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 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.
