[ccp4bb] crystallization of synthetic peptides
Dear crystallographers, Because of the low cost and speed of synthesizing 40- to 60-mer peptides, I wonder whether anyone has (good or bad) experiences crystalizing such peptides. In literature, I've found up to 34-mer synthetic coiled coils, but no other protein class. I can imagine that a protein sample with a few percent random deletion mutants mixed into it won't crystallize easily, but has anyone actually tried? cheers, Hans
Re: [ccp4bb] crystallization of synthetic peptides
Hans, Most natural toxins from snakes, scorpions etc are 50+/-some peptides. And quite a few of those have been studied and crystallized (see pdb for a list). Having worked on one of these structures as a graduate student, I can share my experience: - Purification is harder than you would think. You are talking about 10kD, usually around 5kD. Many methods (size exclusion, even concentration over a simple membrane) don't work as easily as you would like. - I did not have much of a problem crystallizing (i.e. no worse than other proteins, maybe even a little easier) - Crystals tend to diffract well (maybe better than average) - Structures can be hard to solve; MIR is very difficult because ions tend to not go into such crystals easily (because the molecules are small and tightly packed?); MR is hard because (again) it does not work very well on very small systems - Crystallization is not necessarily purification - if you have a mixture of peptides to start with, it may be harder to crystallize, or not: you might get a crystal that is a (random-ish) mixture. - If you have more than two cysteines in your sequence (natural toxins typically do), the additional problem is to get the correct folding and disulphide bridges; alternatively it is very hard to discriminate between correctly and incorrectly linked disulphides Finally: These sequence should be small enough for NMR. That may or may not answer your questions, but it avoids your original question. Mark -Original Message- From: H. Raaijmakers hraaijmak...@xs4all.nl To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Thu, Nov 10, 2011 8:16 am Subject: [ccp4bb] crystallization of synthetic peptides Dear crystallographers, Because of the low cost and speed of synthesizing 40- to 60-mer peptides, I wonder whether anyone has (good or bad) experiences crystalizing such peptides. In literature, I've found up to 34-mer synthetic coiled coils, but no other protein class. I can imagine that a protein sample with a few percent random deletion mutants mixed into it won't crystallize easily, but has anyone actually tried? cheers, Hans
Re: [ccp4bb] crystallization of synthetic peptides
As Mark says, structure solution of smallish peptides is not usually as easy as one might expect. A number of the small (say up to 50 residue) peptides in the PDB were solved by direct methods, but these require native data to 1.2A or (preferably) better. If sulfur is present in the molecule, SAD is a good choice and does not require such a very high resolution, but you need highly redundant data, so a high symmetry space group helps. If even one Met is present in the sequence, since you are synthesizing the peptides anyway, you can replace it with selenomethionine. George On 11/10/2011 09:15 PM, mjvdwo...@netscape.net wrote: Hans, Most natural toxins from snakes, scorpions etc are 50+/-some peptides. And quite a few of those have been studied and crystallized (see pdb for a list). Having worked on one of these structures as a graduate student, I can share my experience: - Purification is harder than you would think. You are talking about 10kD, usually around 5kD. Many methods (size exclusion, even concentration over a simple membrane) don't work as easily as you would like. - I did not have much of a problem crystallizing (i.e. no worse than other proteins, maybe even a little easier) - Crystals tend to diffract well (maybe better than average) - Structures can be hard to solve; MIR is very difficult because ions tend to not go into such crystals easily (because the molecules are small and tightly packed?); MR is hard because (again) it does not work very well on very small systems - Crystallization is not necessarily purification - if you have a mixture of peptides to start with, it may be harder to crystallize, or not: you might get a crystal that is a (random-ish) mixture. - If you have more than two cysteines in your sequence (natural toxins typically do), the additional problem is to get the correct folding and disulphide bridges; alternatively it is very hard to discriminate between correctly and incorrectly linked disulphides Finally: These sequence should be small enough for NMR. That may or may not answer your questions, but it avoids your original question. Mark -Original Message- From: H. Raaijmakers hraaijmak...@xs4all.nl To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Thu, Nov 10, 2011 8:16 am Subject: [ccp4bb] crystallization of synthetic peptides Dear crystallographers, Because of the low cost and speed of synthesizing 40- to 60-mer peptides, I wonder whether anyone has (good or bad) experiences crystalizing such peptides. In literature, I've found up to 34-mer synthetic coiled coils, but no other protein class. I can imagine that a protein sample with a few percent random deletion mutants mixed into it won't crystallize easily, but has anyone actually tried? cheers, Hans
Re: [ccp4bb] crystallization of synthetic peptides
Some HIV protease structures have been done using synthetic HIV protease (99 amino acid monomers). Look at J. Martin et al from UQ in Queensland. I believe this was done with Steve Kent. The protein contains some non-natural amino acids too. Hope this helps -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of H. Raaijmakers Sent: Friday, 11 November 2011 4:17 a.m. To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystallization of synthetic peptides Dear crystallographers, Because of the low cost and speed of synthesizing 40- to 60-mer peptides, I wonder whether anyone has (good or bad) experiences crystalizing such peptides. In literature, I've found up to 34-mer synthetic coiled coils, but no other protein class. I can imagine that a protein sample with a few percent random deletion mutants mixed into it won't crystallize easily, but has anyone actually tried? cheers, Hans
