I am willing to bet that the ketone is covalently reacting with the arginine and the result is what you see :)
Artem - Cosmic Cats approve of this message On Thu, Nov 7, 2019 at 3:33 AM <[email protected]> wrote: > 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. GSK monitoring is conducted with appropriate > >> confidentiality controls and in accordance with local laws and after > >> appropriate consultation. > > > > ###################################################################### > > ## > > > > 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-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=EoLwt0JuWdDOc1BU6xjU4le8GxWW0NB5xhp-20uHVws&s=C6m6fYEOPl79xkEzUw4GMTCpsV1JmEBJmKwXr5Gk4CQ&e= > > > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
