Re: [ccp4bb] Ligand fitting into density
Dipankar, What you describe occurs frequently and some times improves with refinement and other times does not. I suggest not adding ligands until the rest of the structure is well behaved - usually around 25-28% Rfree. This will tend to favor the highest quality electron density and will not have bias from the ligand in the phases. Small proteins with electron rich ligands may need the ligand fitted to cross even 30%, but it is a guideline. Also keep in mind that organic molecules can show true electron density shifts based on the chemistry present. A sulfonamide on a phenyl ring will for instance pull most of the electrons (on average) from the ring, leaving it looking more like a comma than a disc...even at quite high resolution...it is "real" electronics observed visually. Your second question regarding ~100A^2 for the B-factor of the ligand requires further clarification: 1. Is the resolution of your data sufficient to warrant individual B refinement? If so, what is the average B of residues near the ligand? In general, I ligand at roughly 100% occupancy should have B-factors within 10-20% of the value of the protein. Some programs do a poor job of refining B-factors of ligands. X-PLOR, for instance used to have a hard time with them. 2. What is the relative quality of the electron density of the ligand vs protein? Does it suggest less than full occupancy of the ligand in the pocket? If so, the approximate occupancy of the compound can be estimated by the comparison of the ligand B and nearby protein B factors. A ligand B twice the value of the protein residues would roughly suggest an occupancy of 50%. You can try that and see if the Bfactors stabilize. Cheers, Jim On Wed, Apr 11, 2012 at 2:11 AM, Dipankar Manna wrote: > Dear Crystallographers, > > > > The protein I am working with is having SG P3121, Structure is solved at > 2.5A. the protein was soaked with compound, compound density is also > looking prominent except one six membered ring. There is no density at all > for the particular ring, but other parts of the compound is fitting well > enough into the density. The B factor of the ligand is showing >100. How > can I justify this issue. Asking for suggestions. > > > > Regards, > > > > Dipankar Manna > > > > -- > > This e-mail and any files transmitted with it are for the sole use of the > intended recipient(s) and may contain confidential and privileged > information.If you are not the intended recipient, please contact the > sender by reply e-mail and destroy all copies of the original message.Any > unauthorized review, use, disclosure, dissemination, forwarding,printing or > copying of this email or any action taken in reliance on this e-mail is > strictly prohibited and may be unlawful. > > Visit us at http://www.aurigene.com > -- * * * James Kiefer, Ph.D. * Structural Biology Genentech, Inc. 1 DNA Way, Mailstop 27 South San Francisco, CA 94080-4990
Re: [ccp4bb] Ligand fitting into density
Sent from my LG phone Kendall Nettles wrote: >Sometimes you can distinguish disorder from the other possibilities if you >have enough structures with the same protein. We have several examples where >part of the ligand is not visible in the maps, but there is clear distortion >of the ligand binding pocket to accommodate the missing piece. for example, we >have one with a ligand that has an extended linker. You can see a big whole in >the protein where the linker leaves the pocket, but no density for the tether. >Kendall >On Apr 11, 2012, at 8:09 AM, Fischmann, Thierry wrote: > >Dipankar, > >Herman's message describes the most common case, by far. In addition to his >excellent post there are 4 other possibilities which I can think of of why, in >general, the e- density may be missing for part of a ligand: >- the compound solution are never 100% pure. One impurity may be the compound >you're trying to soak but missing a specific moiety. This would be a result of >the chemical synthesis: one of the steps would be incomplete and the impurity >was not separated at a later stage. The impurity is what you'll see in the >electron density if it happens to bind significantly more tightly than the >intact ligand. Sometimes this possibility can be excluded just from the >chemical synthesis (unless the purity of some of the starting reagents is >questionable). Or you can check the inhibitor structure by Mass spec. >- compound is not stable in the soak conditions. For instance it may not be >stable at, say, in water, or in acidic conditions, or exposed to visible >light, etc. >- cleavage by the protein: on occasion the protein may be able to cleave the >ligand. This is usually observed if the ligand is a substrate (say 3rd >phosphoryl missing in a soak with ATP) or a close relative of the true >substrate. >- cleavage by X-ray: the compound gets degraded during data collection, fast >enough that a significant part of the data set is collected with the compound >with the missing piece. > >Thierry > > >From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of >herman.schreu...@sanofi.com<mailto:herman.schreu...@sanofi.com> >Sent: Wednesday, April 11, 2012 5:39 AM >To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> >Subject: Re: [ccp4bb] Ligand fitting into density > >Dear Dipankar, > >I have had a case where I had soaked the same compound in Trypsin (2.2 Å) and >in Factor Xa (2.0 Å). In Trypsin, one six-membered ring was completely >invisible, despite good resolution and phases, whereas this ring was clearly >visible in the Factor Xa structure. The electron density is shown in >J.Med.Chem (2002)45:2749 figures 1A and 1E. > >It does happen that parts of a soaked compound are completely without electron >density. In these cases I assume that this part is disordered and I refine the >compound without the undefined parts, while in contrast to flexible surface >residues, people look closely at bound compounds and use the structures e.g. >to optimize scoring functions for docking programs. Leaving the undefined >parts in the model in a guessed conformation would likely cause people to draw >wrong conclusions. > >For the rest, if the inhibitor is well-defined in the electron density maps, I >would not worry about the high B factors. They may even normalize once you >leave out the undefined part. > >Best regards, >Herman > >________________ >From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dipankar >Manna >Sent: Wednesday, April 11, 2012 11:11 AM >To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> >Subject: [ccp4bb] Ligand fitting into density > >Dear Crystallographers, > >The protein I am working with is having SG P3121, Structure is solved at 2.5A. >the protein was soaked with compound, compound density is also looking >prominent except one six membered ring. There is no density at all for the >particular ring, but other parts of the compound is fitting well enough into >the density. The B factor of the ligand is showing >100. How can I justify >this issue. Asking for suggestions. > >Regards, > >Dipankar Manna > > > > >This e-mail and any files transmitted with it are for the sole use of the >intended recipient(s) and may contain confidential and privileged >information.If you are not the intended recipient, please contact the sender >by reply e-mail and destroy all copies of the original message.Any >unauthorized review, use, disclosure, dissemination, forwarding,printing or >copying of this email or any action taken in reliance on this e-mail is
Re: [ccp4bb] Ligand fitting into density
Sometimes you can distinguish disorder from the other possibilities if you have enough structures with the same protein. We have several examples where part of the ligand is not visible in the maps, but there is clear distortion of the ligand binding pocket to accommodate the missing piece. for example, we have one with a ligand that has an extended linker. You can see a big whole in the protein where the linker leaves the pocket, but no density for the tether. Kendall On Apr 11, 2012, at 8:09 AM, Fischmann, Thierry wrote: Dipankar, Herman's message describes the most common case, by far. In addition to his excellent post there are 4 other possibilities which I can think of of why, in general, the e- density may be missing for part of a ligand: - the compound solution are never 100% pure. One impurity may be the compound you're trying to soak but missing a specific moiety. This would be a result of the chemical synthesis: one of the steps would be incomplete and the impurity was not separated at a later stage. The impurity is what you'll see in the electron density if it happens to bind significantly more tightly than the intact ligand. Sometimes this possibility can be excluded just from the chemical synthesis (unless the purity of some of the starting reagents is questionable). Or you can check the inhibitor structure by Mass spec. - compound is not stable in the soak conditions. For instance it may not be stable at, say, in water, or in acidic conditions, or exposed to visible light, etc. - cleavage by the protein: on occasion the protein may be able to cleave the ligand. This is usually observed if the ligand is a substrate (say 3rd phosphoryl missing in a soak with ATP) or a close relative of the true substrate. - cleavage by X-ray: the compound gets degraded during data collection, fast enough that a significant part of the data set is collected with the compound with the missing piece. Thierry From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of herman.schreu...@sanofi.com<mailto:herman.schreu...@sanofi.com> Sent: Wednesday, April 11, 2012 5:39 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] Ligand fitting into density Dear Dipankar, I have had a case where I had soaked the same compound in Trypsin (2.2 Å) and in Factor Xa (2.0 Å). In Trypsin, one six-membered ring was completely invisible, despite good resolution and phases, whereas this ring was clearly visible in the Factor Xa structure. The electron density is shown in J.Med.Chem (2002)45:2749 figures 1A and 1E. It does happen that parts of a soaked compound are completely without electron density. In these cases I assume that this part is disordered and I refine the compound without the undefined parts, while in contrast to flexible surface residues, people look closely at bound compounds and use the structures e.g. to optimize scoring functions for docking programs. Leaving the undefined parts in the model in a guessed conformation would likely cause people to draw wrong conclusions. For the rest, if the inhibitor is well-defined in the electron density maps, I would not worry about the high B factors. They may even normalize once you leave out the undefined part. Best regards, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dipankar Manna Sent: Wednesday, April 11, 2012 11:11 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Ligand fitting into density Dear Crystallographers, The protein I am working with is having SG P3121, Structure is solved at 2.5A. the protein was soaked with compound, compound density is also looking prominent except one six membered ring. There is no density at all for the particular ring, but other parts of the compound is fitting well enough into the density. The B factor of the ligand is showing >100. How can I justify this issue. Asking for suggestions. Regards, Dipankar Manna This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted a
Re: [ccp4bb] Ligand fitting into density
Dipankar, Herman's message describes the most common case, by far. In addition to his excellent post there are 4 other possibilities which I can think of of why, in general, the e- density may be missing for part of a ligand: - the compound solution are never 100% pure. One impurity may be the compound you're trying to soak but missing a specific moiety. This would be a result of the chemical synthesis: one of the steps would be incomplete and the impurity was not separated at a later stage. The impurity is what you'll see in the electron density if it happens to bind significantly more tightly than the intact ligand. Sometimes this possibility can be excluded just from the chemical synthesis (unless the purity of some of the starting reagents is questionable). Or you can check the inhibitor structure by Mass spec. - compound is not stable in the soak conditions. For instance it may not be stable at, say, in water, or in acidic conditions, or exposed to visible light, etc. - cleavage by the protein: on occasion the protein may be able to cleave the ligand. This is usually observed if the ligand is a substrate (say 3rd phosphoryl missing in a soak with ATP) or a close relative of the true substrate. - cleavage by X-ray: the compound gets degraded during data collection, fast enough that a significant part of the data set is collected with the compound with the missing piece. Thierry From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of herman.schreu...@sanofi.com Sent: Wednesday, April 11, 2012 5:39 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Ligand fitting into density Dear Dipankar, I have had a case where I had soaked the same compound in Trypsin (2.2 Å) and in Factor Xa (2.0 Å). In Trypsin, one six-membered ring was completely invisible, despite good resolution and phases, whereas this ring was clearly visible in the Factor Xa structure. The electron density is shown in J.Med.Chem (2002)45:2749 figures 1A and 1E. It does happen that parts of a soaked compound are completely without electron density. In these cases I assume that this part is disordered and I refine the compound without the undefined parts, while in contrast to flexible surface residues, people look closely at bound compounds and use the structures e.g. to optimize scoring functions for docking programs. Leaving the undefined parts in the model in a guessed conformation would likely cause people to draw wrong conclusions. For the rest, if the inhibitor is well-defined in the electron density maps, I would not worry about the high B factors. They may even normalize once you leave out the undefined part. Best regards, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dipankar Manna Sent: Wednesday, April 11, 2012 11:11 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Ligand fitting into density Dear Crystallographers, The protein I am working with is having SG P3121, Structure is solved at 2.5A. the protein was soaked with compound, compound density is also looking prominent except one six membered ring. There is no density at all for the particular ring, but other parts of the compound is fitting well enough into the density. The B factor of the ligand is showing >100. How can I justify this issue. Asking for suggestions. Regards, Dipankar Manna This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Ligand fitting into density
Dear Dipankar, I have had a case where I had soaked the same compound in Trypsin (2.2 Å) and in Factor Xa (2.0 Å). In Trypsin, one six-membered ring was completely invisible, despite good resolution and phases, whereas this ring was clearly visible in the Factor Xa structure. The electron density is shown in J.Med.Chem (2002)45:2749 figures 1A and 1E. It does happen that parts of a soaked compound are completely without electron density. In these cases I assume that this part is disordered and I refine the compound without the undefined parts, while in contrast to flexible surface residues, people look closely at bound compounds and use the structures e.g. to optimize scoring functions for docking programs. Leaving the undefined parts in the model in a guessed conformation would likely cause people to draw wrong conclusions. For the rest, if the inhibitor is well-defined in the electron density maps, I would not worry about the high B factors. They may even normalize once you leave out the undefined part. Best regards, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dipankar Manna Sent: Wednesday, April 11, 2012 11:11 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Ligand fitting into density Dear Crystallographers, The protein I am working with is having SG P3121, Structure is solved at 2.5A. the protein was soaked with compound, compound density is also looking prominent except one six membered ring. There is no density at all for the particular ring, but other parts of the compound is fitting well enough into the density. The B factor of the ligand is showing >100. How can I justify this issue. Asking for suggestions. Regards, Dipankar Manna This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com
[ccp4bb] Ligand fitting into density
Dear Crystallographers, The protein I am working with is having SG P3121, Structure is solved at 2.5A. the protein was soaked with compound, compound density is also looking prominent except one six membered ring. There is no density at all for the particular ring, but other parts of the compound is fitting well enough into the density. The B factor of the ligand is showing >100. How can I justify this issue. Asking for suggestions. Regards, Dipankar Manna This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com
