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 <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Heng-Keat Tam Sent: Mittwoch, 6. November 2019 07:30 To: CCP4BB@JISCMAIL.AC.UK Subject: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand EXTERNAL : Real sender is owner-ccp...@jiscmail.ac.uk 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 <nestor.o.con...@gsk.com>: > 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 <CCP4BB@JISCMAIL.AC.UK> On Behalf Of > Heng-Keat Tam > Sent: Tuesday, November 5, 2019 10:55 AM > To: CCP4BB@JISCMAIL.AC.UK > 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 <nicho...@mrc-lmb.cam.ac.uk>: > >> 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 <t...@em.uni-frankfurt.de> 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.com >>> _sh_ppmfp5dnpy1b9e9_AAAV79bOzPHQUrVYp9loMwyha-3F&d=DwIBaQ&c=Dbf9zosw >>> cQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8 >>> oHzZyRHMQu1AQ&m=EoLwt0JuWdDOc1BU6xjU4le8GxWW0NB5xhp-20uHVws&s=gY91rA >>> 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.ac >>> .uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwIBaQ&c=Dbf9zosw >>> cQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8 >>> oHzZyRHMQu1AQ&m=EoLwt0JuWdDOc1BU6xjU4le8GxWW0NB5xhp-20uHVws&s=C6m6fY >>> 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=Dbf9zoswcQ >> -CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHz >> ZyRHMQu1AQ&m=EoLwt0JuWdDOc1BU6xjU4le8GxWW0NB5xhp-20uHVws&s=C6m6fYEOPl >> 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_tL8CLLA93vdywyu3qI70R4H8oHzZyR > HMQu1AQ&m=EoLwt0JuWdDOc1BU6xjU4le8GxWW0NB5xhp-20uHVws&s=C6m6fYEOPl79xk > 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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