Dear HK, In your case mass spec would be extremely valuable. Not only could it prove or rule out a covalent link, it will also convince referees that the link is real. There is clearly more density than the degraded product you showed. I would also ask an experienced chemist if a link between your ligand and the arginine would be possible. Finally, I would also do a quality check on the ligand you used. Might it be that some reactive intermediate from the synthesis still is present?
Best, Herman -----Ursprüngliche Nachricht----- Von: [email protected] <[email protected]> Gesendet: Donnerstag, 7. November 2019 07:58 An: Schreuder, Herman /DE <[email protected]> Cc: [email protected] Betreff: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand EXTERNAL : Real sender is [email protected] Hi Herman, Thanks for the suggestion. In principle, I have refined the structure without Arg and ligand, as well as the connected residues to the Arg. However, I still see the connected density of Arg and the ligand (Figure - unmodel1.png - only removal of Arg and ligand). I have tried to refine the degraded product but the positive density in between Arg and the ligand is still there, indicating something should be modeled there (Figure - degraded_product.png and degraded_product2.png - both figures are the same ligand but with different side view). I also tried to model the ligand in different conformation but still the same as shown in degraded product. The positive density is there between Arg and the ligand. For both cases, I refined Arg with full occupancy but ligand with occupancy refinement. Now, Arg did not flip away from the density. I might have a look at the mass-spec to check whether it is covalently linked although I am not aware of the reactivity of the moiety of the ligand and we never see this compound to covalent link with protein in our experience. Thanks for the advice and suggestion. It is very helpful. Thanks. Best HK Quoting [email protected]: > Hi Heng-Keat, > > I had again a look at your electron density pictures and these were my > impressions: > 1) the Arginine as fitted looks fully occupied. There are no hints for > an alternative conformations. > 2) The fit of the ligand, although reasonable, is not extremely good. > > It seems that the only reason to invoke partial occupancies for the > Arginine and ligand are problems to fit both the ligand and the > arginine in the available density. This means that for the overlapping > parts, to sum of the occupancies is maximal 1.0. > However, the ligand and the Arginine do not look half occupied. > > So I would look at other explanations: > - would it be possible that the ligand reacts covalently with the > Arginine? The density looks pretty continuous to me. > - could it be that instead of the intended ligand, some side- or > degradation product has bound? > > What I would also do, but what you probably already did, is to delete > the ligand and the Arginine side chain atoms and run several rounds of > refinement, to get an electron density map with as much bias as > possible removed. > > Good luck! > Herman > > -----Original Message----- > From: CCP4 bulletin board <[email protected]> On Behalf Of > Heng-Keat Tam > Sent: Mittwoch, 6. November 2019 07:30 > To: [email protected] > Subject: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping > electron density of a residue and ligand > > EXTERNAL : Real sender is [email protected] > > > > Hi Nestor, > > I think the reason for Arg side chain to curl up is because I refined > ligand and Arg side chain with occupancy refinement, and the Arg moved > away from the density, most likely due to 'repulsion' from ligand. > > The other question is: Is it possible that the H-bonds stay very > close? As I tried to 'real space refine' in coot, and the Arg side > chain flipped away from the density. > > Thanks for the suggestion. > > Best > HK > > > Quoting Nestor Concha <[email protected]>: > >> Hi Tam, >> The density looks very strong and therefore I'm going to guess that >> the Arg guanidimium stays in contact/interacts with the ligand and >> with the phosphate/sulfate next to it. Perhaps it is one of those >> close interactions with shorter H-bonds that usual given the >> arrangement of ligand-phosphate/sulfate-Arg. I'd try to find a >> rotamer for the Arg that leaves the interactions intact rather than >> refine occupancies. Seems that the Arg side chain is curled up ???? >> Nestor >> >> -----Original Message----- >> From: CCP4 bulletin board <[email protected]> On Behalf Of >> Heng-Keat Tam >> Sent: Tuesday, November 5, 2019 10:55 AM >> To: [email protected] >> Subject: Re: [ccp4bb] Occupancy refinement of overlapping electron >> density of a residue and ligand >> >> EXTERNAL >> >> Dear Rob, >> >> I would like to model the alternative position for the side chain of >> R120 but I don't really know whether the alternative conformation >> exist as shown in the attached figure - density without the ligand >> and >> R120 overlaid with the refined structures of modeled ligand and R120. >> The ligand was modeled in two different conformations. From the >> density, it seems to me that the density is connected or overlapped. >> >> It should not be a post-translation modification as it is well-known >> that there is no post-translation modification for this protein. >> Furthermore, the crystal was obtained by co-crystallization of >> protein with the ligand itself. The density seems to be the expected ligand. >> >> Thanks for the advice. >> >> Best regards >> HK >> >> >> Quoting Robert Nicholls <[email protected]>: >> >>> Dear HK, >>> >>> No that's not quite correct - 'occupancy group alts complete' means >>> that both R120 and the ligand are constrained so that their >>> occupancies sum to unity. In contrast, 'occupancy group alts >>> incomplete' means that the occupancies of R120 and the ligand will >>> not be constrained to sum to unity (but the sum of their occupancies >>> must be less than one). In both cases, R120 and the ligand will "see" >>> each other in a certain sense. But, because they are assigned to >>> different groups, it is assumed that they are present in different >>> parts of the crystal. This means that they can overlap. >>> >>> Assuming that the ligand is in the correct conformation, I suspect >>> the source of your problem is that you are modelling the side chain >>> of >>> R120 as only one conformation. And I would also include the other >>> atoms in the side chain - CB and CG. >>> >>> If you are modelling the sidechain of R120 with partial occupancies, >>> then you should model those side chain atoms in two alternative >>> positions (i.e. representing the portions of the crystal that >>> do/don't have the ligand bound). This will help to ensure that your >>> model makes physical sense. So the ligand plus the alt of R120 in >>> the portion of the crystal that contains the ligand would be >>> assigned to one occupancy group, and the alt of R120 in the portion >>> of the crystal that does not contain the ligand would be assigned to >>> the second group. In this case it would be appropriate to specify >>> 'occupancy group alts complete', because the occupancies for the two >>> alternative conformers of R120 should sum to unity. Although no >>> doubt the estimation of the occupancies would be dominated by the >>> ligand in this case. >>> >>> Be sure to check your B-factors after occupancy refinement to make >>> sure the whole picture makes sense. Assuming your current model is >>> essentially correct, from visual inspection it looks like R120 will >>> have low occupancy and low B-factors when the ligand is not present >>> (or at least B-factors consistent with the environment), but will >>> have high occupancy and high B-factors when the ligand is present. >>> >>> On another note, have you considered whether that part of the ligand >>> could be modelled in a slightly different conformation, or whether >>> there could be a post-translation modification? >>> >>> I hope that helps, >>> Rob >>> >>> >>> Dr Rob Nicholls >>> Senior Investigator Scientist >>> MRC Laboratory of Molecular Biology >>> Francis Crick Avenue >>> Cambridge Biomedical Campus >>> Cambridge CB2 0QH >>> >>> >>> >>>> On 5 Nov 2019, at 13:36, HK <[email protected]> wrote: >>>> >>>> Dear all, >>>> >>>> I have problem with occupancy refinement by Refmac5 for an >>>> overlapping electron density of part of residue (an arginine) and >>>> part of ligand (tetracyclic compound) (attached figures in Dropbox >>>> with a link as shown below). >>>> >>>> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.dropbox.co >>>> m >>>> _sh_ppmfp5dnpy1b9e9_AAAV79bOzPHQUrVYp9loMwyha-3F&d=DwIBaQ&c=Dbf9zos >>>> w >>>> cQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H >>>> 8 >>>> oHzZyRHMQu1AQ&m=EoLwt0JuWdDOc1BU6xjU4le8GxWW0NB5xhp-20uHVws&s=gY91r >>>> A 7hskyc0OM1awqWwiv2ufjEmlQN1QyqosE8YQQ&e= >>>> dl=0 >>>> >>>> I refined part of the side chain of residue 120 and ligand (chain J >>>> residue 1105) with Refmac keyword as shown below. Unfortunately, >>>> the side chain of arginine moves away from the density but the >>>> ligand stays in the density. As far as I understood, occupancy >>>> refinement with keyword 'occupancy group alts complete' means both >>>> R120 and the ligand do not meet each other. Did I miss something >>>> from the occupancy refinement keyword? >>>> >>>> occupancy group id 1 chain A residue 120 atom NE occupancy group id >>>> 1 chain A residue 120 atom CZ occupancy group id 1 chain A residue >>>> 120 atom NH2 occupancy group id 1 chain A residue 120 atom NH1 >>>> occupancy group id 1 chain A residue 120 atom CD occupancy group id >>>> 2 chain J residue 1105 occupancy group alts complete 1 2 occupancy >>>> refine >>>> >>>> Thank you for the advice. >>>> >>>> Best regards >>>> HK >>>> >>>> ################################################################### >>>> # >>>> # >>>> ### >>>> >>>> To unsubscribe from the CCP4BB list, click the following link: >>>> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.a >>>> c >>>> .uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwIBaQ&c=Dbf9zos >>>> w >>>> cQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H >>>> 8 >>>> oHzZyRHMQu1AQ&m=EoLwt0JuWdDOc1BU6xjU4le8GxWW0NB5xhp-20uHVws&s=C6m6f >>>> Y EOPl79xkEzUw4GMTCpsV1JmEBJmKwXr5Gk4CQ&e= >>> >>> >>> #################################################################### >>> # >>> # >>> ## >>> >>> To unsubscribe from the CCP4BB list, click the following link: >>> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac. >>> uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwIBaQ&c=Dbf9zoswc >>> Q >>> -CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oH >>> z >>> ZyRHMQu1AQ&m=EoLwt0JuWdDOc1BU6xjU4le8GxWW0NB5xhp-20uHVws&s=C6m6fYEOP >>> l 79xkEzUw4GMTCpsV1JmEBJmKwXr5Gk4CQ&e= >> >> >> >> ##################################################################### >> # >> ## >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac. >> u >> k_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwIBaQ&c=Dbf9zoswcQ- >> C >> RvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZy >> R >> HMQu1AQ&m=EoLwt0JuWdDOc1BU6xjU4le8GxWW0NB5xhp-20uHVws&s=C6m6fYEOPl79x >> k EzUw4GMTCpsV1JmEBJmKwXr5Gk4CQ&e= GSK monitors email communications >> sent to and from GSK in order to protect GSK, our employees, >> customers, suppliers and business partners, from cyber threats and >> loss of GSK Information. 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