDK) noin...@niddk.nih.govmailto:noin...@niddk.nih.gov Date: Thursday, 12 September 2013 10:21 To: Christopher Browning christopher_b_brown...@vrtx.commailto:christopher_b_brown...@vrtx.com, CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: RE: Adding ligand to crystallization drop Chris, Bottom line is that it is all dependent on how your ligand interacts with your protein, if there are any conformational changes, and how your crystals behave in such a dilution and high concentration of ligand. You just have to test it and see, no way to know for sure until you do the experiments and see. However, there are some things to look for along the way. For example, do your crystals crack after introducing the ligand or do they start to dissolve and lose their nice edges, or turn opaque? you will probably also want to do a buffer only control without ligand to ensure that the crystals are ok with the dilution and change of environment. I assume the native crystals diffract well enough to solve the structure. You would want to test crystals before any soaking and then with buffer only and then with ligand, to ensure diffraction is retained to a level useful enough for structure determination. I did quite a bit of crystal soaking in grad school and it is something you just have to try since every crystal/protein is different. my experience is that soaking can often lead to a slight loss of resolution but typically you still get the information you are seeking if the crystals survive. with that being said, soaking can sometimes lead to partial occupancy of your ligand. So if you are able to do co-xtallization, i think that would be preferred and doesn't seem like too much additional effort assuming you are getting crystals in the same conditions as with native protein only. and with co-xtallization, I often see slightly better resolution that with native protein only (not always the same condition though unfortunately). But again, no way to know how your particular protein/crystal will behave without doing some pilot experiments first. Good luck! Cheers, Nick [ Nicholas Noinaj ] the Buchanan Lab Laboratory of Molecular Biology LMB-NIDDK, NIH 50 South Drive, Room 4505 Bethesda, MD 20892-8030 1-301-594-9230 (lab) 1-859-893-4789 (cell) noin...@niddk.nih.govmailto:noin...@niddk.nih.gov [ the Buchanan Lab ] http://www-mslmb.niddk.nih.gov/buchanan/ From: Christopher Browning [mailto:christopher_b_brown...@vrtx.com] Sent: Thursday, September 12, 2013 4:37 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Adding ligand to crystallization drop Hi, I've just got a quick question about getting ligands bound to proteins in crystals. I've managed to co-crystallize my proteins with the various ligands, and I'm aware that soaking will work as well but I want to speed up the process. Would this work? If I had a bunch of crystals that have grown in their drop, can I pipette the ligand (say 0.6ul to get a desired conc.) onto the drop and just let the crystals soak that way. Any dilution that would have occurred when then readjust as the drop will be sealed again. Perhaps the crystals would get a little stressed when adding the high concentrated ligand and this method would not work…? Thanks, Chris -- Christopher Browning, Ph. D Vertex Pharmaceuticals (Europe) Ltd 86-88 Jubilee Avenue Milton Park Abingdon Oxfordshire OX14 4RW United Kingdom Tel +44 (0) 1235 438327 christopher_b_brown...@vrtx.commailto:christopher_b_brown...@vrtx.com www.vrtx.comhttp://www.vrtx.com/ This email message and any attachments are confidential and intended for use by the addressee(s) only. If you are not the intended recipient, please notify me immediately by replying to this message, and destroy all copies of this message and any attachments. Thank you. Vertex Pharmaceuticals (Europe) Ltd. Registered in England and Wales, company no. 2907620 Registered Office: 86 - 88 Jubilee Avenue, Milton Park, Abingdon, Oxfordshire, OX14 4RW, UK This email message and any attachments are confidential and intended for use by the addressee(s) only. If you are not the intended recipient, please notify me immediately by replying to this
[ccp4bb] Data Fitting for protein-ligand interaction.
Dear All, Firstly sorry for asking a non-crystallography question, but i want help in understanding the data analysis for fitting a protein-ligand binding data. Actually i have a protein which is a tetramer in solution and i have done its flourescence binding with a ligand. I am trying to fit the data to a 4-site binding model in scientist. But i donot have a correct model to fit in the data for identical or non-identical, co-operative or sequential binding. Can anyone help me in analysing the binding data. Any help will be highly appreciated. Thankyou !
[ccp4bb] Data Fitting for protein-ligand interaction.
Dear All I've been reading several mails that adress the problem of acetylated N-termini when refining peptide ligands with refmac. I managed to include LINKR records after running refmacs review restraints as suggested by Eleanor Dodson in one of the mails I found: LINKRC ACE I 0 N TRP I 1ACE_C-N But the records themself are obviously not sufficient to maintain the ACE linked to TRP during refinement and ACE moves away. Eleanor Dodson wrote about that topic: If you run [...] review restraints, it will detect and make a LINK entry for you Then you will need to use the GUI task - merge monomer library to combine your corrected MAL with the new LINK Run refmac again with XYZIN the output from review restraints task (that will include a LINK record) and it should/might! work... If I read that correctly, Review restraints should produce a LIBOUT that can be loaded as .cif in a later step for refinement? Such a file was not produced. I tried afterwards: 1) To regularize and safe an ACE-TRP monomer in JLIgand and load the .cif as LIBIN for refmac. The link is found, but ACE still moves away with the same messages: WARNING : link:ACE_C-N is found dist = 1.361 ideal_dist= 1.329 ch:II res: 1 TRP at:N .-Ia res: 0 ACE at:C . Even though XYZOUT still contains the LINKR records. 2) As I suspected, refmac needs except for the LINKR record a LIBIN that should be produced when running Review Restraints. As this .cif file is not produced, I sent the whole ligand to prodrug and safed the .cif from there. But that did not work out either. 3) I also tried to change the LINKR record into LINK I failed until now to tell refmac to maintain ACE linked to the TRP. I know this issues have been discussed before, but none of the suggestions helped yet. Any help would be highly appriciated.
Re: [ccp4bb] Adding ligand to crystallization drop
Have a look at these two papers: http://www.ncbi.nlm.nih.gov/pubmed/17004709 http://www.ncbi.nlm.nih.gov/pubmed/19929835 When you say high DMSO, how much is that in % ? Do you know if your crystals survive that much percentage DMSO even without ligand ? Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu On Sep 12, 2013, at 6:25 AM, Christopher Browning wrote: Hi Again, Thanks for the suggestions. I just tried it at 2 ligand concentrations and the crystals seem not to mind. The drop does go a little cloudy but I can't say if this is protein precipitation, or the ligand coming out of solution. My feeling its a bit of protein because of the high DMSO conc. being added. I'll co-crystallize just to have a backup! Cheers C -- Christopher Browning, Ph. D Vertex Pharmaceuticals (Europe) Ltd 86-88 Jubilee Avenue Milton Park Abingdon Oxfordshire OX14 4RW United Kingdom Tel +44 (0) 1235 438327 christopher_b_brown...@vrtx.com www.vrtx.comhttp://www.vrtx.com/ From: Noinaj, Nicholas [E] (NIH/NIDDK) noin...@niddk.nih.govmailto:noin...@niddk.nih.gov Date: Thursday, 12 September 2013 10:21 To: Christopher Browning christopher_b_brown...@vrtx.commailto:christopher_b_brown...@vrtx.com, CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: RE: Adding ligand to crystallization drop Chris, Bottom line is that it is all dependent on how your ligand interacts with your protein, if there are any conformational changes, and how your crystals behave in such a dilution and high concentration of ligand. You just have to test it and see, no way to know for sure until you do the experiments and see. However, there are some things to look for along the way. For example, do your crystals crack after introducing the ligand or do they start to dissolve and lose their nice edges, or turn opaque? you will probably also want to do a buffer only control without ligand to ensure that the crystals are ok with the dilution and change of environment. I assume the native crystals diffract well enough to solve the structure. You would want to test crystals before any soaking and then with buffer only and then with ligand, to ensure diffraction is retained to a level useful enough for structure determination. I did quite a bit of crystal soaking in grad school and it is something you just have to try since every crystal/protein is different. my experience is that soaking can often lead to a slight loss of resolution but typically you still get the information you are seeking if the crystals survive. with that being said, soaking can sometimes lead to partial occupancy of your ligand. So if you are able to do co-xtallization, i think that would be preferred and doesn't seem like too much additional effort assuming you are getting crystals in the same conditions as with native protein only. and with co-xtallization, I often see slightly better resolution that with native protein only (not always the same condition though unfortunately). But again, no way to know how your particular protein/crystal will behave without doing some pilot experiments first. Good luck! Cheers, Nick [ Nicholas Noinaj ] the Buchanan Lab Laboratory of Molecular Biology LMB-NIDDK, NIH 50 South Drive, Room 4505 Bethesda, MD 20892-8030 1-301-594-9230 (lab) 1-859-893-4789 (cell) noin...@niddk.nih.govmailto:noin...@niddk.nih.gov [ the Buchanan Lab ] http://www-mslmb.niddk.nih.gov/buchanan/ From: Christopher Browning [mailto:christopher_b_brown...@vrtx.com] Sent: Thursday, September 12, 2013 4:37 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Adding ligand to crystallization drop Hi, I've just got a quick question about getting ligands bound to proteins in crystals. I've managed to co-crystallize my proteins with the various ligands, and I'm aware that soaking will work as well but I want to speed up the process. Would this work? If I had a bunch of crystals that have grown in their drop, can I pipette the ligand (say 0.6ul to get a desired conc.) onto the drop and just let the crystals soak that way. Any dilution that would have occurred when then readjust as the drop will be sealed again. Perhaps the crystals would get a little stressed when adding the high concentrated ligand and this method would not work…? Thanks, Chris -- Christopher Browning, Ph. D Vertex Pharmaceuticals (Europe) Ltd 86-88 Jubilee Avenue Milton Park Abingdon Oxfordshire OX14 4RW United Kingdom Tel +44 (0) 1235 438327 christopher_b_brown...@vrtx.commailto:christopher_b_brown...@vrtx.com www.vrtx.comhttp://www.vrtx.com/
Re: [ccp4bb] over-expression strategies
Is your gene of interest smaller than 750bp ? Then I would synthesize the gene with IDT and optimize it for E.coli that's 139$ Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://lupo.jhsph.edu On Sep 12, 2013, at 10:23, Elias Fernandez efern...@utk.edumailto:efern...@utk.edu wrote: Dear CCP4ers, We’ve been struggling with little (nearly none) expression of our protein, in both E coli and with in vitro transcription/translation methods. It appears that our mRNA has high 2’ structure with a low dG (theoretically ~760kcal/mol). If this is indeed the source of our problem, are there any potential strategies to either disrupt the mRNA structure chemically (in E coli or in vitro) or with thermophile expression systems for expression at higher temperatures? Regards, Elias
[ccp4bb] over-expression strategies
Dear CCP4ers, We've been struggling with little (nearly none) expression of our protein, in both E coli and with in vitro transcription/translation methods. It appears that our mRNA has high 2' structure with a low dG (theoretically ~760kcal/mol). If this is indeed the source of our problem, are there any potential strategies to either disrupt the mRNA structure chemically (in E coli or in vitro) or with thermophile expression systems for expression at higher temperatures? Regards, Elias
Re: [ccp4bb] Adding ligand to crystallization drop
Thanks for the really helpful tips. Right now I've tried 5, 10 and 16% DMSO (due to variations in the final ligand conc.) and the crystals visually look happy. I'll definitely give the cryo/ligand mix method a go as this seems to do the job all at once. I already know what cryo to use. Thanks, Chris -- Christopher Browning, Ph. D Vertex Pharmaceuticals (Europe) Ltd 86-88 Jubilee Avenue Milton Park Abingdon Oxfordshire OX14 4RW United Kingdom Tel +44 (0) 1235 438327 christopher_b_brown...@vrtx.com www.vrtx.comhttp://www.vrtx.com/ From: Bosch, Juergen jubo...@jhsph.edumailto:jubo...@jhsph.edu Date: Thursday, 12 September 2013 15:09 To: Christopher Browning christopher_b_brown...@vrtx.commailto:christopher_b_brown...@vrtx.com Cc: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Adding ligand to crystallization drop Have a look at these two papers: http://www.ncbi.nlm.nih.gov/pubmed/17004709 http://www.ncbi.nlm.nih.gov/pubmed/19929835 When you say high DMSO, how much is that in % ? Do you know if your crystals survive that much percentage DMSO even without ligand ? Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu On Sep 12, 2013, at 6:25 AM, Christopher Browning wrote: Hi Again, Thanks for the suggestions. I just tried it at 2 ligand concentrations and the crystals seem not to mind. The drop does go a little cloudy but I can't say if this is protein precipitation, or the ligand coming out of solution. My feeling its a bit of protein because of the high DMSO conc. being added. I'll co-crystallize just to have a backup! Cheers C -- Christopher Browning, Ph. D Vertex Pharmaceuticals (Europe) Ltd 86-88 Jubilee Avenue Milton Park Abingdon Oxfordshire OX14 4RW United Kingdom Tel +44 (0) 1235 438327 christopher_b_brown...@vrtx.commailto:christopher_b_brown...@vrtx.com www.vrtx.comhttp://www.vrtx.com/ From: Noinaj, Nicholas [E] (NIH/NIDDK) noin...@niddk.nih.govmailto:noin...@niddk.nih.gov Date: Thursday, 12 September 2013 10:21 To: Christopher Browning christopher_b_brown...@vrtx.commailto:christopher_b_brown...@vrtx.com, CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: RE: Adding ligand to crystallization drop Chris, Bottom line is that it is all dependent on how your ligand interacts with your protein, if there are any conformational changes, and how your crystals behave in such a dilution and high concentration of ligand. You just have to test it and see, no way to know for sure until you do the experiments and see. However, there are some things to look for along the way. For example, do your crystals crack after introducing the ligand or do they start to dissolve and lose their nice edges, or turn opaque? you will probably also want to do a buffer only control without ligand to ensure that the crystals are ok with the dilution and change of environment. I assume the native crystals diffract well enough to solve the structure. You would want to test crystals before any soaking and then with buffer only and then with ligand, to ensure diffraction is retained to a level useful enough for structure determination. I did quite a bit of crystal soaking in grad school and it is something you just have to try since every crystal/protein is different. my experience is that soaking can often lead to a slight loss of resolution but typically you still get the information you are seeking if the crystals survive. with that being said, soaking can sometimes lead to partial occupancy of your ligand. So if you are able to do co-xtallization, i think that would be preferred and doesn't seem like too much additional effort assuming you are getting crystals in the same conditions as with native protein only. and with co-xtallization, I often see slightly better resolution that with native protein only (not always the same condition though unfortunately). But again, no way to know how your particular protein/crystal will behave without doing some pilot experiments first. Good luck! Cheers, Nick [ Nicholas Noinaj ] the Buchanan Lab Laboratory of Molecular Biology LMB-NIDDK, NIH 50 South Drive, Room 4505 Bethesda, MD 20892-8030 1-301-594-9230 (lab) 1-859-893-4789 (cell) noin...@niddk.nih.govmailto:noin...@niddk.nih.gov [ the Buchanan Lab ] http://www-mslmb.niddk.nih.gov/buchanan/ From: Christopher Browning [mailto:christopher_b_brown...@vrtx.com] Sent: Thursday, September 12, 2013 4:37 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Adding ligand to crystallization
Re: [ccp4bb] Data Fitting for protein-ligand interaction.
Hi Monica If protein is Homo-tetramer then one can expect the identical binding sites. I am also working on homo-dimeric protein which binds to DNA. I used PRISM to estimate the binding affinity through flourescence bindingmethod using “SATURATION and NON-LINEAR REGRESSION and ONE SITE binding Model’ considering protein concentration as dimer or monomer. Then I looked for the sensibility of the model by analyzing (comparing) best fit values (like SD, Bmax) of the parameters with reasonable certainty. Unlike ITC binding model fitting (gives good estimate of stoichiometry, cooperativity and also binding sites (one, two or sequential binding sites)), flourescence binding models do not give very good estimate of these parameters. Thus, based on protein you should assume the model which fits to the properties of the protein. Good luck Raj On Thu, Sep 12, 2013 at 12:59 PM, MONICA MITTAL monica.mitta...@gmail.comwrote: Dear All, Firstly sorry for asking a non-crystallography question, but i want help in understanding the data analysis for fitting a protein-ligand binding data. Actually i have a protein which is a tetramer in solution and i have done its flourescence binding with a ligand. I am trying to fit the data to a 4-site binding model in scientist. But i donot have a correct model to fit in the data for identical or non-identical, co-operative or sequential binding. Can anyone help me in analysing the binding data. Any help will be highly appreciated. Thankyou !
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 dkgh...@cdfd.org.inwrote: 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
[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] Adding ligand to crystallization drop
But you have not tested in the beam if the visually happy crystals diffract right ? I would do this first before consuming your precious ligands in worthless drops. Jürgen On Sep 12, 2013, at 10:34 AM, Christopher Browning wrote: Right now I've tried 5, 10 and 16% DMSO (due to variations in the final ligand conc.) and the crystals visually look happy. .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
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 dkgh...@cdfd.org.in 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
[ccp4bb] About molecular replacement
Dear crystallographers, I have solved a structure of a glucose binding protein of CE4 family. When I try to solve the structure using the same CE4 family enzyme as search model, it failed for many case. Finally, I solved the with a same family enzyme used as search model. As soon as I solved the structure, I superposed my final refined model with structures of CE4 family enzymes which did not produce the good molecular replacement solution for my enzyme. I found that all are having (Beta/alpha)7 fold and superpose very well with my model. Whereas, some loop region are not superpose very well. My doubt is why molecular replacement failed thought over-all fold is same?. -- *Dhanasekaran Varudharasu* Post-Doctoral Fellow Department of Oral Biology Rutgers school of Dental Medicine Rutgers Biomedical and Health Sciences Newark, NJ 07103 USA
Re: [ccp4bb] About molecular replacement
Perhaps some correct solutions were thrown out due to packing considerations. There are a few methods to address that possibility. You could use a search model with large loops trimmed, especially is a sequence comparison shows they are probably not conserved. Or you could search with a suite of structures from the family. On 09/12/13 17:21, Dhanasekaran Varudharasu wrote: Dear crystallographers, I have solved a structure of a glucose binding protein of CE4 family. When I try to solve the structure using the same CE4 family enzyme as search model, it failed for many case. Finally, I solved the with a same family enzyme used as search model. As soon as I solved the structure, I superposed my final refined model with structures of CE4 family enzymes which did not produce the good molecular replacement solution for my enzyme. I found that all are having (Beta/alpha)7 fold and superpose very well with my model. Whereas, some loop region are not superpose very well. My doubt is why molecular replacement failed thought over-all fold is same?. -- *Dhanasekaran Varudharasu* Post-Doctoral Fellow Department of Oral Biology Rutgers school of Dental Medicine Rutgers Biomedical and Health Sciences Newark, NJ 07103 USA -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] About molecular replacement
Dear Dhanasekaran, There are many examples of molecular replacement failing even in cases where the model and target structure share 100% sequence identity. These examples illuminate several factors that MR search strategies are sensitive to, including percent sequence identity and related parameters like RMSD from target, the impact of loops, small domain movements etc. So if you want to learn in more depth about what makes a good model and what makes or breaks an MR search, perhaps some googling combined with a bit of obsessive reading might help. I actually found a ton of thoroughly helpful articles and reviews for MR on the web without too much sweat. Best wishes, Raji On Thu, Sep 12, 2013 at 5:21 PM, Dhanasekaran Varudharasu dhana...@gmail.com wrote: Dear crystallographers, I have solved a structure of a glucose binding protein of CE4 family. When I try to solve the structure using the same CE4 family enzyme as search model, it failed for many case. Finally, I solved the with a same family enzyme used as search model. As soon as I solved the structure, I superposed my final refined model with structures of CE4 family enzymes which did not produce the good molecular replacement solution for my enzyme. I found that all are having (Beta/alpha)7 fold and superpose very well with my model. Whereas, some loop region are not superpose very well. My doubt is why molecular replacement failed thought over-all fold is same?. -- *Dhanasekaran Varudharasu* Post-Doctoral Fellow Department of Oral Biology Rutgers school of Dental Medicine Rutgers Biomedical and Health Sciences Newark, NJ 07103 USA -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
