Re: [ccp4bb] Stabilization of crystals and ligand exchange
Hi Sabine, The easy experiment to start with, is to take your best conditions (nice looking crystals, no diffraction) and instead of overnight wait 4-8 weeks. Sometimes ligand exchange is slow, or the ligand induces a conformational change which takes a long time to complete in the crystals. There are cases that after a number of weeks, diffraction came back. However, even if the above experiment works, there will be the nagging uncertainty that the crystal packing may have prevented some completely unexpected, nature or science publication worthy conformational change. There will be no way around at least trying to cocrystallize your ligand. Your chance of success will depend on how your protein and ligand behave: -your ligand causes your protein to precipitate. Here your chances are slim. You could try to use the ligand as a precipitant by slowly diffusing it in (e.g. in a capillary with some gel to separate the protein and ligand solutions). -your ligand is poorly soluble. Here you have better chances. As you mentioned, one can add the dilute ligand to a dilute protein solution and then concentrate the complex. The amount of ligand needed depends on the affinity of the ligand for the protein. To get 90% occupancy, you need a free ligand concentration at least 10 times over the Kd (or ~IC50). To give an example: if you use for crystallization 10 mg/ml of a 30 kDa protein, your protein concentration is ~0.33 mM. If your ligand has an affinity of 100 nM, you need a free ligand concentration of 1 µM, which is 300 fold less than what you need to saturate all binding sites in the protein. To account for uncertainties in protein concentrations, I would add 0.5 - 1.0 mM Ligand. If you dilute 10 fold, you have 33 µM protein and I would add 50-100 µM ligand, which is still well above the 1 µM free concentration needed. Even with 100 fold dilution, you still just can dilute the ligand with the same factor as the protein and still be well above the required free concentration and you do not need more ligand. Of course, this only works for high-affinity ligands, for low affinity ligands it is quite a different story. I also would try to use the highest possible ligand concentration, since in many cases, although the ligand should bind in theory, in practise it is quite a different story. Good luck! Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sabine Schneider Sent: Wednesday, October 17, 2012 6:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Stabilization of crystals and ligand exchange Hi everyone, I am trying to get the structure of a protein-ligand complex were I need to exchange the ligand which it co-crystallises nicely with. Problem: either they crack, disolve, turn brown,... OR they still look very nice, well shaped but do not show a single reflection at the synchrotron!!! Here is what I tried so far: 1) initially stabilising with higher precipitant (here PEG1500) before slowly transferring (*) it to the ligand-removal solution (= artifical mother liquor with higher PEG, ethylen glycol or glucose, but without initial ligand) (*) by slow exchange I mean : initially mixing drop solution with stabilising/ligand-removal solution and adding it back to the drop stepwise before fully transferring it. Or calculation wise I have fully exchange the solution to the new solution 2) here I let them ist over night (if they did not disolve, crack or whatever) 3) slow exchange transfer to the artificial ML with the new ligand (10mM), left them over night and directly froze them 'Best' so far (crystals still looking nice but no reflection...) was slow exchange into higher PEG, than to higher PEG with ethylenglycol (30% and also adding ethylenglycol to the reservoir), let them sit for over night, before again slow exchange to the solution with the new ligand in higher PEG and 30% ethylen glycol. As I said here the crystals keep shape, but don't diffract at all anymore. Just freezing them with 30% ethylen glycol they diffract nicely to 2.5A on a home source. But already after step one they are sometimes not happy anymore. Co-crystallisation failed since when I add the ligand, which is not that soluble to the purified protein, everything crashed out of solution. I am thinking about to test adding the ligand to the diluted protein and concentrate it together. But I don't have that much ligand, since the synthesis is quite tedious The ligand can be dissolved in 30% ethylenglycol to ~50mM Thus I was wondering if someone has done successfully ligand exchange with glutaraldehyd stabilised xtals? Or any ideas how to stabilise them? I appreciate any ideas or comments! Sorry for the lengthy email! Best, Sabine
Re: [ccp4bb] Stabilization of crystals and ligand exchange
Hi Sabine, On top of the excellent suggestions of Herman, I was just wondering. Do you have a structure of the ligand-bound protein? If you do and the ligand bound at crystal contact with another molecule, I would think that it would be hard to get it out without harming the crystals (although not impossible). This may help you decide which way to go for obtaining the ligand-free structure. Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of herman.schreu...@sanofi.com [herman.schreu...@sanofi.com] Sent: Thursday, October 18, 2012 10:32 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Stabilization of crystals and ligand exchange Hi Sabine, The easy experiment to start with, is to take your best conditions (nice looking crystals, no diffraction) and instead of overnight wait 4-8 weeks. Sometimes ligand exchange is slow, or the ligand induces a conformational change which takes a long time to complete in the crystals. There are cases that after a number of weeks, diffraction came back. However, even if the above experiment works, there will be the nagging uncertainty that the crystal packing may have prevented some completely unexpected, nature or science publication worthy conformational change. There will be no way around at least trying to cocrystallize your ligand. Your chance of success will depend on how your protein and ligand behave: -your ligand causes your protein to precipitate. Here your chances are slim. You could try to use the ligand as a precipitant by slowly diffusing it in (e.g. in a capillary with some gel to separate the protein and ligand solutions). -your ligand is poorly soluble. Here you have better chances. As you mentioned, one can add the dilute ligand to a dilute protein solution and then concentrate the complex. The amount of ligand needed depends on the affinity of the ligand for the protein. To get 90% occupancy, you need a free ligand concentration at least 10 times over the Kd (or ~IC50). To give an example: if you use for crystallization 10 mg/ml of a 30 kDa protein, your protein concentration is ~0.33 mM. If your ligand has an affinity of 100 nM, you need a free ligand concentration of 1 µM, which is 300 fold less than what you need to saturate all binding sites in the protein. To account for uncertainties in protein concentrations, I would add 0.5 - 1.0 mM Ligand. If you dilute 10 fold, you have 33 µM protein and I would add 50-100 µM ligand, which is still well above the 1 µM free concentration needed. Even with 100 fold dilution, you still just can dilute the ligand with the same factor as the protein and still be well above the required free concentration and you do not need more ligand. Of course, this only works for high-affinity ligands, for low affinity ligands it is quite a different story. I also would try to use the highest possible ligand concentration, since in many cases, although the ligand should bind in theory, in practise it is quite a different story. Good luck! Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sabine Schneider Sent: Wednesday, October 17, 2012 6:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Stabilization of crystals and ligand exchange Hi everyone, I am trying to get the structure of a protein-ligand complex were I need to exchange the ligand which it co-crystallises nicely with. Problem: either they crack, disolve, turn brown,... OR they still look very nice, well shaped but do not show a single reflection at the synchrotron!!! Here is what I tried so far: 1) initially stabilising with higher precipitant (here PEG1500) before slowly transferring (*) it to the ligand-removal solution (= artifical mother liquor with higher PEG, ethylen glycol or glucose, but without initial ligand) (*) by slow exchange I mean : initially mixing drop solution with stabilising/ligand-removal solution and adding it back to the drop stepwise before fully transferring it. Or calculation wise I have fully exchange the solution to the new solution 2) here I let them ist over night (if they did not disolve, crack or whatever) 3) slow exchange transfer to the artificial ML with the new ligand (10mM), left them over night and directly froze them 'Best' so far (crystals still looking nice but no reflection...) was slow exchange into higher PEG, than to higher PEG with ethylenglycol (30% and also adding ethylenglycol to the reservoir), let them sit for over night, before again slow exchange to the solution with the new ligand in higher PEG and 30% ethylen glycol. As I said here the crystals keep shape, but don't diffract at all anymore. Just
Re: [ccp4bb] Stabilization of crystals and ligand exchange
Thanks a lot for all the excellent suggestions! Lots more things to try now! Cheers, Sabine On 10/18/2012 12:18 PM, Boaz Shaanan wrote: Hi Sabine, On top of the excellent suggestions of Herman, I was just wondering. Do you have a structure of the ligand-bound protein? If you do and the ligand bound at crystal contact with another molecule, I would think that it would be hard to get it out without harming the crystals (although not impossible). This may help you decide which way to go for obtaining the ligand-free structure. Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of herman.schreu...@sanofi.com [herman.schreu...@sanofi.com] Sent: Thursday, October 18, 2012 10:32 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Stabilization of crystals and ligand exchange Hi Sabine, The easy experiment to start with, is to take your best conditions (nice looking crystals, no diffraction) and instead of overnight wait 4-8 weeks. Sometimes ligand exchange is slow, or the ligand induces a conformational change which takes a long time to complete in the crystals. There are cases that after a number of weeks, diffraction came back. However, even if the above experiment works, there will be the nagging uncertainty that the crystal packing may have prevented some completely unexpected, nature or science publication worthy conformational change. There will be no way around at least trying to cocrystallize your ligand. Your chance of success will depend on how your protein and ligand behave: -your ligand causes your protein to precipitate. Here your chances are slim. You could try to use the ligand as a precipitant by slowly diffusing it in (e.g. in a capillary with some gel to separate the protein and ligand solutions). -your ligand is poorly soluble. Here you have better chances. As you mentioned, one can add the dilute ligand to a dilute protein solution and then concentrate the complex. The amount of ligand needed depends on the affinity of the ligand for the protein. To get 90% occupancy, you need a free ligand concentration at least 10 times over the Kd (or ~IC50). To give an example: if you use for crystallization 10 mg/ml of a 30 kDa protein, your protein concentration is ~0.33 mM. If your ligand has an affinity of 100 nM, you need a free ligand concentration of 1 µM, which is 300 fold less than what you need to saturate all binding sites in the protein. To account for uncertainties in protein concentrations, I would add 0.5 - 1.0 mM Ligand. If you dilute 10 fold, you have 33 µM protein and I would add 50-100 µM ligand, which is still well above the 1 µM free concentration needed. Even with 100 fold dilution, you still just can dilute the ligand with the same factor as the protein and still be well above the required free concentration and you do not need more ligand. Of course, this only works for high-affinity ligands, for low affinity ligands it is quite a different story. I also would try to use the highest possible ligand concentration, since in many cases, although the ligand should bind in theory, in practise it is quite a different story. Good luck! Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sabine Schneider Sent: Wednesday, October 17, 2012 6:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Stabilization of crystals and ligand exchange Hi everyone, I am trying to get the structure of a protein-ligand complex were I need to exchange the ligand which it co-crystallises nicely with. Problem: either they crack, disolve, turn brown,... OR they still look very nice, well shaped but do not show a single reflection at the synchrotron!!! Here is what I tried so far: 1) initially stabilising with higher precipitant (here PEG1500) before slowly transferring (*) it to the ligand-removal solution (= artifical mother liquor with higher PEG, ethylen glycol or glucose, but without initial ligand) (*) by slow exchange I mean : initially mixing drop solution with stabilising/ligand-removal solution and adding it back to the drop stepwise before fully transferring it. Or calculation wise I have fully exchange the solution to the new solution 2) here I let them ist over night (if they did not disolve, crack or whatever) 3) slow exchange transfer to the artificial ML with the new ligand (10mM), left them over night and directly froze them 'Best' so far (crystals still looking nice but no reflection...) was slow exchange into higher PEG, than to higher PEG with ethylenglycol (30% and also adding ethylenglycol to the reservoir), let them sit for over night, before again slow exchange to the solution
Re: [ccp4bb] Stabilization of crystals and ligand exchange
Hi Sabine, Glutaraldehyde crosslinking worked pretty good for various soaks in my experience. J. Appl. Cryst. (1999). 32, 106-112[ doi:10.1107/S002188989801053X ] A gentle vapor-diffusion technique for cross-linking of protein crystals for cryocrystallography C. J. Lusty Best regards, Dmitry On 2012-10-17, at 12:26 PM, Sabine Schneider wrote: Hi everyone, I am trying to get the structure of a protein-ligand complex were I need to exchange the ligand which it co-crystallises nicely with. Problem: either they crack, disolve, turn brown,... OR they still look very nice, well shaped but do not show a single reflection at the synchrotron!!! Here is what I tried so far: 1) initially stabilising with higher precipitant (here PEG1500) before slowly transferring (*) it to the ligand-removal solution (= artifical mother liquor with higher PEG, ethylen glycol or glucose, but without initial ligand) (*) by slow exchange I mean : initially mixing drop solution with stabilising/ligand-removal solution and adding it back to the drop stepwise before fully transferring it. Or calculation wise I have fully exchange the solution to the new solution 2) here I let them ist over night (if they did not disolve, crack or whatever) 3) slow exchange transfer to the artificial ML with the new ligand (10mM), left them over night and directly froze them 'Best' so far (crystals still looking nice but no reflection...) was slow exchange into higher PEG, than to higher PEG with ethylenglycol (30% and also adding ethylenglycol to the reservoir), let them sit for over night, before again slow exchange to the solution with the new ligand in higher PEG and 30% ethylen glycol. As I said here the crystals keep shape, but don't diffract at all anymore. Just freezing them with 30% ethylen glycol they diffract nicely to 2.5A on a home source. But already after step one they are sometimes not happy anymore. Co-crystallisation failed since when I add the ligand, which is not that soluble to the purified protein, everything crashed out of solution. I am thinking about to test adding the ligand to the diluted protein and concentrate it together. But I don't have that much ligand, since the synthesis is quite tedious The ligand can be dissolved in 30% ethylenglycol to ~50mM Thus I was wondering if someone has done successfully ligand exchange with glutaraldehyd stabilised xtals? Or any ideas how to stabilise them? I appreciate any ideas or comments! Sorry for the lengthy email! Best, Sabine
Re: [ccp4bb] Stabilization of crystals and ligand exchange
I say (of course I would!) why not try co-crystallization with random microseeding using the crystals with the original ligand? It usually allows you to control the number of crystals per drop too On 18 October 2012 15:59, Dmitry Rodionov d.rodio...@gmail.com wrote: Hi Sabine, Glutaraldehyde crosslinking worked pretty good for various soaks in my experience. J. Appl. Cryst. (1999). 32, 106-112[ doi:10.1107/S002188989801053X ] A gentle vapor-diffusion technique for cross-linking of protein crystals for cryocrystallography C. J. Lusty Best regards, Dmitry On 2012-10-17, at 12:26 PM, Sabine Schneider wrote: Hi everyone, I am trying to get the structure of a protein-ligand complex were I need to exchange the ligand which it co-crystallises nicely with. Problem: either they crack, disolve, turn brown,... OR they still look very nice, well shaped but do not show a single reflection at the synchrotron!!! Here is what I tried so far: 1) initially stabilising with higher precipitant (here PEG1500) before slowly transferring (*) it to the ligand-removal solution (= artifical mother liquor with higher PEG, ethylen glycol or glucose, but without initial ligand) (*) by slow exchange I mean : initially mixing drop solution with stabilising/ligand-removal solution and adding it back to the drop stepwise before fully transferring it. Or calculation wise I have fully exchange the solution to the new solution 2) here I let them ist over night (if they did not disolve, crack or whatever) 3) slow exchange transfer to the artificial ML with the new ligand (10mM), left them over night and directly froze them 'Best' so far (crystals still looking nice but no reflection...) was slow exchange into higher PEG, than to higher PEG with ethylenglycol (30% and also adding ethylenglycol to the reservoir), let them sit for over night, before again slow exchange to the solution with the new ligand in higher PEG and 30% ethylen glycol. As I said here the crystals keep shape, but don't diffract at all anymore. Just freezing them with 30% ethylen glycol they diffract nicely to 2.5A on a home source. But already after step one they are sometimes not happy anymore. Co-crystallisation failed since when I add the ligand, which is not that soluble to the purified protein, everything crashed out of solution. I am thinking about to test adding the ligand to the diluted protein and concentrate it together. But I don't have that much ligand, since the synthesis is quite tedious The ligand can be dissolved in 30% ethylenglycol to ~50mM Thus I was wondering if someone has done successfully ligand exchange with glutaraldehyd stabilised xtals? Or any ideas how to stabilise them? I appreciate any ideas or comments! Sorry for the lengthy email! Best, Sabine -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, 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
[ccp4bb] Stabilization of crystals and ligand exchange
Hi everyone, I am trying to get the structure of a protein-ligand complex were I need to exchange the ligand which it co-crystallises nicely with. Problem: either they crack, disolve, turn brown,... OR they still look very nice, well shaped but do not show a single reflection at the synchrotron!!! Here is what I tried so far: 1) initially stabilising with higher precipitant (here PEG1500) before slowly transferring (*) it to the ligand-removal solution (= artifical mother liquor with higher PEG, ethylen glycol or glucose, but without initial ligand) (*) by slow exchange I mean : initially mixing drop solution with stabilising/ligand-removal solution and adding it back to the drop stepwise before fully transferring it. Or calculation wise I have fully exchange the solution to the new solution 2) here I let them ist over night (if they did not disolve, crack or whatever) 3) slow exchange transfer to the artificial ML with the new ligand (10mM), left them over night and directly froze them 'Best' so far (crystals still looking nice but no reflection...) was slow exchange into higher PEG, than to higher PEG with ethylenglycol (30% and also adding ethylenglycol to the reservoir), let them sit for over night, before again slow exchange to the solution with the new ligand in higher PEG and 30% ethylen glycol. As I said here the crystals keep shape, but don't diffract at all anymore. Just freezing them with 30% ethylen glycol they diffract nicely to 2.5A on a home source. But already after step one they are sometimes not happy anymore. Co-crystallisation failed since when I add the ligand, which is not that soluble to the purified protein, everything crashed out of solution. I am thinking about to test adding the ligand to the diluted protein and concentrate it together. But I don't have that much ligand, since the synthesis is quite tedious The ligand can be dissolved in 30% ethylenglycol to ~50mM Thus I was wondering if someone has done successfully ligand exchange with glutaraldehyd stabilised xtals? Or any ideas how to stabilise them? I appreciate any ideas or comments! Sorry for the lengthy email! Best, Sabine
