Re: [ccp4bb] Trying to digest PISA results
Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it is easy to get things wrong.. But isnt a buried surface area of 720 rather small for a stable interface? If there is other confirming evidence like 2 diff space groups then you feel more secure!! On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system)
Re: [ccp4bb] Trying to digest PISA results
720 is not an impressive size for a stable interface, but it may do depending on molecule size and exact chemistry of the interface (h-bonds, salt bridges, disulphides, charges etc etc). Everything is subject to chemical environment and concentration, as usual. For these entries, PISA gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy of PISA, this may or may not be a stable thing. And yes, it has about 70-80% chances to be simply an artefact of crystal packing, according to some sort of derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last year. Having said all this, PISA is not an oracle and does not pretend to be correct in 100% of instances. Eugene. On 5 Sep 2011, at 10:14, Eleanor Dodson wrote: Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it is easy to get things wrong.. But isnt a buried surface area of 720 rather small for a stable interface? If there is other confirming evidence like 2 diff space groups then you feel more secure!! On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system)
Re: [ccp4bb] Trying to digest PISA results
I get confused by these figures. As I understand it the interface area given in Pisa is half the loss of accessible area on forming the complex: is that right? We're working on a complex with interface area ~500A^2, where the complex is stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa estimate of DelG -2.3. Does that sound sensible? Phil On 5 Sep 2011, at 10:33, Eugene Krissinel wrote: 720 is not an impressive size for a stable interface, but it may do depending on molecule size and exact chemistry of the interface (h-bonds, salt bridges, disulphides, charges etc etc). Everything is subject to chemical environment and concentration, as usual. For these entries, PISA gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy of PISA, this may or may not be a stable thing. And yes, it has about 70-80% chances to be simply an artefact of crystal packing, according to some sort of derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last year. Having said all this, PISA is not an oracle and does not pretend to be correct in 100% of instances. Eugene. On 5 Sep 2011, at 10:14, Eleanor Dodson wrote: Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it is easy to get things wrong.. But isnt a buried surface area of 720 rather small for a stable interface? If there is other confirming evidence like 2 diff space groups then you feel more secure!! On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system)
Re: [ccp4bb] Trying to digest PISA results
Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK On Mon, Sep 5, 2011 at 6:45 AM, Phil Evans p...@mrc-lmb.cam.ac.uk wrote: I get confused by these figures. As I understand it the interface area given in Pisa is half the loss of accessible area on forming the complex: is that right? We're working on a complex with interface area ~500A^2, where the complex is stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa estimate of DelG -2.3. Does that sound sensible? Phil On 5 Sep 2011, at 10:33, Eugene Krissinel wrote: 720 is not an impressive size for a stable interface, but it may do depending on molecule size and exact chemistry of the interface (h-bonds, salt bridges, disulphides, charges etc etc). Everything is subject to chemical environment and concentration, as usual. For these entries, PISA gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy of PISA, this may or may not be a stable thing. And yes, it has about 70-80% chances to be simply an artefact of crystal packing, according to some sort of derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last year. Having said all this, PISA is not an oracle and does not pretend to be correct in 100% of instances. Eugene. On 5 Sep 2011, at 10:14, Eleanor Dodson wrote: Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it is easy to get things wrong.. But isnt a buried surface area of 720 rather small for a stable interface? If there is other confirming evidence like 2 diff space groups then you feel more secure!! On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system) -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Trying to digest PISA results
Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK On Mon, Sep 5, 2011 at 6:45 AM, Phil Evans p...@mrc-lmb.cam.ac.ukmailto:p...@mrc-lmb.cam.ac.uk wrote: I get confused by these figures. As I understand it the interface area given in Pisa is half the loss of accessible area on forming the complex: is that right? We're working on a complex with interface area ~500A^2, where the complex is stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa estimate of DelG -2.3. Does that sound sensible? Phil On 5 Sep 2011, at 10:33, Eugene Krissinel wrote: 720 is not an impressive size for a stable interface, but it may do depending on molecule size and exact chemistry of the interface (h-bonds, salt bridges, disulphides, charges etc etc). Everything is subject to chemical environment and concentration, as usual. For these entries, PISA gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy of PISA, this may or may not be a stable thing. And yes, it has about 70-80% chances to be simply an artefact of crystal packing, according to some sort of derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last year. Having said all this, PISA is not an oracle and does not pretend to be correct in 100% of instances. Eugene. On 5 Sep 2011, at 10:14, Eleanor Dodson wrote: Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it is easy to get things wrong.. But isnt a buried surface area of 720 rather small for a stable interface? If there is other confirming evidence like 2 diff space groups then you feel more secure!! On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system) -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] Trying to digest PISA results
mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen jubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK On Mon, Sep 5, 2011 at 6:45 AM, Phil Evans p...@mrc-lmb.cam.ac.uk wrote: I get confused by these figures. As I understand it the interface area given in Pisa is half the loss of accessible area on forming the complex: is that right? We're working on a complex with interface area ~500A^2, where the complex is stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa estimate of DelG -2.3. Does that sound sensible? Phil On 5 Sep 2011, at 10:33, Eugene Krissinel wrote: 720 is not an impressive size for a stable interface, but it may do depending on molecule size and exact chemistry of the interface (h-bonds, salt bridges, disulphides, charges etc etc). Everything is subject to chemical environment and concentration, as usual. For these entries, PISA gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy of PISA, this may or may not be a stable thing. And yes, it has about 70-80% chances to be simply an artefact of crystal packing, according to some sort of derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last year. Having said all this, PISA is not an oracle and does not pretend to be correct in 100% of instances. Eugene. On 5 Sep 2011, at 10:14, Eleanor Dodson wrote: Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it is easy to get things wrong.. But isnt a buried surface area of 720 rather small for a stable interface? If there is other confirming evidence like 2 diff space groups then you feel more secure!! On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system) -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/ -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Trying to digest PISA results
AUC ! Andreas On 05/09/2011 6:00, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergenjubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
Re: [ccp4bb] Trying to digest PISA results
NMR...take that! JPK 2011/9/5 Andreas Förster docandr...@gmail.com: AUC ! Andreas On 05/09/2011 6:00, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergenjubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Trying to digest PISA results
Free flow electrophoresis would be another option, by the way anybody on the East Coast who has one of those instruments ? I'd be interested to get an email directly. Thanks, Jürgen On Sep 5, 2011, at 1:00 PM, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen jubo...@jhsph.edumailto:jubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK On Mon, Sep 5, 2011 at 6:45 AM, Phil Evans p...@mrc-lmb.cam.ac.ukmailto:p...@mrc-lmb.cam.ac.uk wrote: I get confused by these figures. As I understand it the interface area given in Pisa is half the loss of accessible area on forming the complex: is that right? We're working on a complex with interface area ~500A^2, where the complex is stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa estimate of DelG -2.3. Does that sound sensible? Phil On 5 Sep 2011, at 10:33, Eugene Krissinel wrote: 720 is not an impressive size for a stable interface, but it may do depending on molecule size and exact chemistry of the interface (h-bonds, salt bridges, disulphides, charges etc etc). Everything is subject to chemical environment and concentration, as usual. For these entries, PISA gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy of PISA, this may or may not be a stable thing. And yes, it has about 70-80% chances to be simply an artefact of crystal packing, according to some sort of derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last year. Having said all this, PISA is not an oracle and does not pretend to be correct in 100% of instances. Eugene. On 5 Sep 2011, at 10:14, Eleanor Dodson wrote: Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it is easy to get things wrong.. But isnt a buried surface area of 720 rather small for a stable interface? If there is other confirming evidence like 2 diff space groups then you feel more secure!! On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system) -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/ -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] Trying to digest PISA results
Jacob, You may also try DARTS (drug affinity responsive target stability). In this method, small molecule (or protein) binding leads to specific protection of its target protein from protease digestion. It is completely label free, and appears to be able to detect very low affinity interactions (high microM for small molecules). We'd be happy to send detailed protocols if you are interested. http://www.pnas.org/content/106/51/21984.full Best, Jing -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 10:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results NMR...take that! JPK 2011/9/5 Andreas Förster docandr...@gmail.com: AUC ! Andreas On 05/09/2011 6:00, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergenjubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer.
Re: [ccp4bb] Trying to digest PISA results
I did a similar assay years ago, but since the results were negative, never published anything--it was seeing whether nucleotides bound to my protein of interest by time courses of proteolysis +/- nucleotide. One tricky part of the assay, however, is to be sure that the compound of interest doesn't inhibit the protease--did you address that? I guess you would have to have some control proteins for that... Jacob g -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 10:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results NMR...take that! JPK 2011/9/5 Andreas Förster docandr...@gmail.com: AUC ! Andreas On 05/09/2011 6:00, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergenjubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer. -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Trying to digest PISA results
Excellent point indeed. We always include (at least one, preferably multiple) control proteins that are proteolysed equally across to make sure that the observed target stabilization is not due to fortuitous protease inhibition. What protease did you use for your nucleotide binding case? The protease sometimes matters. For example, thermolysin mainly only digests proteins that are unfolded, whereas pronase, which is a mixture of various proteases, can digest both folded and unfolded proteins. Best, Jing -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 12:18 PM To: Huang, Jing Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results I did a similar assay years ago, but since the results were negative, never published anything--it was seeing whether nucleotides bound to my protein of interest by time courses of proteolysis +/- nucleotide. One tricky part of the assay, however, is to be sure that the compound of interest doesn't inhibit the protease--did you address that? I guess you would have to have some control proteins for that... Jacob g -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 10:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results NMR...take that! JPK 2011/9/5 Andreas Förster docandr...@gmail.com: AUC ! Andreas On 05/09/2011 6:00, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergenjubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer. -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer.
Re: [ccp4bb] Trying to digest PISA results
Good ol' trypsin--any reason why not? JPK On Mon, Sep 5, 2011 at 5:40 PM, Huang, Jing jinghu...@mednet.ucla.edu wrote: Excellent point indeed. We always include (at least one, preferably multiple) control proteins that are proteolysed equally across to make sure that the observed target stabilization is not due to fortuitous protease inhibition. What protease did you use for your nucleotide binding case? The protease sometimes matters. For example, thermolysin mainly only digests proteins that are unfolded, whereas pronase, which is a mixture of various proteases, can digest both folded and unfolded proteins. Best, Jing -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 12:18 PM To: Huang, Jing Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results I did a similar assay years ago, but since the results were negative, never published anything--it was seeing whether nucleotides bound to my protein of interest by time courses of proteolysis +/- nucleotide. One tricky part of the assay, however, is to be sure that the compound of interest doesn't inhibit the protease--did you address that? I guess you would have to have some control proteins for that... Jacob g -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 10:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results NMR...take that! JPK 2011/9/5 Andreas Förster docandr...@gmail.com: AUC ! Andreas On 05/09/2011 6:00, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergenjubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer. -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer. -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Trying to digest PISA results
Ideally, the more nonspecific the protease, the better. (That would be a separate protein engineering project, or chemical catalyst project..) For now, we found that Pronase works well enough. Cheers, Jing -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 3:44 PM To: Huang, Jing Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results Good ol' trypsin--any reason why not? JPK On Mon, Sep 5, 2011 at 5:40 PM, Huang, Jing jinghu...@mednet.ucla.edu wrote: Excellent point indeed. We always include (at least one, preferably multiple) control proteins that are proteolysed equally across to make sure that the observed target stabilization is not due to fortuitous protease inhibition. What protease did you use for your nucleotide binding case? The protease sometimes matters. For example, thermolysin mainly only digests proteins that are unfolded, whereas pronase, which is a mixture of various proteases, can digest both folded and unfolded proteins. Best, Jing -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 12:18 PM To: Huang, Jing Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results I did a similar assay years ago, but since the results were negative, never published anything--it was seeing whether nucleotides bound to my protein of interest by time courses of proteolysis +/- nucleotide. One tricky part of the assay, however, is to be sure that the compound of interest doesn't inhibit the protease--did you address that? I guess you would have to have some control proteins for that... Jacob g -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 10:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results NMR...take that! JPK 2011/9/5 Andreas Förster docandr...@gmail.com: AUC ! Andreas On 05/09/2011 6:00, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergenjubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer. -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure
Re: [ccp4bb] Trying to digest PISA results
Wasn't the original question directed to our (growing) feeling that many times PISA says No obvious oligomerization pattern but we already have evidence of dimer formation etc.. This should happen occasionally as the approach implied in the calculations is statistical in a sense. We should not be getting such contradictions on a regular basis. Possible I misunderstood the original point ... Jan On Thu, Sep 1, 2011 at 7:46 AM, Karthik S biokart...@gmail.com wrote: http://www.ebi.ac.uk/msd-srv/prot_int/pi_ilist_css.html so it depends on how many 'stable assemblies' pisa can find i suppose. more interfaces and especially if stable enough will make your fraction go down. i would have been more surprised or worried if that conservative mutation showed radically different CSS scores say one close to zero and the other one or close to it. so the exclamation marks here are really pointless (since both values are close to zero). hence i would ignore the CSS in these two cases. CSS is a statistical measure and does not imply biological meaning. in making me (us) assume the latter through this one singular value leads to all misconceptions. -- Karthik On Wed, Aug 31, 2011 at 7:52 PM, Yuri Pompeu yuri.pom...@ufl.edu wrote: I was playing around with PDBe PISA and came across the following: For pdb entry 1OYA. The most promising interface has an area bury of around 720A^2 and DeltaG of -10.6Kcal/mol. sym_op(y,x,-z+1) and CSS of 0.039! Assembly analysis says it has no strong indications that point to stable quaternary structure. This protein has been extensively studied and determined to be a dimer. Entry 3RND is the same protein with one single conservative mutation deep in the active site. They align with a RMSD of 0.3 A, 99.8% sequence identity. Superposition and inspection of the regions that contact the adjacent monomer shows they are basically identical. The interface here shows Area bury of 760 A^2 and DeltaG = -6.6Kcal/mol. sym_op (-y,-x,-z-1/2) CSS=0.00 ! Assembly analysis basically says no stable oligomers form. This enzyme also is dimer according to gel filtration. Could anyone ellaborate on this please, if they feel like they have the time... Cheers -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 390 Fax: +420 296 809 410
Re: [ccp4bb] Trying to digest PISA results
On Wednesday, 31 August 2011, Jan Dohnalek wrote: Wasn't the original question directed to our (growing) feeling that many times PISA says No obvious oligomerization pattern but we already have evidence of dimer formation etc.. This should happen occasionally as the approach implied in the calculations is statistical in a sense. We should not be getting such contradictions on a regular basis. I think there are at least two possibilities 1) the interface seen in the crystal is a real dimer interface, but the PISA score fails to rate it as significant 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I have no idea which, if either, of these might be the case for 1OYA. Ethan Possible I misunderstood the original point ... Jan On Thu, Sep 1, 2011 at 7:46 AM, Karthik S biokart...@gmail.com wrote: http://www.ebi.ac.uk/msd-srv/prot_int/pi_ilist_css.html so it depends on how many 'stable assemblies' pisa can find i suppose. more interfaces and especially if stable enough will make your fraction go down. i would have been more surprised or worried if that conservative mutation showed radically different CSS scores say one close to zero and the other one or close to it. so the exclamation marks here are really pointless (since both values are close to zero). hence i would ignore the CSS in these two cases. CSS is a statistical measure and does not imply biological meaning. in making me (us) assume the latter through this one singular value leads to all misconceptions. -- Karthik On Wed, Aug 31, 2011 at 7:52 PM, Yuri Pompeu yuri.pom...@ufl.edu wrote: I was playing around with PDBe PISA and came across the following: For pdb entry 1OYA. The most promising interface has an area bury of around 720A^2 and DeltaG of -10.6Kcal/mol. sym_op(y,x,-z+1) and CSS of 0.039! Assembly analysis says it has no strong indications that point to stable quaternary structure. This protein has been extensively studied and determined to be a dimer. Entry 3RND is the same protein with one single conservative mutation deep in the active site. They align with a RMSD of 0.3 A, 99.8% sequence identity. Superposition and inspection of the regions that contact the adjacent monomer shows they are basically identical. The interface here shows Area bury of 760 A^2 and DeltaG = -6.6Kcal/mol. sym_op (-y,-x,-z-1/2) CSS=0.00 ! Assembly analysis basically says no stable oligomers form. This enzyme also is dimer according to gel filtration. Could anyone ellaborate on this please, if they feel like they have the time... Cheers
Re: [ccp4bb] Trying to digest PISA results
I guess both of the mentioned possibilities occur and it is hard to judge which one it is for a particular case. PISA is extremely useful for clear-cut cases to judge them quick. In the borderline ones it remains to be the task of the research teams to prove what sort of oligomerisation state is biologically relevant. I wish we had a method that delivers a reliable answer regarding the real state of any protein studied... Jan On Thu, Sep 1, 2011 at 8:41 AM, Ethan Merritt merr...@u.washington.eduwrote: On Wednesday, 31 August 2011, Jan Dohnalek wrote: Wasn't the original question directed to our (growing) feeling that many times PISA says No obvious oligomerization pattern but we already have evidence of dimer formation etc.. This should happen occasionally as the approach implied in the calculations is statistical in a sense. We should not be getting such contradictions on a regular basis. I think there are at least two possibilities 1) the interface seen in the crystal is a real dimer interface, but the PISA score fails to rate it as significant 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I have no idea which, if either, of these might be the case for 1OYA. Ethan Possible I misunderstood the original point ... Jan On Thu, Sep 1, 2011 at 7:46 AM, Karthik S biokart...@gmail.com wrote: http://www.ebi.ac.uk/msd-srv/prot_int/pi_ilist_css.html so it depends on how many 'stable assemblies' pisa can find i suppose. more interfaces and especially if stable enough will make your fraction go down. i would have been more surprised or worried if that conservative mutation showed radically different CSS scores say one close to zero and the other one or close to it. so the exclamation marks here are really pointless (since both values are close to zero). hence i would ignore the CSS in these two cases. CSS is a statistical measure and does not imply biological meaning. in making me (us) assume the latter through this one singular value leads to all misconceptions. -- Karthik On Wed, Aug 31, 2011 at 7:52 PM, Yuri Pompeu yuri.pom...@ufl.edu wrote: I was playing around with PDBe PISA and came across the following: For pdb entry 1OYA. The most promising interface has an area bury of around 720A^2 and DeltaG of -10.6Kcal/mol. sym_op(y,x,-z+1) and CSS of 0.039! Assembly analysis says it has no strong indications that point to stable quaternary structure. This protein has been extensively studied and determined to be a dimer. Entry 3RND is the same protein with one single conservative mutation deep in the active site. They align with a RMSD of 0.3 A, 99.8% sequence identity. Superposition and inspection of the regions that contact the adjacent monomer shows they are basically identical. The interface here shows Area bury of 760 A^2 and DeltaG = -6.6Kcal/mol. sym_op (-y,-x,-z-1/2) CSS=0.00 ! Assembly analysis basically says no stable oligomers form. This enzyme also is dimer according to gel filtration. Could anyone ellaborate on this please, if they feel like they have the time... Cheers -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 390 Fax: +420 296 809 410
Re: [ccp4bb] Trying to digest PISA results
This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system)
[ccp4bb] Trying to digest PISA results
I was playing around with PDBe PISA and came across the following: For pdb entry 1OYA. The most promising interface has an area bury of around 720A^2 and DeltaG of -10.6Kcal/mol. sym_op(y,x,-z+1) and CSS of 0.039! Assembly analysis says it has no strong indications that point to stable quaternary structure. This protein has been extensively studied and determined to be a dimer. Entry 3RND is the same protein with one single conservative mutation deep in the active site. They align with a RMSD of 0.3 A, 99.8% sequence identity. Superposition and inspection of the regions that contact the adjacent monomer shows they are basically identical. The interface here shows Area bury of 760 A^2 and DeltaG = -6.6Kcal/mol. sym_op (-y,-x,-z-1/2) CSS=0.00 ! Assembly analysis basically says no stable oligomers form. This enzyme also is dimer according to gel filtration. Could anyone ellaborate on this please, if they feel like they have the time... Cheers
