[ccp4bb] Fab:antigen complex crystallization!!!
Hi everyone, I have been trying to crystallize Fab:antigen complex( 50kda:90kDa) complex and initially got needle clusters which after microseeding gave me single crystals but they are very small and I could not repeat the results. I have been using HEPES buffer at pH 6.8 to do the final SEC purification step of the complex before setting trays. I was wondering whether there are some other buffers (that one could suggest eg tris-hcl etc) which have given decent positive results when crystallizing Fab complexes.Though I have gone through individual papers (case by case) to get some idea, It would be great if anyone could direct me to a comprehensive literature towards studying the crystatllization conditions of Fab complexes. Equally, people who have considerable experience could suggest a list of must do steps for such problems which have routinely been practiced in their lab Also what is a good storage condition for the excess complex that you want to use later? I would really appreciate any suggestion,help, direction. Thanks ivan
Re: [ccp4bb] Fab:antigen complex crystallization!!!
Dear Ivan, If you take a look at the MPCD ( http://www.cinam.univ-mrs.fr/mpcd/ ) and search for Fab it brings up 172 crystallization conditions. The names should help you narrow down, which conditions are of a complex. Hopefully, you can use those conditions to help you decide, which direction you want to head. Spoiler - Fabs like ammonium sulfate. Take Care, Sean P212121 http://store.p212121.com/
Re: [ccp4bb] Fab:antigen complex crystallization!!!
Hi Ivan, you might also want to find out what buffers your particular system likes. Jancarik et al. Optimum solubility (OS) screening: an efficient method to optimize buffer conditions for homogeneity and crystallization of proteins. Acta Crystallogr D Biol Crystallogr (2004) vol. 60 (Pt 9) pp. 1670-3 Andreas On 28/07/2011 4:40, xaravich ivan wrote: Hi everyone, I have been trying to crystallize Fab:antigen complex( 50kda:90kDa) complex and initially got needle clusters which after microseeding gave me single crystals but they are very small and I could not repeat the results. I have been using HEPES buffer at pH 6.8 to do the final SEC purification step of the complex before setting trays. I was wondering whether there are some other buffers (that one could suggest eg tris-hcl etc) which have given decent positive results when crystallizing Fab complexes.Though I have gone through individual papers (case by case) to get some idea, It would be great if anyone could direct me to a comprehensive literature towards studying the crystatllization conditions of Fab complexes. Equally, people who have considerable experience could suggest a list of must do steps for such problems which have routinely been practiced in their lab Also what is a good storage condition for the excess complex that you want to use later? I would really appreciate any suggestion,help, direction. Thanks ivan -- Andreas Förster, Research Associate Paul Freemont & Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
Re: [ccp4bb] Fab:antigen complex crystallization!!!
Hi Ivan Did you use microseeding with *random *solutions? If not see the following paper by Obmolova and Co about exactly this, microseeding with Fab complexes, http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf For more subtle variations in using microseeding with complexes see http://pubs.acs.org/doi/abs/10.1021/cg2001442 Good luck Patrick On Thu, Jul 28, 2011 at 4:41 AM, xaravich ivan wrote: > Hi everyone, > I have been trying to crystallize Fab:antigen complex( 50kda:90kDa) complex > and initially got needle clusters which after microseeding gave me single > crystals but they are very small and I could not repeat the results. I have > been using HEPES buffer at pH 6.8 to do the final SEC purification step of > the complex before setting trays. > I was wondering whether there are some other buffers (that one could suggest > eg tris-hcl etc) which have given decent positive results when crystallizing > Fab complexes.Though I have gone through individual papers (case by case) to > get some idea, It would be great if anyone could direct me to a > comprehensive literature towards studying the crystatllization conditions of > Fab complexes. > Equally, people who have considerable experience could suggest a list of > must do steps for such problems which have routinely been practiced in their > lab > > Also what is a good storage condition for the excess complex that you want > to use later? > I would really appreciate any suggestion,help, direction. > Thanks > ivan -- patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Fab:antigen complex crystallization!!!
Hi Ivan, Here is another example of a method to crystallize antibody/antigene complexes. It uses a limited proteolysis step to generate crystals of poor quality, which are then used as seeds for an MMS screening... http://www.ncbi.nlm.nih.gov/pubmed/21536542 Good luck, Alex 2011/7/28 xaravich ivan > Hi everyone, > I have been trying to crystallize Fab:antigen complex( 50kda:90kDa) complex > and initially got needle clusters which after microseeding gave me single > crystals but they are very small and I could not repeat the results. I have > been using HEPES buffer at pH 6.8 to do the final SEC purification step of > the complex before setting trays. > I was wondering whether there are some other buffers (that one could > suggest eg tris-hcl etc) which have given decent positive results when > crystallizing Fab complexes.Though I have gone through individual papers > (case by case) to get some idea, It would be great if anyone could direct me > to a comprehensive literature towards studying the crystatllization > conditions of Fab complexes. > Equally, people who have considerable experience could suggest a list of > must do steps for such problems which have routinely been practiced in their > lab > > > Also what is a good storage condition for the excess complex that you want > to use later? > > I would really appreciate any suggestion,help, direction. > > Thanks > ivan >
Re: [ccp4bb] Fab:antigen complex crystallization!!!
On Thu, 2011-07-28 at 05:07 +0100, Sean Seaver wrote: > Spoiler - Fabs like ammonium sulfate. Not really - in my hands the ammonium sulfate was one hit out of 7. While Ivan's question is about Fab complexes with protein antigen, I think it brings up a more general question of protein class-dependent crystallization bias. While some general trends exist for classes of biopolymers (e.g. MPD is number one precipitant for DNA; protein:DNA complexes tend to crystallize in PEG-based conditions), a general idea of assigning a preferred precipitant to a protein class is, imho, pointless. Fabs are a good example - one would think that with half of the protein more or less the same in all instances some general trends should exist. And perhaps they do, as this http://scripts.iucr.org/cgi-bin/paper?S0907444999016224 seems to suggest. But alas, Fab crystallization conditions, once you look into it, appear to be just as diverse as the same for proteins in general. Crystallization conditions may change radically upon point mutation, so why would one expect that a class of proteins sharing some 50% identity will show unusual love for PEG, ammonium sulfate, sodium malonate or any other "miracle precipitant"? Consider this. Thanks to great engineering at the Douglas Instruments, we can routinely set up ~1000 drops for a given protein. If one of them shows a crystalline shower, we celebrate. To me, the fact that we try wrong crystallization conditions 99.9% of the time, proves that any attempt to predict crystallization conditions beyond vague things like "keep pH close to protein pI", "sodium malonate is cool", "PEG and ammonium sulfate are two most successful precipitants in history of protein crystallography", etc., is futile. Time wasted on looking into what is the most common precipitant for a particular class of proteins is better spent on setting up more trays. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] Fab:antigen complex crystallization!!!
Ed (and Ivan) Peter Sun and colleagues published two papers where they show that crystallization conditions for protein-protein complexes are strongly biased towards PEG-based rather than high-salt or organic-solvent-based conditions. This includes antibody-antigen complexes. http://www.ncbi.nlm.nih.gov/pubmed/16699187 http://scripts.iucr.org/cgi-bin/paper?do0016 I have heard anecdotally that the same is true of protein-peptide and protein-small molecule complexes, although I don't know of any systematic study. Can anyone shed light on this? I guess we can look in the Marseilles database Best wishes to all Patrick On Thu, Jul 28, 2011 at 2:32 PM, Ed Pozharski wrote: > > On Thu, 2011-07-28 at 05:07 +0100, Sean Seaver wrote: > > Spoiler - Fabs like ammonium sulfate. > > Not really - in my hands the ammonium sulfate was one hit out of 7. > > While Ivan's question is about Fab complexes with protein antigen, I > think it brings up a more general question of protein class-dependent > crystallization bias. While some general trends exist for classes of > biopolymers (e.g. MPD is number one precipitant for DNA; protein:DNA > complexes tend to crystallize in PEG-based conditions), a general idea > of assigning a preferred precipitant to a protein class is, imho, > pointless. Fabs are a good example - one would think that with half of > the protein more or less the same in all instances some general trends > should exist. And perhaps they do, as this > > http://scripts.iucr.org/cgi-bin/paper?S0907444999016224 > > seems to suggest. But alas, Fab crystallization conditions, once you > look into it, appear to be just as diverse as the same for proteins in > general. Crystallization conditions may change radically upon point > mutation, so why would one expect that a class of proteins sharing some > 50% identity will show unusual love for PEG, ammonium sulfate, sodium > malonate or any other "miracle precipitant"? > > Consider this. Thanks to great engineering at the Douglas Instruments, > we can routinely set up ~1000 drops for a given protein. If one of them > shows a crystalline shower, we celebrate. To me, the fact that we try > wrong crystallization conditions 99.9% of the time, proves that any > attempt to predict crystallization conditions beyond vague things like > "keep pH close to protein pI", "sodium malonate is cool", "PEG and > ammonium sulfate are two most successful precipitants in history of > protein crystallography", etc., is futile. Time wasted on looking into > what is the most common precipitant for a particular class of proteins > is better spent on setting up more trays. > > Cheers, > > Ed. > > -- > Oh, suddenly throwing a giraffe into a volcano to make water is crazy? > Julian, King of Lemurs -- patr...@douglas.co.uk Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Fab:antigen complex crystallization!!!
Thanks everyone, I got my crystals with PEG 8000 at first and after micro-seeding aith PEG 3350.Now I would work with all your suggestions and references with new vigor. ivan On Thu, Jul 28, 2011 at 9:43 AM, Patrick Shaw Stewart wrote: > Ed (and Ivan) > > Peter Sun and colleagues published two papers where they show that > crystallization conditions for protein-protein complexes are strongly > biased towards PEG-based rather than high-salt or > organic-solvent-based conditions. This includes antibody-antigen > complexes. > > http://www.ncbi.nlm.nih.gov/pubmed/16699187 > http://scripts.iucr.org/cgi-bin/paper?do0016 > > I have heard anecdotally that the same is true of protein-peptide and > protein-small molecule complexes, although I don't know of any > systematic study. > > Can anyone shed light on this? > > I guess we can look in the Marseilles database > > Best wishes to all > > Patrick > > > > On Thu, Jul 28, 2011 at 2:32 PM, Ed Pozharski > wrote: > > > > On Thu, 2011-07-28 at 05:07 +0100, Sean Seaver wrote: > > > Spoiler - Fabs like ammonium sulfate. > > > > Not really - in my hands the ammonium sulfate was one hit out of 7. > > > > While Ivan's question is about Fab complexes with protein antigen, I > > think it brings up a more general question of protein class-dependent > > crystallization bias. While some general trends exist for classes of > > biopolymers (e.g. MPD is number one precipitant for DNA; protein:DNA > > complexes tend to crystallize in PEG-based conditions), a general idea > > of assigning a preferred precipitant to a protein class is, imho, > > pointless. Fabs are a good example - one would think that with half of > > the protein more or less the same in all instances some general trends > > should exist. And perhaps they do, as this > > > > http://scripts.iucr.org/cgi-bin/paper?S0907444999016224 > > > > seems to suggest. But alas, Fab crystallization conditions, once you > > look into it, appear to be just as diverse as the same for proteins in > > general. Crystallization conditions may change radically upon point > > mutation, so why would one expect that a class of proteins sharing some > > 50% identity will show unusual love for PEG, ammonium sulfate, sodium > > malonate or any other "miracle precipitant"? > > > > Consider this. Thanks to great engineering at the Douglas Instruments, > > we can routinely set up ~1000 drops for a given protein. If one of them > > shows a crystalline shower, we celebrate. To me, the fact that we try > > wrong crystallization conditions 99.9% of the time, proves that any > > attempt to predict crystallization conditions beyond vague things like > > "keep pH close to protein pI", "sodium malonate is cool", "PEG and > > ammonium sulfate are two most successful precipitants in history of > > protein crystallography", etc., is futile. Time wasted on looking into > > what is the most common precipitant for a particular class of proteins > > is better spent on setting up more trays. > > > > Cheers, > > > > Ed. > > > > -- > > Oh, suddenly throwing a giraffe into a volcano to make water is crazy? > > Julian, King of Lemurs > > > > -- > patr...@douglas.co.ukDouglas Instruments Ltd. > DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK > Directors: Peter Baldock, Patrick Shaw Stewart > > http://www.douglas.co.uk > Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 > Regd. England 2177994, VAT Reg. GB 480 7371 36 >
