[ccp4bb] AW: [ccp4bb] crystallization of complex and ...
Dear Min, regarding #1 some things come to my mind: - What is the Kd that you got from the Biacore? And did you make sure that the sample is concentrated enough (both on gel filtration and in crystallization) to have a sufficient amount in the complex. - did you use the same buffer systems? Your Kd might be different in different buffers. Best Alex Dr. Alexander Pautsch Boehringer Ingelheim Pharma GmbH Co. KG Dept. Lead Identific. and Optim. Sup. Ge Tel.: +49 (7351) 54-4683 Fax: +49 (7351) 54-97924 mailto:alexander.paut...@boehringer-ingelheim.com mailto:alexander.paut...@boehringer-ingelheim.com Boehringer Ingelheim Pharma GmbH Co. KG, Sitz: Ingelheim am Rhein; Registergericht Mainz: HR A 22206; Komplementär Boehringer Ingelheim Deutschland GmbH; Geschäftsführung: Dr. Engelbert Günster (Vorsitzender), Ralf Gorniak, Mark Hagmann, Michael Klein, Dr. Martin Wanning; Vorsitzender des Aufsichtsrates: Prof. Dr. Dr. Andreas Barner; Sitz: Ingelheim am Rhein; Registergericht Mainz: HR B 23260 Diese E-Mail ist vertraulich zu behandeln. Sie kann besonderem rechtlichen Schutz unterliegen. Wenn Sie nicht der richtige Adressat sind, senden Sie bitte diese E-Mail an den Absender zurück, löschen die eingegangene E-Mail und geben den Inhalt der E-Mail nicht weiter. Jegliche unbefugte Bearbeitung, Nutzung, Vervielfältigung oder Verbreitung ist verboten. / This e-mail is confidential and may also be legally privileged. If you are not the intended recipient please reply to sender, delete the e-mail and do not disclose its contents to any person. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited. Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von m zhang Gesendet: Freitag, 16. September 2011 04:20 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] crystallization of complex and ... Dear all, I have two questions: First, I was trying to crystallize a complex of two proteins. Both proteins has been crystallized before. The two proteins bind to each other based on Biacore study, but they didn't form a single peak on gel filtration. When I mixed them at 1:1 ratio, the crystals I got contain only one of the two proteins. I was suggested to increase the ratio, for example 1.5:1, to increase the probability of co-crystallization which I will try. But I do want to hear if there are other possible ways to try. What would you try if you were in my situation? Second is about reusing of Ni-NTA resin. According to Qiagen's instruction, after using fresh Ni-NTA resin, one only needs to wash the used Ni resin first with 0.5M NaOH, then with your own buffer. After that the resin is ready to be reused until it needs being recharged. But my question is: Once immidazole competes with His-tagged protein and binds to Ni-resin, how can immidazole be rinsed off with the same buffer(usually pH is above 7) one uses to purify the protein? Thank you for any suggestion or comment. Min
[ccp4bb] Silver staining Coomassie stained gels
Dear All, Can anyone suggest me a protocol for silver-staining the PAGE that is already stained with Coomassie. Kris
Re: [ccp4bb] crystallization of complex and ...
Dear Min, Regarding the stoichiometry that you should use in crystallizing two proteins that form a complex. I have looked at this question before. See: Stura, E.A., Graille, M., Taussig, M.J., Sutton, B.J. Gore, M.G., Silverman, G.J., Charbonnier, J.-B. (2001) Crystallization of macromolecular complexes: Stoichiometric variation screening. J. Cryst. Growth 232:580-590. http://www.sciencedirect.com/science/article/pii/S0022024801011721 Briefly: The stoichiometry can, and should, be varied in your screening. The relative protein solubility of the two individual proteins and the solubility of the complex should be analysed under various potential crystallization conditions. Conditions where the complex is less soluble than the individual ptoteins should be chosen if the complex has a tendency to dissociate. Since both proteins have been crystallized before you may also use crystals of both the free forms of the two proteins to stimulate nucleation of the complex The final composition of the asymmetric unit may include free poteins as well as the complex. The paper will give you a lot more methodology to use to obtain crystals of the complex, if such complex really exists, is homegeneous and relatively stable. Enrico. On Fri, 16 Sep 2011 04:20:26 +0200, m zhang mzhang...@hotmail.com wrote: Dear all, I have two questions: First, I was trying to crystallize a complex of two proteins. Both proteins has been crystallized before. The two proteins bind to each other based on Biacore study, but they didn't form a single peak on gel filtration. When I mixed them at 1:1 ratio, the crystals I got contain only one of the two proteins. I was suggested to increase the ratio, for example 1.5:1, to increase the probability of co-crystallization which I will try. But I do want to hear if there are other possible ways to try. What would you try if you were in my situation? Second is about reusing of Ni-NTA resin. According to Qiagen's instruction, after using fresh Ni-NTA resin, one only needs to wash the used Ni resin first with 0.5M NaOH, then with your own buffer. After that the resin is ready to be reused until it needs being recharged. But my question is: Once immidazole competes with His-tagged protein and binds to Ni-resin, how can immidazole be rinsed off with the same buffer(usually pH is above 7) one uses to purify the protein? Thank you for any suggestion or comment. Min -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] Why Does Cross-linking Mean Anything?
Jacob, One of the problems with glutaraldehyde is the its chemistry is so bizarre. It actually forms quite long transient polymers in solution. You also have to ask yourself why formaldehyde also fixes tissues. This is why glutaraldehyde works so well for tissue fixation for EM as opposed to our usual bivalent crosslinkers we use in biochemistry experiments. Check out the old EM literature about discussions of glutaraldehyde chemistry. Moreover, the Schiff's base linkage glutaraldehyde is slowly reversible. You need to reduce it to make it permanent. I think that glutaraldehyde is a very poor choice for a precise bivalent crosslinker, but as a broad spectrum crosslinker (hitting lysines and a free amino terminii that are different distances apart), glutaraldehyde is great. As it is highly volatile (its unique smell tells you you've had the bottle open too long), you can crosslink crystals by vapor diffusion in an hour. So I would be cautious about interpreting any crosslinking results using glutaraldehyde, except the obvious (i.e., oligomers may indicate the native tertiary state of a protein or complex). Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Sep 15, 2011, at 4:10 PM, Jacob Keller wrote: Dear Crystallographers and Biochemists, cross-linking, say with gluteraldehyde, is an oft-used method of demonstrating a protein's oligomeric state in solution. I have a difficulty with this, however: theoretically (and in practice!), one can tune the amount of cross-linker to get what ever result is desired, such that any protein with some exposed lysines can be cross-linked in any oligomeric state. How, then, does one evaluate the power of this evidence? Maybe one should do a gradient of gluteraldehyde concentrations, then plot the deviation of the observed cross-linked oligomerization from a theoretical null hypothesis? Seems like this could be done, but I have never seen this in the literature... Best, Jacob Keller -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Mac OSX 10.7 Lion
I use my MBP with external screen, keyboard mouse all the time. The new ones are fast, mine should easily cope with Lion My question about Lion was because 1. on the one hand as far as I can see Bill Scott only builds latest stand-alone Coots (0.7...) for Lion, and these don't work on 10.6 (is this true Bill?) 2. on the other hand, I had a report that ccp4mg doesn't work on Lion (is that true Stuart?) and I need both of these (and don't fancy building either myself) Phil (still on 10.6) On 11 Sep 2011, at 17:55, Sean Seaver wrote: Dear Herbert, I've come across quite a few people that are using mac books as their main development computer. This site ( http://usesthis.com/ ) can be an good way to learn about various setups. A popular trend seems to be using a mac book along with the apple thunderbolt display for more screen real estate. Take Care, Sean Seaver P212121 http://store.p212121.com/
Re: [ccp4bb] Mac OSX 10.7 Lion
Dear Phil (and everyone): 1. I've now got automated build systems for coot for 10.7.1 and 10.6.8. I just haven't had a chance to get the 10.6.8 one on line. I'll try to do this today. (Also, the last few haven't build due to a change in the code with which the compiler can't cope, but hopefully that will be a thing of the past soon.) 2. Most things build on 10.6.X will run on 10.7.X. I haven't opened ccp4mg, but will give it a try after I wake up. 3. I've had trouble with stereo on coot with 10.7.1, but, oddly, the problem goes away if I have a second monitor. If you update to 10.7, keep a clone of 10.6 just in case it drives you nuts. There are all sorts of perverse changes, and (unlike the reversal in scrolling direction) not a lot of over-ride options. I guess this is a glimpse of the post-Jobs era, albeit one provided from an inconvenient vista. Peace and joy, Bill On Sep 16, 2011, at 6:33 AM, William G. Scott wrote: Dear Phil (and everyone): 1. I've now got automated build systems for coot for 10.7.1 and 10.6.8. I just haven't had a chance to get the 10.6.8 one on line. I'll try to do this today. (Also, the last few haven't build due to a change in the code with which the compiler can't cope, but hopefully that will be a thing of the past soon.) 2. Most things build on 10.6.X will run on 10.7.X. I haven't opened ccp4mg, but will give it a try after I wake up. 3. I've had trouble with stereo on coot with 10.7.1, but, oddly, the problem goes away if I have a second monitor. If you update to 10.7, keep a clone of 10.6 just in case it drives you nuts. There are all sorts of perverse changes, and (unlike the reversal in scrolling direction) not a lot of over-ride options. I guess this is a glimpse of the post-Jobs era, albeit one provided from an inconvenient vista. Peace and joy, Bill On Sep 16, 2011, at 5:48 AM, Phil Evans wrote: I use my MBP with external screen, keyboard mouse all the time. The new ones are fast, mine should easily cope with Lion My question about Lion was because 1. on the one hand as far as I can see Bill Scott only builds latest stand-alone Coots (0.7...) for Lion, and these don't work on 10.6 (is this true Bill?) 2. on the other hand, I had a report that ccp4mg doesn't work on Lion (is that true Stuart?) and I need both of these (and don't fancy building either myself) Phil (still on 10.6) On 11 Sep 2011, at 17:55, Sean Seaver wrote: Dear Herbert, I've come across quite a few people that are using mac books as their main development computer. This site ( http://usesthis.com/ ) can be an good way to learn about various setups. A popular trend seems to be using a mac book along with the apple thunderbolt display for more screen real estate. Take Care, Sean Seaver P212121 http://store.p212121.com/
Re: [ccp4bb] crystallization of complex and ...
On Thu, 2011-09-15 at 22:20 -0400, m zhang wrote: Second is about reusing of Ni-NTA resin. According to Qiagen's instruction, after using fresh Ni-NTA resin, one only needs to wash the used Ni resin first with 0.5M NaOH, then with your own buffer. After that the resin is ready to be reused until it needs being recharged. But my question is: Once immidazole competes with His-tagged protein and binds to Ni-resin, how can immidazole be rinsed off with the same buffer(usually pH is above 7) one uses to purify the protein? Imidazole binds much weaker than a his-tag, and thus more of it goes into the buffer when you wash the column. In theory, if you wash a column with protein bound long enough, it will all slowly come off. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] Silver staining Coomassie stained gels
On Fri, 2011-09-16 at 15:28 +0530, K Singh wrote: Dear All, Can anyone suggest me a protocol for silver-staining the PAGE that is already stained with Coomassie. Kris Just destain it and then use the standard silver-staining protocol. If for some reason you want to have both stainings superimposed, take two pictures and then combine them - it should be easy to do by superimposing the ladders, just make sure that pictures are of the same size (maybe tricky if you don't have an imager, but nothing GIMP couldn't overcome). -- Hurry up before we all come back to our senses! Julian, King of Lemurs
[ccp4bb] about alternate conformations
Hi, I was using Refmac from CCP4 to refine a protein's crystal structure. The methionine has half selenium and half sulfur. I was trying to make alternate conformations and let refmac do the refinement. But it keeps giving me error message as follows: There is an error in the input coordinate file At least one the chains has 2 residues with the same number Check above to see error === Error: Problem with coordinate file BFONT COLOR=#FF!--SUMMARY_BEGIN-- Refmac_5.5.0109: Problem with coordinate file And here is my modified pdb file. HETATM 403 N AMSE A 129 *** N HETATM 404 CA AMSE A 129 *** C HETATM 405 CB AMSE A 129 *** C HETATM 406 CG AMSE A 129 *** C HETATM 407 SE AMSE A 129 *** SE HETATM 408 CE AMSE A 129 *** C HETATM 409 C AMSE A 129 *** C HETATM 410 O AMSE A 129 *** O ATOM411 N BMET A 129 *** N ATOM412 CA BMET A 129 *** C ATOM413 CB BMET A 129 *** C ATOM414 CG BMET A 129 *** C ATOM415 SD BMET A 129 *** S ATOM416 CE BMET A 129 *** C ATOM417 C BMET A 129 *** C ATOM418 O BMET A 129 *** O I already confirmed with pdb that this is the right format for this case. But refmac doesn't work with it. I wonder if there is any other changes I should make for refmac? Thank you, Ming
[ccp4bb] about alternate conformations
Hi all, I was using Refmac from CCP4 to refine a protein's crystal structure. The methionine has half selenium and half sulfur. I was trying to make alternate conformations and let refmac do the refinement. But it keeps giving me error message as follows: There is an error in the input coordinate file At least one the chains has 2 residues with the same number Check above to see error === Error: Problem with coordinate file BFONT COLOR=#FF!--SUMMARY_BEGIN-- Refmac_5.5.0109: Problem with coordinate file And here is my modified pdb file. HETATM 403 N AMSE A 129 *** N HETATM 404 CA AMSE A 129 *** C HETATM 405 CB AMSE A 129 *** C HETATM 406 CG AMSE A 129 *** C HETATM 407 SE AMSE A 129 *** SE HETATM 408 CE AMSE A 129 *** C HETATM 409 C AMSE A 129 *** C HETATM 410 O AMSE A 129 *** O ATOM411 N BMET A 129 *** N ATOM412 CA BMET A 129 *** C ATOM413 CB BMET A 129 *** C ATOM414 CG BMET A 129 *** C ATOM415 SD BMET A 129 *** S ATOM416 CE BMET A 129 *** C ATOM417 C BMET A 129 *** C ATOM418 O BMET A 129 *** O I already confirmed with pdb that this is the right format for this case. But refmac doesn't work with it. I wonder if there is any other changes I should make for refmac? Thank you, Ming
Re: [ccp4bb] Silver staining Coomassie stained gels
Destain it really well. One easy peasy option is to put the gel in water and add 1 or 2 ml of cheap Q sepharose to it then boil. Gel will destain in 2-3 minutes of boiling. On Sep 16, 2011 5:10 AM, K Singh ksc...@gmail.com wrote: Dear All, Can anyone suggest me a protocol for silver-staining the PAGE that is already stained with Coomassie. Kris
Re: [ccp4bb] crystallization of complex and ...
1 imidazole affinity is not high which is why you use 200 mM or more to elute. So it comes off by itself. 2 you can wash with low ph and then recharge this is somewhat easier on the resin On Sep 15, 2011 9:25 PM, m zhang mzhang...@hotmail.com wrote: Dear all, I have two questions: First, I was trying to crystallize a complex of two proteins. Both proteins has been crystallized before. The two proteins bind to each other based on Biacore study, but they didn't form a single peak on gel filtration. When I mixed them at 1:1 ratio, the crystals I got contain only one of the two proteins. I was suggested to increase the ratio, for example 1.5:1, to increase the probability of co-crystallization which I will try. But I do want to hear if there are other possible ways to try. What would you try if you were in my situation? Second is about reusing of Ni-NTA resin. According to Qiagen's instruction, after using fresh Ni-NTA resin, one only needs to wash the used Ni resin first with 0.5M NaOH, then with your own buffer. After that the resin is ready to be reused until it needs being recharged. But my question is: Once immidazole competes with His-tagged protein and binds to Ni-resin, how can immidazole be rinsed off with the same buffer(usually pH is above 7) one uses to purify the protein? Thank you for any suggestion or comment. Min
Re: [ccp4bb] Silver staining Coomassie stained gels
Now that's a nifty trick I hadn't heard of before! From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Artem Evdokimov Sent: Friday, September 16, 2011 10:27 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Silver staining Coomassie stained gels Destain it really well. One easy peasy option is to put the gel in water and add 1 or 2 ml of cheap Q sepharose to it then boil. Gel will destain in 2-3 minutes of boiling. On Sep 16, 2011 5:10 AM, K Singh ksc...@gmail.com wrote: Dear All, Can anyone suggest me a protocol for silver-staining the PAGE that is already stained with Coomassie. Kris Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Silver staining Coomassie stained gels
It is nice indeed, that's how we stainand destain our gels in less than 8 minutes :-) there was at one time a proprietary product like tis called magic beads or somesuch. But regular resin works ;-) On Sep 16, 2011 9:31 AM, Soisson, Stephen M stephen_sois...@merck.com wrote: Now that's a nifty trick I hadn't heard of before! From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Artem Evdokimov Sent: Friday, September 16, 2011 10:27 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Silver staining Coomassie stained gels Destain it really well. One easy peasy option is to put the gel in water and add 1 or 2 ml of cheap Q sepharose to it then boil. Gel will destain in 2-3 minutes of boiling. On Sep 16, 2011 5:10 AM, K Singh ksc...@gmail.com wrote: Dear All, Can anyone suggest me a protocol for silver-staining the PAGE that is already stained with Coomassie. Kris Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] about alternate conformations
On Fri, 2011-09-16 at 10:08 -0400, Ming wrote: The methionine has half selenium and half sulfur. Do you have the evidence that your incorporation ration was 50%? On a practical side, try giving the SeMET a different chain ID. You can change it back manually after refinement. Assuming that the side chain conformation is the same, you can probably just have the selenium atom added and hope that due to partial occupancy refmac will not apply any vdw terms. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] about alternate conformations
I did not make it clear. The protocol we used for protein expression always limit the occ of MSE as 0.75. You may estimate your occ of MSE by ED. You can check PDB for JCSG structures. Cheers, Kevin On Fri, Sep 16, 2011 at 7:09 AM, Ming dongm...@udel.edu wrote: Hi all, I was using Refmac from CCP4 to refine a protein's crystal structure. The methionine has half selenium and half sulfur. I was trying to make alternate conformations and let refmac do the refinement. But it keeps giving me error message as follows: There is an error in the input coordinate file At least one the chains has 2 residues with the same number Check above to see error === Error: Problem with coordinate file BFONT COLOR=#FF!--SUMMARY_BEGIN-- Refmac_5.5.0109: Problem with coordinate file And here is my modified pdb file. HETATM 403 N AMSE A 129 *** N HETATM 404 CA AMSE A 129 *** C HETATM 405 CB AMSE A 129 *** C HETATM 406 CG AMSE A 129 *** C HETATM 407 SE AMSE A 129 *** SE HETATM 408 CE AMSE A 129 *** C HETATM 409 C AMSE A 129 *** C HETATM 410 O AMSE A 129 *** O ATOM411 N BMET A 129 *** N ATOM412 CA BMET A 129 *** C ATOM413 CB BMET A 129 *** C ATOM414 CG BMET A 129 *** C ATOM415 SD BMET A 129 *** S ATOM416 CE BMET A 129 *** C ATOM417 C BMET A 129 *** C ATOM418 O BMET A 129 *** O I already confirmed with pdb that this is the right format for this case. But refmac doesn't work with it. I wonder if there is any other changes I should make for refmac? Thank you, Ming
[ccp4bb] DNA cif files
I have a question about the bond angle restraints in the DNA cif files. I recently submitted a protein-DNA complex to the PDB, and found out that many of the glycosidic bond angles (atoms O4'-C1'-N9/N1) were outside the accepted range. I switched from CCP4 6.1.13 to 6.2.0 (installed/updated with fink on Mac OSX 10.6.8), and this fixed the problem in that now there is a narrower distribution of bond angles, but the target/ideal values are different in these two CCP4 monomer libraries (108.4 versus 107.8): = /sw64/lib/ccp4-6.1.13/data/monomers file=a/AD.cif Ad A 'Adenosine ' DNA32 21 . Ad N9 C1*O4* 108.4003.000 file=c/CD.cif Cd C 'Cytidine' DNA30 19 . Cd N1 C1*O4* 108.4003.000 file=g/GD.cif Gd G 'Guanosine ' DNA33 22 . Gd N9 C1*O4* 108.4003.000 file=t/TD.cif Td T 'Thymidine ' DNA32 20 . Td N1 C1*O4* 108.4003.000 = /sw64/lib/ccp4-6.2.0/data/monomers file=d/DA.cif DA DA '2'-DEOXYADENOSINE-5'-MONOPHOSPHATE ' DNA34 22 . DA O4' C1' N9 107.8000.800 file=d/DC.cif DC DC '2'-DEOXYCYTIDINE-5'-MONOPHOSPHATE ' DNA32 20 . DC O4' C1' N1 107.8000.800 file=d/DG.cif DG DG '2'-DEOXYGUANOSINE-5'-MONOPHOSPHATE ' DNA35 23 . DG O4' C1' N9 107.8000.800 file=d/DT.cif DT DT 'THYMIDINE-5'-MONOPHOSPHATE ' DNA34 21 . DT O4' C1' N1 107.8000.800 Why are these values different in the two libraries? Am I looking at the wrong files or the wrong bonds? Thanks! Greg
Re: [ccp4bb] Silver staining Coomassie stained gels
Can anyone suggest me a protocol for silver-staining the PAGE that is already stained with Coomassie. There is absolutely nothing wrong with silver staining of a Coomassie-stained gel without destaining. It only prevents blowout of most intense bands already well-visible with Coomassie. The only thing to do is to equlibrate the gel in water before going with silver. At least that's my experinece with the silver staining protocol that is based on tungstosilicic acid (aka Bio-Rad Silver Stain Plus described here for DNA in agarose but works equally well for proteins in PAAG: http://www.ncbi.nlm.nih.gov/pubmed/2446526 ). - Dima
Re: [ccp4bb] DNA cif files
Hi, it depends what you mean by 'different'. The SUs of the new set are much lower than the old values (0.8 deg vs. 3.0) so one would assume that these are improved estimates based on new data. The difference between the old and the new is (108.4 - 107.8) = 0.6 deg with a SU of the difference of sqrt(3^2 + 0.8^2) = 3.1 deg, so difference/SU = 0.6/3.1 = 0.2 sigma - hardly what one could call 'significantly different'. Cheers -- Ian On Fri, Sep 16, 2011 at 4:26 PM, Gregory Bowman gdbow...@jhu.edu wrote: I have a question about the bond angle restraints in the DNA cif files. I recently submitted a protein-DNA complex to the PDB, and found out that many of the glycosidic bond angles (atoms O4'-C1'-N9/N1) were outside the accepted range. I switched from CCP4 6.1.13 to 6.2.0 (installed/updated with fink on Mac OSX 10.6.8), and this fixed the problem in that now there is a narrower distribution of bond angles, but the target/ideal values are different in these two CCP4 monomer libraries (108.4 versus 107.8): = /sw64/lib/*ccp4-6.1.13*/data/monomers file=a/AD.cif Ad A 'Adenosine ' DNA32 21 . Ad N9 C1*O4* 108.4003.000 file=c/CD.cif Cd C 'Cytidine' DNA30 19 . Cd N1 C1*O4* 108.4003.000 file=g/GD.cif Gd G 'Guanosine ' DNA33 22 . Gd N9 C1*O4* 108.4003.000 file=t/TD.cif Td T 'Thymidine ' DNA32 20 . Td N1 C1*O4* 108.4003.000 = /sw64/lib/*ccp4-6.2.0*/data/monomers file=d/DA.cif DA DA '2'-DEOXYADENOSINE-5'-MONOPHOSPHATE ' DNA34 22 . DA O4' C1' N9 107.8000.800 file=d/DC.cif DC DC '2'-DEOXYCYTIDINE-5'-MONOPHOSPHATE ' DNA32 20 . DC O4' C1' N1 107.8000.800 file=d/DG.cif DG DG '2'-DEOXYGUANOSINE-5'-MONOPHOSPHATE ' DNA35 23 . DG O4' C1' N9 107.8000.800 file=d/DT.cif DT DT 'THYMIDINE-5'-MONOPHOSPHATE ' DNA34 21 . DT O4' C1' N1 107.8000.800 Why are these values different in the two libraries? Am I looking at the wrong files or the wrong bonds? Thanks! Greg
[ccp4bb] Insoluble ligand?
Hi All I have a ligand that is dissolved in 100% DMSO. The ligand crashes out even when diluted to 20% final DMSO concentration in the protein buffer. I have heard of people using detergents such as n octyl Beta D glucoside to keep the ligand solubilized at lower DMSO concentrations. Can someone give me pointers in this direction? Any suggestions, references etc. Thanks much Ritcha
Re: [ccp4bb] Insoluble ligand?
Hi Ritcha, Did you try Methanol or Ethanol to see if your ligand dissolve in these solvents? You should try these solvents first before adding any detergents. Good luck. Smita Mohanty Chaudhary, Ritcha chaudha...@missouri.edu 9/16/2011 11:04 AM Hi All I have a ligand that is dissolved in 100% DMSO. The ligand crashes out even when diluted to 20% final DMSO concentration in the protein buffer. I have heard of people using detergents such as n octyl Beta D glucoside to keep the ligand solubilized at lower DMSO concentrations. Can someone give me pointers in this direction? Any suggestions, references etc. Thanks much Ritcha
Re: [ccp4bb] AW: [ccp4bb] crystallization of complex and ...
To all and Alex, -The Kd is around 500nM from Biacore. -One of my protein tends to precipitate and is only stable below 3mg/m. If I concentrated it to higher concentration, it will precipitate a lot after 1hr. And it was previously crystallized at low concentration. But when I inject on gel filtration, I do try to load large amount both proteins. Then this leads to another question: How high should the concentration of complex be in co-crystallization or gel filtration? I set mine at 6mg/ml for the complex and I get some precipitate. What concentration of complex would people start with to co-crystallize? -Yes, I use the same buffer system for both proteins and complex. Thank you all for your suggestions. Min Date: Fri, 16 Sep 2011 09:13:46 +0200 From: alexander.paut...@boehringer-ingelheim.com Subject: [ccp4bb] AW: [ccp4bb] crystallization of complex and ... To: CCP4BB@JISCMAIL.AC.UK Dear Min,regarding #1 some things come to my mind:- What is the Kd that you got from the Biacore? And did you make sure that the sample is concentrated enough (both on gel filtration and in crystallization) to have a “sufficient” amount in the complex.- did you use the same buffer systems? Your Kd might be different in different buffers.BestAlex Dr. Alexander Pautsch Boehringer Ingelheim Pharma GmbH Co. KG Dept. Lead Identific. and Optim. Sup. Ge Tel.: +49 (7351) 54-4683 Fax: +49 (7351) 54-97924 mailto:alexander.paut...@boehringer-ingelheim.com Boehringer Ingelheim Pharma GmbH Co. KG, Sitz: Ingelheim am Rhein; Registergericht Mainz: HR A 22206; Komplementär Boehringer Ingelheim Deutschland GmbH; Geschäftsführung: Dr. Engelbert Günster (Vorsitzender), Ralf Gorniak, Mark Hagmann, Michael Klein, Dr. Martin Wanning; Vorsitzender des Aufsichtsrates: Prof. Dr. Dr. Andreas Barner; Sitz: Ingelheim am Rhein; Registergericht Mainz: HR B 23260 Diese E-Mail ist vertraulich zu behandeln. Sie kann besonderem rechtlichen Schutz unterliegen. Wenn Sie nicht der richtige Adressat sind, senden Sie bitte diese E-Mail an den Absender zurück, löschen die eingegangene E-Mail und geben den Inhalt der E-Mail nicht weiter. Jegliche unbefugte Bearbeitung, Nutzung, Vervielfältigung oder Verbreitung ist verboten. / This e-mail is confidential and may also be legally privileged. If you are not the intended recipient please reply to sender, delete the e-mail and do not disclose its contents to any person. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited.Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von m zhang Gesendet: Freitag, 16. September 2011 04:20 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] crystallization of complex and ... Dear all, I have two questions: First, I was trying to crystallize a complex of two proteins. Both proteins has been crystallized before. The two proteins bind to each other based on Biacore study, but they didn't form a single peak on gel filtration. When I mixed them at 1:1 ratio, the crystals I got contain only one of the two proteins. I was suggested to increase the ratio, for example 1.5:1, to increase the probability of co-crystallization which I will try. But I do want to hear if there are other possible ways to try. What would you try if you were in my situation? Second is about reusing of Ni-NTA resin. According to Qiagen's instruction, after using fresh Ni-NTA resin, one only needs to wash the used Ni resin first with 0.5M NaOH, then with your own buffer. After that the resin is ready to be reused until it needs being recharged. But my question is: Once immidazole competes with His-tagged protein and binds to Ni-resin, how can immidazole be rinsed off with the same buffer(usually pH is above 7) one uses to purify the protein? Thank you for any suggestion or comment. Min
Re: [ccp4bb] Insoluble ligand?
Keep in mind it may also be possible to soak in ligand from the solid if the binding constant is tight enough. Le Chatelier's principle will drag it from the undissolved solid into the complex. This process will be improved if you can increase solubility in solution, e.g. by incuding 20% DMSO or some other cosolvent in your drop. If you crystals will tolerate some cosolvent without cracking or dissolving, the equilibrium concentration of ligand in solution may still be high enough to populate your protein. This won't work for a loose-binding ligand. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 9/16/2011 12:04 PM, Chaudhary, Ritcha wrote: Hi All I have a ligand that is dissolved in 100% DMSO. The ligand crashes out even when diluted to 20% final DMSO concentration in the protein buffer. I have heard of people using detergents such as n octyl Beta D glucoside to keep the ligand solubilized at lower DMSO concentrations. Can someone give me pointers in this direction? Any suggestions, references etc. Thanks much Ritcha
Re: [ccp4bb] UV imaging of crystals
Would be curious to know the current limitations on UV microscopy employed for screening protein crystals - such as content of aromatic amino acids, protein size etc. Cheers, Shiva On Fri, Sep 16, 2011 at 1:19 AM, Klaus Fütterer k.futte...@bham.ac.ukwrote: From the experience when our (commercial) UV imaging system was set up, I can confirm that signal-to-noise is a non-trivial parameter for imaging in the UV range. I find the additional info gained from the UV capability very useful, not just to distinguish salt from protein crystals, but also to tell protein from buffer precipitate, buffer phase separation from protein phase separation, etc. Klaus === Klaus Fütterer, Ph.D. Reader in Structural Biology Undergraduate Admissions School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: k.futte...@bham.ac.uk Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab === On 16 Sep 2011, at 04:57, Nagarajan V wrote: Typically, what you image is Trp fluorescence by exciting at around 280 nm and observing at around 350 nm. Standard silicon based detectors do fine at the detection wavelength, although, as you can imagine, increased sensitivity in the UV means increase in the price of the detector. If your excitation and emission light paths do not overlap, you also can get by with standard glass (crown, flint, etc.) optics since they do allow some of the 350-nm light to get through. Therefore, yes, it is possible to build an inexpensive UV imager based on inexpensive excitation light source (Douglas Instruments offers a pen light), and standard lab microscope. Of course, for increased sensitivity and contrast you need a very good light source, optics made of quartz and calcium fluoride that let almost all the UV light through, highly discriminating filters and a sensitive detector. V. Nagarajan JANSi http://janscientific.com On Thu, Sep 15, 2011 at 7:07 PM, Edward A. Berry ber...@upstate.edu wrote: A real UV microscope requires quartz optics, right? Probably conventional microscopes use glass. And you can't see 280 nm (and its not good for your eyes) so you need some kind of phosphor screen to view the image? Bosch, Juergen wrote: I'm replying here to myself :-) So in an off-board discussion it turns out that the microscope in question was a special emitted light and not a UV microscope. So real UV microscopes might be better for the purpose of detecting real crystals. Sorry for the confusion - had too much sun today :-) Jürgen On Sep 15, 2011, at 4:19 PM, Jürgen Bosch wrote: I once tested such a commercial system in Seattle about 4 years ago. It did not impress me. In particular the discrimination between salt and protein did not work for about 10 different proteins from which we already had collected data. sure those were small between 10 and 100 micrometer. Excuse was to few tryptophans So in theory it is nice but a cheaper variant might be to add Gfp to your protein and screen for something green. 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://web.mac.com/bosch_lab/ On Sep 15, 2011, at 16:03, Frank von Delft frank.vonde...@sgc.ox.ac.uk wrote: A while ago I was trying to be cheap, so we played around with it quite a bit in the lab. After rediscovering some of the basics of signal-to-noise and microscope transmission efficiency and that sort of rot, I realised that the commercial systems may not be all that ridiculously overpriced after all. Not if one wants to be able to say something useful about really really small crystals -- the only ones that really matter in the grand scheme of things (big ones are quick to test; little ones must first be optimized = money+time). But maybe I was just being incompetent. Happens. phx. On 15/09/2011 20:50, Andrew Purkiss-Trew wrote: Quoting Harman, Christinechristine.har...@fda.hhs.gov: Hi All, I was curious if any of you have tried or even know if it is possible to adapt a stereoscope (in my case an Olympus SZX10 model) so as to view protein crystals with UV illumination. Basically, I want a cheap manual version of what a Rock UV Imager does. I know this is probably a crazy dream. However, I would greatly appreciate any comments, advice or experience any of you may have. Molecular Dimension do such an adaptor which fits to existing
[ccp4bb] density modification help
Dear BB, I hope you can help. I have been trying to density modify my potential SAD solutions but I think it would help if I could conceptualize the arrangement of molecules in my crystals and it is making my head hurt. I have 2 crystal forms. I only obtained a single crystal for the first form which I have never been able to reproduce. It processes well to ~3A in P6122/P6522 (a=52.3, b=52.3, c=216.9; 1 mol/AU) and C2221 (a=52.3, b=90.5, c=216.9; 3 mol/AU), systematic absences are convincing and there does not look like any twinning issues. MR has not worked but the closest model has less than 20% similarity. A self rotation function shows strong peaks along x, y and z (k=180) and weaker ones along z (k=60 and 120). In a slightly altered condition I can obtain reproducible crystals although the space group has now changed. I have collected native and iodide-SAD data to ~3.2A on these crystals which now index in P222. Systematic absences suggest they are P212121 (a=52.61, b=88.261, c=212.072 in the SAD data), however, they contain pseudotranslational NCS (0.5,0.5,0.03) which is ~40% of the origin, so it may be another choice of P222. The self rotation function is similar to the previous data. As these cell dimensions and that of the C2221 data are almost identical it looks like there has been a slight shift in the lattice arrangement causing the pseudotranslational symmetry (therefore 6 mols/AU). The native and SAD data are isomorphous within 1% but I get much better statistics from phasing with SAD alone. The anomalous signal is reasonable to 4.5-5A but I do get some potential solutions using PHENIX (FOMs ~0.5, BAYES-CC ~40, scew 10-20). Whilst I can see solvent/protein boundaries, at this resolution it is hard to tell what is going on and I am finding it hard to trace any sheets (it is an Ig-like fold) and I think some NCS averaging may do a world of good. The problem is how do I apply this with improper NCS - i.e. how is the 6-fold NCS from the self rotation related to the translational NCS. Also am I missing something which is blatantly obvious which can help me improve these maps and hopefully push the resolution out to its full potential. I might be asking for a lot with this data but if you can help it would be much appreciated - and if I have left out any details please ask away. Many thanks James Dr James Garnett Division of Molecular Biosciences, Imperial College London Level 5, Biochemistry Building, South Kensington, LONDON, SW7 2AZ, UK.
[ccp4bb] Paper describing the structure of LFA-1
Dear All: I would like to know the literature on the crystal structure of Leukocyte Function-Associated Antigen One (LFA-1, CD11a/CD18). I have the structure of I domain but not for the entire molecule. I would greatly appreciate if people can point me to the right article/reference. Thanks Subbu
Re: [ccp4bb] stereo
Hi, On Thu, Sep 15, 2011 at 1:55 PM, Hena Dutta hdutt...@gmail.com wrote: Hi Sabuj, Thanks for all your answers. I finally came to this plan. Please tell me if I am doing anything wrong. Option 1. HP Workstation Z400 FL998U8#ABA Desktop PC - Intel Xeon W3550 3.06GHz, 8GB DDR3, 160GB 10k RPM HDD, DVDRW, NVIDIA Quadro FX3800, Windows 7 Professional 32-bit $1000 Acer HN274H bmiiid 27 Class Widescreen 3D LED HD Monitor - 1920 x 1080, 16:9, 1:1 Dynamic, 1000:1 Native, 120Hz, 2ms, HDMI, DVI-D, VGA, NVIDIA 3D Glasses, Energy Star $670 I highly doubt this monitor will work in Linux unless you buy the 3d vision kit which comes with the emitter that you can hook into the 3 pin mini din port of your quadro (btw your quadro 3800 requires an extra 3 pin mini din bracket, more on that below). This monitor has a built in emitter that works with the pair of nvidia (these are not generic active shutter goggles) 3d vision goggles it comes bundled with : http://3dvision-blog.com/review-of-the-27-acer-hn274h-3d-vision-ready-lcd-monitor/ ...but does not have an input for the 3 pin mini din coming off a proper quadro. The latest nvidia driver for linux still mentions that you need the 3 pin mini din for nvidia 3d vision stereo in linux: http://us.download.nvidia.com/XFree86/Linux-x86_64/280.13/README/xconfigoptions.html Option Stereo integer 10 NVIDIA 3D Vision mode for use with NVIDIA 3D Vision glasses. The NVIDIA 3D Vision infrared emitter must be connected to a USB port of your computer, and to the 3-pin DIN connector of a Quadro graphics board (based on G8xGL or higher GPU) before starting the X server. Hot-plugging the USB infrared stereo emitter is not yet supported. Also, 3D Vision Stereo Linux support requires a Linux kernel built with USB device filesystem (usbfs) and USB 2.0 support. Not presently supported on FreeBSD or Solaris. Having just the USB connected to the emitter still only works in windows with the standard emitter. I also know that the windows nvidia driver explicitly checks to see what sort of monitor you have before enabling 3d vision (see the nvidia 3d vision wizard in the nvidia control panel), i.e. that's probably why you don't need a USB cable connected from your computer to this particular monitor where the emitter is housed in the bezel. So, basically if you decide to go with this monitor you'll still probably need to get the 3D vision kit with the proper emitter and you'll also need to get the 3 pin mini din bracket for your Quadro 3800 since it doesn't have the 3 pin mini din output: There's a forum about it here : http://forums.nvidia.com/index.php?showtopic=96163 which leads to this PNY part # 900-50762--000 : http://www.google.com/products/catalog?q=900-50762--000cid=17694678402912029306ei=OoVzTvKqH6mExgXwi5HRAQved=0CAkQgggwAA#scoring=tp Ofcourse I have to install linux. Do I need to buy anything else(say emiter or connector) for 3D stereo set up in linux distribution. What linux you like to suggest? I am familiar with open suse or ubuntu. Either would be fine. Lenovo IdeaCentre K330B 7747-1GU Desktop PC - Intel Core i7-2600 3.40GHz, 8GB DDR3, 1.5TB HDD, DVDRW, ATI Radeon HD 6450, Windows 7 Home Premium 64-bit $750 But, then I have to buy the right graphics card. Yes. I think buying the NVIDIA Quadro FX 3800 graphics card will be much costly than to increase the hard drive. Which one is better processor? Intel Xeon W3550 3.06GHz or Intel Core i7-2600 3.40GHz i'd say the i7 2600 @ 3.4GHz, uses less power too. If they are not big difference, I will go with the first one and increase the hard drive. What do you think? Buy the 2nd one, get a quadro 3700, get the 3d vision kit, and if you want a 120Hz 27 LED monitor, here's one : http://www.buy.com/prod/samsung-syncmaster-s27a750d-27-3d-led-lcd-monitor-16-9-2-ms-adjustable/223430191.html that doesn't have a built in emitter which as mentioned above probably isn't going to work in linux. HTH, Sabuj Many thanks for your time. Hena On Wed, Sep 14, 2011 at 5:56 PM, Sabuj Pattanayek sab...@gmail.com wrote: On Wed, Sep 14, 2011 at 3:23 PM, Hena Dutta hdutt...@gmail.com wrote: Hi, Isn't that an LCD monitor (Acer HS244HQ)? It's edge LED backlit. I don't know if there are any direct LED backlit 120Hz monitors. Couldn't find much information on those types.
Re: [ccp4bb] Paper describing the structure of LFA-1
On Fri, 2011-09-16 at 14:10 -0400, Narayanan Ramasubbu wrote: Dear All: I would like to know the literature on the crystal structure of Leukocyte Function-Associated Antigen One (LFA-1, CD11a/CD18). I have the structure of I domain but not for the entire molecule. I would greatly appreciate if people can point me to the right article/reference. Thanks Subbu One way to get that kind of information is to run your sequence against pdb using NCBI BLAST, and then follow up the papers associated with the PDB entries that come up. PubMed search is also not a bad starting point. If all else fails, there is always google. I am sure that with right keywords (e.g. structure of Leukocyte Function-Associated Antigen One) a review article will pop up (if it exists) among the first five or so hits. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] Protein preps become a jelly
Gamma delta resolvase catalytic domain stock solutions used to make a nice clear jelly at 4 degrees, but it was perfectly reversible by warming the sample to room T. In fact, one mutant crystallized in the stock tube after a few trips in and out of the fridge. The crystals didn't diffract very well (so we never published that), but it was a fun way to grow them. Phoebe = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Tue, 30 Aug 2011 23:31:20 +0800 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of aidong a...@xmu.edu.cn) Subject: [ccp4bb] Protein preps become a jelly To: CCP4BB@JISCMAIL.AC.UK Dear Buddies, Sorry for bothering you with an off-ccp4 question. We recently are experiencing a very strange phenomena. A couple of protein preps with reasonably high concentration (10-20mg/ml) become a jelly after storages for overnight or a couple of days at 4C. All of them have been purified by gel filtration. Some of these proteins behave like this from very first preps but some of them had been very kind to us previously. We have googled extensively in CCP4BB and www but it appears this only happens to us. It would be highly appreciated that you could exchange their experiences or provide your suggestions. Aidong Han, Ph.D Department of Biomedical Sciences School of Life Sciences Xiamen University Xiamen, Fujian 361005 China Phone: 0592-218-8172 (O) 0592-218-8173 (L) Web: http://life.xmu.edu.cn/adhanlab/
Re: [ccp4bb] Protein preps become a jelly
I think I know another protein that does this: gelatin! (Well, not the crystallization part...) Jacob On Fri, Sep 16, 2011 at 2:41 PM, Phoebe Rice pr...@uchicago.edu wrote: Gamma delta resolvase catalytic domain stock solutions used to make a nice clear jelly at 4 degrees, but it was perfectly reversible by warming the sample to room T. In fact, one mutant crystallized in the stock tube after a few trips in and out of the fridge. The crystals didn't diffract very well (so we never published that), but it was a fun way to grow them. Phoebe = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Tue, 30 Aug 2011 23:31:20 +0800 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of aidong a...@xmu.edu.cn) Subject: [ccp4bb] Protein preps become a jelly To: CCP4BB@JISCMAIL.AC.UK Dear Buddies, Sorry for bothering you with an off-ccp4 question. We recently are experiencing a very strange phenomena. A couple of protein preps with reasonably high concentration (10-20mg/ml) become a jelly after storages for overnight or a couple of days at 4C. All of them have been purified by gel filtration. Some of these proteins behave like this from very first preps but some of them had been very kind to us previously. We have googled extensively in CCP4BB and www but it appears this only happens to us. It would be highly appreciated that you could exchange their experiences or provide your suggestions. Aidong Han, Ph.D Department of Biomedical Sciences School of Life Sciences Xiamen University Xiamen, Fujian 361005 China Phone: 0592-218-8172 (O) 0592-218-8173 (L) Web: http://life.xmu.edu.cn/adhanlab/ -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
