Re: [ccp4bb] relationship between B factors and Koff
Although Bfactors and occupancies are correlated, occupancy refinement is quite feasible for the following reasons: 1) Modern maximum likelyhood programs like Buster do a decent job in separating occupancies and Bfactors. It shows up as shells of negative difference density e.g. around partially occupied metal ions which have been refined with full occupancy and inflated Bfactors. 2) A ligand is either completely there or it is completely gone, i.e. it is not that one could refine a group occupancy, one should refine a group occupancy and one has only one parameter to refine for say a ligand of 10+ atoms. In my experience, refining group occupancies even at moderate resolution, is stable. I now refine by default (group) occupancies for all ligands. These include the inhibitor soaked in, but also items like bound sulfate and phosphate ions, Tris, Hepes molecules and bound metal ions like zinc or cadmium. Only waters I refine at full occupancy and let the Bfactors soak up any partial occupancy and other errors since here the data do not allow occupancy refinement. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of James Holton Sent: Friday, November 19, 2010 9:20 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] relationship between B factors and Koff I don't think there is any relationship between rate constants and B factors. Yes, there is the hand-wavy argument of disorder begets disorder (and people almost always LITERALLY wave their hands when they propose this), but you have to be much more careful than that when it comes to thermodynamics. Yes, disorder and entropy are related, but just because a ligand is disordered does not mean that the delta-S term of the binding delta-G is higher. Now, there is a relationship between equilibrium constants and occupancies, since the occupancy is really just the ratio of the concentration of protein-bound-to-ligand to the total protein concentration, and an equilibrium exists between these two species. NB all the usual caveats of how crystal packing could change binding constants, etc. You could logically extend this to B factors by invoking a property of refinement: Specifically, if the true occupancy is less than 1, but modeled as 1.00, your refinement program will give you a B factor that is larger than the true atomic B factor. However, if you try to make this claim, then the obvious cantankerous reviewer suggestion would be to refine the occupancy. Problem is, refining both occupancy and B at the same time is usually unstable at moderate resolution. In general, it is hard to distinguish between something that is flopping around (high B factor) and something that is simply not there part of the time (low occupancy). I know it is tempting to try and relate B factors directly to entropy, but the disorder that leads to large B factors has a lot more to do with crystals than it has to do with proteins. For example, there are plenty of tightly-bound complexes that don't diffract well at all. If you refine these structures, you will get big B factors (roughly, B = 4*d^2+12 where d is the resolution in Angstrom). You may even have several crystal forms of the same thing with different Wilson B factors, but that is in no way evidence that the proteins in the two crystal forms somehow have different binding constants or rate constants. On the bright side, in your case it sounds like you have an entire protomer that is disorered relative to the rest of the crystal lattice. We have seen a few cases now like this where dehydrating the crystal (with an FMS or similar procedure) causes the unit cell to shrink and this locks the wobbly molecule into place. I think this is the principle mechanism of improved diffraction from dehydration. No, it does not work very often! But sometimes it does. -James Holton MAD Scientist On 11/19/2010 4:58 AM, Sebastiano Pasqualato wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure
Re: [ccp4bb] relationship between B factors and Koff
I agree that refining a single occupancy parameter for the ligand is a good approach. Because of the correlation with the B-values, it is essential to refine occupancy and isotropic or anisotropic B-values together, not in separate steps. SHELX has been able to do this correctly for the last 40 years. One factor that can affect the results is that if the ligand isn't there, something else (e.g. water) probably is. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Mon, 22 Nov 2010, herman.schreu...@sanofi-aventis.com wrote: Although Bfactors and occupancies are correlated, occupancy refinement is quite feasible for the following reasons: 1) Modern maximum likelyhood programs like Buster do a decent job in separating occupancies and Bfactors. It shows up as shells of negative difference density e.g. around partially occupied metal ions which have been refined with full occupancy and inflated Bfactors. 2) A ligand is either completely there or it is completely gone, i.e. it is not that one could refine a group occupancy, one should refine a group occupancy and one has only one parameter to refine for say a ligand of 10+ atoms. In my experience, refining group occupancies even at moderate resolution, is stable. I now refine by default (group) occupancies for all ligands. These include the inhibitor soaked in, but also items like bound sulfate and phosphate ions, Tris, Hepes molecules and bound metal ions like zinc or cadmium. Only waters I refine at full occupancy and let the Bfactors soak up any partial occupancy and other errors since here the data do not allow occupancy refinement. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of James Holton Sent: Friday, November 19, 2010 9:20 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] relationship between B factors and Koff I don't think there is any relationship between rate constants and B factors. Yes, there is the hand-wavy argument of disorder begets disorder (and people almost always LITERALLY wave their hands when they propose this), but you have to be much more careful than that when it comes to thermodynamics. Yes, disorder and entropy are related, but just because a ligand is disordered does not mean that the delta-S term of the binding delta-G is higher. Now, there is a relationship between equilibrium constants and occupancies, since the occupancy is really just the ratio of the concentration of protein-bound-to-ligand to the total protein concentration, and an equilibrium exists between these two species. NB all the usual caveats of how crystal packing could change binding constants, etc. You could logically extend this to B factors by invoking a property of refinement: Specifically, if the true occupancy is less than 1, but modeled as 1.00, your refinement program will give you a B factor that is larger than the true atomic B factor. However, if you try to make this claim, then the obvious cantankerous reviewer suggestion would be to refine the occupancy. Problem is, refining both occupancy and B at the same time is usually unstable at moderate resolution. In general, it is hard to distinguish between something that is flopping around (high B factor) and something that is simply not there part of the time (low occupancy). I know it is tempting to try and relate B factors directly to entropy, but the disorder that leads to large B factors has a lot more to do with crystals than it has to do with proteins. For example, there are plenty of tightly-bound complexes that don't diffract well at all. If you refine these structures, you will get big B factors (roughly, B = 4*d^2+12 where d is the resolution in Angstrom). You may even have several crystal forms of the same thing with different Wilson B factors, but that is in no way evidence that the proteins in the two crystal forms somehow have different binding constants or rate constants. On the bright side, in your case it sounds like you have an entire protomer that is disorered relative to the rest of the crystal lattice. We have seen a few cases now like this where dehydrating the crystal (with an FMS or similar procedure) causes the unit cell to shrink and this locks the wobbly molecule into place. I think this is the principle mechanism of improved diffraction from dehydration. No, it does not work very often! But sometimes it does. -James Holton MAD Scientist On 11/19/2010 4:58 AM, Sebastiano Pasqualato wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence
Re: [ccp4bb] relationship between B factors and Koff
I can direct you to PDB entry 1EWY, where the average isotropic temperature factor for the major component of the complex is ca. 47 A**2 and that for the smaller component is ca. 69 A**2. Similar values than the ones you are reporting. I am assuming some sort of disorder, or if you prefer, wobbling of the smaller component at the lever of the binding site. Fred. Sebastiano Pasqualato wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s
Re: [ccp4bb] relationship between B factors and Koff
Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might be tempting for you to draw this comparison in your structure, I would warn that this is too large a leap without further (non-anecdotal) evidence. As a further note, during SEC purification of complexes, I have observed that you generally have to have the complexes at at least 5 to 10-fold higher initial concentration if you want to purify the complex, which you are only pushing with your 80-100 uM high end concentration. A colleague of mine once told me this is due to a 5 to 10-fold dilution effect upon addition to the column, but I have never verified this nor read any primary source that validated this so I cannot supply a reference (others might be able to help here). Good luck and cheers~ ~Justin Quoting Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990
Re: [ccp4bb] relationship between B factors and Koff
A suggestion for purifying the complex: let's say there is a 5mL gap between the complex and one of its (smaller)constituents A. You can pre-load the column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to be injected right after the pre-load. This should provide approximately equilibrium conditions, so that the complex should be basically 1:1 when it comes out, even with a high Koff. (Alternatively, for true equilibrium conditions, just equilibrate the entire column in A, then inject the complex.) JPK - Original Message - From: Justin Hall hallj...@onid.orst.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 7:32 AM Subject: Re: [ccp4bb] relationship between B factors and Koff Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might be tempting for you to draw this comparison in your structure, I would warn that this is too large a leap without further (non-anecdotal) evidence. As a further note, during SEC purification of complexes, I have observed that you generally have to have the complexes at at least 5 to 10-fold higher initial concentration if you want to purify the complex, which you are only pushing with your 80-100 uM high end concentration. A colleague of mine once told me this is due to a 5 to 10-fold dilution effect upon addition to the column, but I have never verified this nor read any primary source that validated this so I cannot supply a reference (others might be able to help here). Good luck and cheers~ ~Justin Quoting Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990 *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] relationship between B factors and Koff
Dear Jacob, The SEC is generally run to separate the complex from the unbound components. If run the way your propose, the peak of unbound preinjected smaller component coincides with the peak of the complex and the final stochiometry is not better than by just mixing the components without SEC. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Friday, November 19, 2010 3:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] relationship between B factors and Koff A suggestion for purifying the complex: let's say there is a 5mL gap between the complex and one of its (smaller)constituents A. You can pre-load the column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to be injected right after the pre-load. This should provide approximately equilibrium conditions, so that the complex should be basically 1:1 when it comes out, even with a high Koff. (Alternatively, for true equilibrium conditions, just equilibrate the entire column in A, then inject the complex.) JPK - Original Message - From: Justin Hall hallj...@onid.orst.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 7:32 AM Subject: Re: [ccp4bb] relationship between B factors and Koff Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might be tempting for you to draw this comparison in your structure, I would warn that this is too large a leap without further (non-anecdotal) evidence. As a further note, during SEC purification of complexes, I have observed that you generally have to have the complexes at at least 5 to 10-fold higher initial concentration if you want to purify the complex, which you are only pushing with your 80-100 uM high end concentration. A colleague of mine once told me this is due to a 5 to 10-fold dilution effect upon addition to the column, but I have never verified this nor read any primary source that validated this so I cannot supply a reference (others might be able to help here). Good luck and cheers~ ~Justin Quoting Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990 *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] relationship between B factors and Koff
Well, mixing together has a much bigger pipetting error (to get, say, 500uM, you have to add 1000uM proteins A and B together, so with a pipetting error of ~1% even (and concentrated protein solutions seem to be tricky to pipette accurately), there would be an error of 10uM. Also, there are the errors associated with concentration determination, which are probably not trivial, especially with low EC. If, however, one of the components A is preloaded at low concentration (1-5uM, say), as I have recommended, the excess of that component with be exactly 1-5uM, assuming the complex was loaded with a slight excess of A. And this, as a percentage of the total 500uM, is much less than the various errors involved in mixing the two together. So this would be the procedure: Mix A with B at a calculated stoichiometric ratio of 1.1:1.0 Preload the column with a low level of A just before injecting the complex Inject the complex Collect fractions, solve structure, publish in your favorite venue JPK On Fri, Nov 19, 2010 at 8:30 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Jacob, The SEC is generally run to separate the complex from the unbound components. If run the way your propose, the peak of unbound preinjected smaller component coincides with the peak of the complex and the final stochiometry is not better than by just mixing the components without SEC. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Friday, November 19, 2010 3:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] relationship between B factors and Koff A suggestion for purifying the complex: let's say there is a 5mL gap between the complex and one of its (smaller)constituents A. You can pre-load the column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to be injected right after the pre-load. This should provide approximately equilibrium conditions, so that the complex should be basically 1:1 when it comes out, even with a high Koff. (Alternatively, for true equilibrium conditions, just equilibrate the entire column in A, then inject the complex.) JPK - Original Message - From: Justin Hall hallj...@onid.orst.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 7:32 AM Subject: Re: [ccp4bb] relationship between B factors and Koff Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might be tempting for you to draw this comparison in your structure, I would warn that this is too large a leap without further (non-anecdotal) evidence. As a further note, during SEC purification of complexes, I have observed that you generally have to have the complexes at at least 5 to 10-fold higher initial concentration if you want to purify the complex, which you are only pushing with your 80-100 uM high end concentration. A colleague of mine once told me this is due to a 5 to 10-fold dilution effect upon addition to the column, but I have never verified this nor read any primary source that validated this so I cannot supply a reference (others might be able to help here). Good luck and cheers~ ~Justin Quoting Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much
Re: [ccp4bb] relationship between B factors and Koff
Hi Sebastiano, I don't see how the k-off would influence this, given the timescale of growing crystals. An explanation in terms of high Kd and relative lack of crystal contacts for the component with higher temperature factors would sound more convincing to me. Mark Quoting Vellieux Frederic: I can direct you to PDB entry 1EWY, where the average isotropic temperature factor for the major component of the complex is ca. 47 A**2 and that for the smaller component is ca. 69 A**2. Similar values than the ones you are reporting. I am assuming some sort of disorder, or if you prefer, wobbling of the smaller component at the lever of the binding site. Fred. Sebastiano Pasqualato wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] relationship between B factors and Koff
I should add that this procedure is really only advantageous for high Koff complexes. If the complex does not dissociate appreciably in the time required for SEC, I agree that there is no great benefit for doing it my way. I have been working recently, however, on a high Koff complex, so have been thinking about how to get exactly the right ratio (other suggestions welcome!) Jacob On Fri, Nov 19, 2010 at 8:45 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Well, mixing together has a much bigger pipetting error (to get, say, 500uM, you have to add 1000uM proteins A and B together, so with a pipetting error of ~1% even (and concentrated protein solutions seem to be tricky to pipette accurately), there would be an error of 10uM. Also, there are the errors associated with concentration determination, which are probably not trivial, especially with low EC. If, however, one of the components A is preloaded at low concentration (1-5uM, say), as I have recommended, the excess of that component with be exactly 1-5uM, assuming the complex was loaded with a slight excess of A. And this, as a percentage of the total 500uM, is much less than the various errors involved in mixing the two together. So this would be the procedure: Mix A with B at a calculated stoichiometric ratio of 1.1:1.0 Preload the column with a low level of A just before injecting the complex Inject the complex Collect fractions, solve structure, publish in your favorite venue JPK On Fri, Nov 19, 2010 at 8:30 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Jacob, The SEC is generally run to separate the complex from the unbound components. If run the way your propose, the peak of unbound preinjected smaller component coincides with the peak of the complex and the final stochiometry is not better than by just mixing the components without SEC. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Friday, November 19, 2010 3:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] relationship between B factors and Koff A suggestion for purifying the complex: let's say there is a 5mL gap between the complex and one of its (smaller)constituents A. You can pre-load the column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to be injected right after the pre-load. This should provide approximately equilibrium conditions, so that the complex should be basically 1:1 when it comes out, even with a high Koff. (Alternatively, for true equilibrium conditions, just equilibrate the entire column in A, then inject the complex.) JPK - Original Message - From: Justin Hall hallj...@onid.orst.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 7:32 AM Subject: Re: [ccp4bb] relationship between B factors and Koff Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might be tempting for you to draw this comparison in your structure, I would warn that this is too large a leap without further (non-anecdotal) evidence. As a further note, during SEC purification of complexes, I have observed that you generally have to have the complexes at at least 5 to 10-fold higher initial concentration if you want to purify the complex, which you are only pushing with your 80-100 uM high end concentration. A colleague of mine once told me this is due to a 5 to 10-fold dilution effect upon addition to the column, but I have never verified this nor read any primary source that validated this so I cannot supply a reference (others might be able to help here). Good luck and cheers~ ~Justin Quoting Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going
Re: [ccp4bb] relationship between B factors and Koff
The amount of small component one looses from the complex depends on the Koff and the local concentrations during the SEC run, so I doubt if one could estimate those losses with an error less than the 1% pipetting error you quote. What I would do is to pipet dilute solutions, and concentrate afterwards. If there are conditions which give bad crystals, one could also harvest these crystals, redissolve them and set up crystallizations with those. My two cents, Herman -Original Message- From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu] Sent: Friday, November 19, 2010 3:57 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] relationship between B factors and Koff I should add that this procedure is really only advantageous for high Koff complexes. If the complex does not dissociate appreciably in the time required for SEC, I agree that there is no great benefit for doing it my way. I have been working recently, however, on a high Koff complex, so have been thinking about how to get exactly the right ratio (other suggestions welcome!) Jacob On Fri, Nov 19, 2010 at 8:45 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Well, mixing together has a much bigger pipetting error (to get, say, 500uM, you have to add 1000uM proteins A and B together, so with a pipetting error of ~1% even (and concentrated protein solutions seem to be tricky to pipette accurately), there would be an error of 10uM. Also, there are the errors associated with concentration determination, which are probably not trivial, especially with low EC. If, however, one of the components A is preloaded at low concentration (1-5uM, say), as I have recommended, the excess of that component with be exactly 1-5uM, assuming the complex was loaded with a slight excess of A. And this, as a percentage of the total 500uM, is much less than the various errors involved in mixing the two together. So this would be the procedure: Mix A with B at a calculated stoichiometric ratio of 1.1:1.0 Preload the column with a low level of A just before injecting the complex Inject the complex Collect fractions, solve structure, publish in your favorite venue JPK On Fri, Nov 19, 2010 at 8:30 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Jacob, The SEC is generally run to separate the complex from the unbound components. If run the way your propose, the peak of unbound preinjected smaller component coincides with the peak of the complex and the final stochiometry is not better than by just mixing the components without SEC. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Friday, November 19, 2010 3:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] relationship between B factors and Koff A suggestion for purifying the complex: let's say there is a 5mL gap between the complex and one of its (smaller)constituents A. You can pre-load the column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to be injected right after the pre-load. This should provide approximately equilibrium conditions, so that the complex should be basically 1:1 when it comes out, even with a high Koff. (Alternatively, for true equilibrium conditions, just equilibrate the entire column in A, then inject the complex.) JPK - Original Message - From: Justin Hall hallj...@onid.orst.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 7:32 AM Subject: Re: [ccp4bb] relationship between B factors and Koff Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might be tempting for you to draw this comparison in your structure, I would warn that this is too large a leap without further (non-anecdotal) evidence. As a further note, during SEC purification of complexes, I have observed that you generally have to have the complexes at at least 5 to 10-fold higher initial concentration if you want to purify the complex, which you are only pushing with your 80-100 uM high end concentration. A colleague of mine once told me this is due to a 5 to 10-fold dilution effect upon addition to the column, but I have never verified this nor read any primary source that validated this so I cannot supply a reference (others might be able to help
Re: [ccp4bb] relationship between B factors and Koff
Well, if one of the components is a small peptide and the solutions are dilute, it is hard to concentrate the complex, as the free peptide will go through the concentrator! It's a tricky problem. JPK On Fri, Nov 19, 2010 at 9:38 AM, herman.schreu...@sanofi-aventis.com wrote: The amount of small component one looses from the complex depends on the Koff and the local concentrations during the SEC run, so I doubt if one could estimate those losses with an error less than the 1% pipetting error you quote. What I would do is to pipet dilute solutions, and concentrate afterwards. If there are conditions which give bad crystals, one could also harvest these crystals, redissolve them and set up crystallizations with those. My two cents, Herman -Original Message- From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu] Sent: Friday, November 19, 2010 3:57 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] relationship between B factors and Koff I should add that this procedure is really only advantageous for high Koff complexes. If the complex does not dissociate appreciably in the time required for SEC, I agree that there is no great benefit for doing it my way. I have been working recently, however, on a high Koff complex, so have been thinking about how to get exactly the right ratio (other suggestions welcome!) Jacob On Fri, Nov 19, 2010 at 8:45 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Well, mixing together has a much bigger pipetting error (to get, say, 500uM, you have to add 1000uM proteins A and B together, so with a pipetting error of ~1% even (and concentrated protein solutions seem to be tricky to pipette accurately), there would be an error of 10uM. Also, there are the errors associated with concentration determination, which are probably not trivial, especially with low EC. If, however, one of the components A is preloaded at low concentration (1-5uM, say), as I have recommended, the excess of that component with be exactly 1-5uM, assuming the complex was loaded with a slight excess of A. And this, as a percentage of the total 500uM, is much less than the various errors involved in mixing the two together. So this would be the procedure: Mix A with B at a calculated stoichiometric ratio of 1.1:1.0 Preload the column with a low level of A just before injecting the complex Inject the complex Collect fractions, solve structure, publish in your favorite venue JPK On Fri, Nov 19, 2010 at 8:30 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Jacob, The SEC is generally run to separate the complex from the unbound components. If run the way your propose, the peak of unbound preinjected smaller component coincides with the peak of the complex and the final stochiometry is not better than by just mixing the components without SEC. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Friday, November 19, 2010 3:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] relationship between B factors and Koff A suggestion for purifying the complex: let's say there is a 5mL gap between the complex and one of its (smaller)constituents A. You can pre-load the column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to be injected right after the pre-load. This should provide approximately equilibrium conditions, so that the complex should be basically 1:1 when it comes out, even with a high Koff. (Alternatively, for true equilibrium conditions, just equilibrate the entire column in A, then inject the complex.) JPK - Original Message - From: Justin Hall hallj...@onid.orst.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 7:32 AM Subject: Re: [ccp4bb] relationship between B factors and Koff Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might be tempting for you to draw this comparison in your structure, I would warn that this is too large a leap without further (non-anecdotal) evidence. As a further note, during SEC purification of complexes, I have observed that you generally have to have the complexes at at least 5 to 10-fold higher initial concentration if you want to purify the complex, which you are only pushing with your 80-100 uM high end
Re: [ccp4bb] relationship between B factors and Koff
If the peptide is really small compared to the protein, I would just add excess peptide and not worry about the stochiometry. Best, Herman -Original Message- From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu] Sent: Friday, November 19, 2010 5:03 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] relationship between B factors and Koff Well, if one of the components is a small peptide and the solutions are dilute, it is hard to concentrate the complex, as the free peptide will go through the concentrator! It's a tricky problem. JPK On Fri, Nov 19, 2010 at 9:38 AM, herman.schreu...@sanofi-aventis.com wrote: The amount of small component one looses from the complex depends on the Koff and the local concentrations during the SEC run, so I doubt if one could estimate those losses with an error less than the 1% pipetting error you quote. What I would do is to pipet dilute solutions, and concentrate afterwards. If there are conditions which give bad crystals, one could also harvest these crystals, redissolve them and set up crystallizations with those. My two cents, Herman -Original Message- From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu] Sent: Friday, November 19, 2010 3:57 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] relationship between B factors and Koff I should add that this procedure is really only advantageous for high Koff complexes. If the complex does not dissociate appreciably in the time required for SEC, I agree that there is no great benefit for doing it my way. I have been working recently, however, on a high Koff complex, so have been thinking about how to get exactly the right ratio (other suggestions welcome!) Jacob On Fri, Nov 19, 2010 at 8:45 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Well, mixing together has a much bigger pipetting error (to get, say, 500uM, you have to add 1000uM proteins A and B together, so with a pipetting error of ~1% even (and concentrated protein solutions seem to be tricky to pipette accurately), there would be an error of 10uM. Also, there are the errors associated with concentration determination, which are probably not trivial, especially with low EC. If, however, one of the components A is preloaded at low concentration (1-5uM, say), as I have recommended, the excess of that component with be exactly 1-5uM, assuming the complex was loaded with a slight excess of A. And this, as a percentage of the total 500uM, is much less than the various errors involved in mixing the two together. So this would be the procedure: Mix A with B at a calculated stoichiometric ratio of 1.1:1.0 Preload the column with a low level of A just before injecting the complex Inject the complex Collect fractions, solve structure, publish in your favorite venue JPK On Fri, Nov 19, 2010 at 8:30 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Jacob, The SEC is generally run to separate the complex from the unbound components. If run the way your propose, the peak of unbound preinjected smaller component coincides with the peak of the complex and the final stochiometry is not better than by just mixing the components without SEC. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Friday, November 19, 2010 3:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] relationship between B factors and Koff A suggestion for purifying the complex: let's say there is a 5mL gap between the complex and one of its (smaller)constituents A. You can pre-load the column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to be injected right after the pre-load. This should provide approximately equilibrium conditions, so that the complex should be basically 1:1 when it comes out, even with a high Koff. (Alternatively, for true equilibrium conditions, just equilibrate the entire column in A, then inject the complex.) JPK - Original Message - From: Justin Hall hallj...@onid.orst.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 7:32 AM Subject: Re: [ccp4bb] relationship between B factors and Koff Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might
Re: [ccp4bb] relationship between B factors and Koff
This doesn't directly address your question, but since the subject of analyzing protein-protein interactions with gel filtration is raised on this bb occasionally, I thought I would mention that there are cases in which conventional gel filtration chromatography fails to provide evidence of a known protein:protein interaction. In such cases, the Hummel-Dreyer gel filtration method is sometimes used. It involves supplementing the running buffer with one of the proteins, so you need a lot of protein. Here are two references: Proc. Natl. Acad. Sci. USA 88 (1991) Biochemisrry (1993) 32, 11124-11131 On 11/19/10 6:58 AM, Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990
Re: [ccp4bb] relationship between B factors and Koff
Yes, this is where I heard of the technique, and my suggestion was a modification of that, but my references were: Hummel JP Dreyer WJ (1962) Measurement of protein-binding phenomena by gel filtration. Biochim Biophys Acta 63:530-532. Ratner D (1974) The interaction bacterial and phage proteins with immobilized Escherichia coli RNA polymerase. J Mol Biol 88(2):373-383. JPK - Original Message - From: Tanner, John J. tanne...@missouri.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 11:25 AM Subject: Re: [ccp4bb] relationship between B factors and Koff This doesn't directly address your question, but since the subject of analyzing protein-protein interactions with gel filtration is raised on this bb occasionally, I thought I would mention that there are cases in which conventional gel filtration chromatography fails to provide evidence of a known protein:protein interaction. In such cases, the Hummel-Dreyer gel filtration method is sometimes used. It involves supplementing the running buffer with one of the proteins, so you need a lot of protein. Here are two references: Proc. Natl. Acad. Sci. USA 88 (1991) Biochemisrry (1993) 32, 11124-11131 On 11/19/10 6:58 AM, Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990 *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] relationship between B factors and Koff
Sebastiano, We observed similar phenomenon in our protein-protein complex. This reference will give you details (Crystal Structure of an Electron Transfer Complex between Aromatic Amine Dehydrogenase- Azurin from Alcaligenes faecalis, Biochemistry, 45, 13500-13510, 2006). Sukumar Narayanasami Sukumar NE-CAT, Building 436E Advanced Photon Source Argonne National Laboratory 9700 S.Cass Ave Argonne, IL 60439-4800 USA Tel: 630-252-0681 Fax: 630-252-0687 e-mail: suku...@aps.anl.gov On Fri, Nov 19, 2010 at 6:58 AM, Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990
Re: [ccp4bb] relationship between B factors and Koff
I don't think there is any relationship between rate constants and B factors. Yes, there is the hand-wavy argument of disorder begets disorder (and people almost always LITERALLY wave their hands when they propose this), but you have to be much more careful than that when it comes to thermodynamics. Yes, disorder and entropy are related, but just because a ligand is disordered does not mean that the delta-S term of the binding delta-G is higher. Now, there is a relationship between equilibrium constants and occupancies, since the occupancy is really just the ratio of the concentration of protein-bound-to-ligand to the total protein concentration, and an equilibrium exists between these two species. NB all the usual caveats of how crystal packing could change binding constants, etc. You could logically extend this to B factors by invoking a property of refinement: Specifically, if the true occupancy is less than 1, but modeled as 1.00, your refinement program will give you a B factor that is larger than the true atomic B factor. However, if you try to make this claim, then the obvious cantankerous reviewer suggestion would be to refine the occupancy. Problem is, refining both occupancy and B at the same time is usually unstable at moderate resolution. In general, it is hard to distinguish between something that is flopping around (high B factor) and something that is simply not there part of the time (low occupancy). I know it is tempting to try and relate B factors directly to entropy, but the disorder that leads to large B factors has a lot more to do with crystals than it has to do with proteins. For example, there are plenty of tightly-bound complexes that don't diffract well at all. If you refine these structures, you will get big B factors (roughly, B = 4*d^2+12 where d is the resolution in Angstrom). You may even have several crystal forms of the same thing with different Wilson B factors, but that is in no way evidence that the proteins in the two crystal forms somehow have different binding constants or rate constants. On the bright side, in your case it sounds like you have an entire protomer that is disorered relative to the rest of the crystal lattice. We have seen a few cases now like this where dehydrating the crystal (with an FMS or similar procedure) causes the unit cell to shrink and this locks the wobbly molecule into place. I think this is the principle mechanism of improved diffraction from dehydration. No, it does not work very often! But sometimes it does. -James Holton MAD Scientist On 11/19/2010 4:58 AM, Sebastiano Pasqualato wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s
Re: [ccp4bb] relationship between B factors and Koff
Maybe you should look at: Rotation barriers in crystals from atomic displacement parameters Emily Maverick and Jack Dunitz (1987) Vol 62(2), 451-459. They reported that the libration amplitude of one part of a crystalline small molecule relative to another part is strongly correlated to bond rotation barriers measured by other means. The discussion at the top of page 458 seems to be about off rates. It's not up to me if a multi-temperature diffraction experiment and a lot of TLS analyses will be worth the effort relative to purchasing equipment to measure a binding parameter. -Dan James Holton wrote: I don't think there is any relationship between rate constants and B factors. Yes, there is the hand-wavy argument of disorder begets disorder (and people almost always LITERALLY wave their hands when they propose this), but you have to be much more careful than that when it comes to thermodynamics. Yes, disorder and entropy are related, but just because a ligand is disordered does not mean that the delta-S term of the binding delta-G is higher. Now, there is a relationship between equilibrium constants and occupancies, since the occupancy is really just the ratio of the concentration of protein-bound-to-ligand to the total protein concentration, and an equilibrium exists between these two species. NB all the usual caveats of how crystal packing could change binding constants, etc. You could logically extend this to B factors by invoking a property of refinement: Specifically, if the true occupancy is less than 1, but modeled as 1.00, your refinement program will give you a B factor that is larger than the true atomic B factor. However, if you try to make this claim, then the obvious cantankerous reviewer suggestion would be to refine the occupancy. Problem is, refining both occupancy and B at the same time is usually unstable at moderate resolution. In general, it is hard to distinguish between something that is flopping around (high B factor) and something that is simply not there part of the time (low occupancy). I know it is tempting to try and relate B factors directly to entropy, but the disorder that leads to large B factors has a lot more to do with crystals than it has to do with proteins. For example, there are plenty of tightly-bound complexes that don't diffract well at all. If you refine these structures, you will get big B factors (roughly, B = 4*d^2+12 where d is the resolution in Angstrom). You may even have several crystal forms of the same thing with different Wilson B factors, but that is in no way evidence that the proteins in the two crystal forms somehow have different binding constants or rate constants. On the bright side, in your case it sounds like you have an entire protomer that is disorered relative to the rest of the crystal lattice. We have seen a few cases now like this where dehydrating the crystal (with an FMS or similar procedure) causes the unit cell to shrink and this locks the wobbly molecule into place. I think this is the principle mechanism of improved diffraction from dehydration. No, it does not work very often! But sometimes it does. -James Holton MAD Scientist On 11/19/2010 4:58 AM, Sebastiano Pasqualato wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s
Re: [ccp4bb] relationship between B factors and Koff
The journal was Molecular Physics (1987) Vol 62(2), 451-459. (my brain librates when I type) Daniel Anderson wrote: Maybe you should look at: Rotation barriers in crystals from atomic displacement parameters Emily Maverick and Jack Dunitz (1987) Vol 62(2), 451-459. They reported that the libration amplitude of one part of a crystalline small molecule relative to another part is strongly correlated to bond rotation barriers measured by other means. The discussion at the top of page 458 seems to be about off rates. It's not up to me if a multi-temperature diffraction experiment and a lot of TLS analyses will be worth the effort relative to purchasing equipment to measure a binding parameter. -Dan James Holton wrote: I don't think there is any relationship between rate constants and B factors. Yes, there is the hand-wavy argument of disorder begets disorder (and people almost always LITERALLY wave their hands when they propose this), but you have to be much more careful than that when it comes to thermodynamics. Yes, disorder and entropy are related, but just because a ligand is disordered does not mean that the delta-S term of the binding delta-G is higher. Now, there is a relationship between equilibrium constants and occupancies, since the occupancy is really just the ratio of the concentration of protein-bound-to-ligand to the total protein concentration, and an equilibrium exists between these two species. NB all the usual caveats of how crystal packing could change binding constants, etc. You could logically extend this to B factors by invoking a property of refinement: Specifically, if the true occupancy is less than 1, but modeled as 1.00, your refinement program will give you a B factor that is larger than the true atomic B factor. However, if you try to make this claim, then the obvious cantankerous reviewer suggestion would be to refine the occupancy. Problem is, refining both occupancy and B at the same time is usually unstable at moderate resolution. In general, it is hard to distinguish between something that is flopping around (high B factor) and something that is simply not there part of the time (low occupancy). I know it is tempting to try and relate B factors directly to entropy, but the disorder that leads to large B factors has a lot more to do with crystals than it has to do with proteins. For example, there are plenty of tightly-bound complexes that don't diffract well at all. If you refine these structures, you will get big B factors (roughly, B = 4*d^2+12 where d is the resolution in Angstrom). You may even have several crystal forms of the same thing with different Wilson B factors, but that is in no way evidence that the proteins in the two crystal forms somehow have different binding constants or rate constants. On the bright side, in your case it sounds like you have an entire protomer that is disorered relative to the rest of the crystal lattice. We have seen a few cases now like this where dehydrating the crystal (with an FMS or similar procedure) causes the unit cell to shrink and this locks the wobbly molecule into place. I think this is the principle mechanism of improved diffraction from dehydration. No, it does not work very often! But sometimes it does. -James Holton MAD Scientist On 11/19/2010 4:58 AM, Sebastiano Pasqualato wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s
