Dear HK, I agree with Artem, I would put the ketone in the green density below in your pictures and make a covalent link with the arginine. The single oxygen I would put in the density currently occupied by the ketone and add one water there as well. In your refined omit maps, the current ketone density is rather weak and weakly connected with the ligand. This is more reminiscent of a bound water than of a covalent part of the ligand.
Best, Herman -----Ursprüngliche Nachricht----- Von: t...@em.uni-frankfurt.de <t...@em.uni-frankfurt.de> Gesendet: Donnerstag, 7. November 2019 13:51 An: Dale Tronrud <de...@daletronrud.com>; Seijo, Jose A. <josea.cuesta.se...@carlsberg.com>; Schreuder, Herman /DE <herman.schreu...@sanofi.com> Cc: CCP4BB@jiscmail.ac.uk Betreff: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand EXTERNAL : Real sender is prvs=1214477c0d=t...@em.uni-frankfurt.de Dear Dale, Jose, Herman, For Dale, The contour level of residual positive density in the map is 0.28 e/A^3 and 3 sigma as shown in Coot, when it is refined in full occupancy. I tried to model the amino-sugar (flip the ligand) to the mysterious density but it has no space for it as it clashes with the surrounding amino acids. For Herman, I noticed that there are some impurities of the ligand from the literature and it also mentions that the ligand tends to be degraded due to pH and oxidation. It might be a degraded product. For Jose, I tried to refine it with one water molecule instead of two (Figure - fullocc1.png and fullocc2.png. They are the same but with different view) and positive density seems to be reduced with full occupancy refinement of ligand and water. I also performed a refinement with occupancy refinement with ligand and water (occupancy_ligand_water.png) but it seems to me that positive density appears again in comparison with full occupancy refinement of water. But the B-factor of water is too low if refine it with a occupancy refinement. If occupancy of water is set to 1 then the B-factor seems to be correlated well with the surroundings. Thanks Best HK Quoting Dale Tronrud <de...@daletronrud.com>: > Hi, > > I'm curious. What is the contour level of the residual positive > density in the maps with full occupancy ligand and Arg, and how does > that level compare to the level of the contour of your omit map? > e/A^3 would be the most useful. > > Without the contour levels it isn't possible to distinguish between > the absence of fully occupied atoms and something more minor. > > Could your ligand be spending a small part of its time flipped over > on the long axis of your fused ring system? This would put the > sugar-like group over by your mystery density. > > Dale Tronrud > > On 11/6/2019 10:58 PM, Heng-Keat Tam wrote: >> 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 herman.schreu...@sanofi.com: >> >>> 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=Dbf9z >>>>>> osw >>>>>> cQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R >>>>>> 4H8 >>>>>> oHzZyRHMQu1AQ&m=EoLwt0JuWdDOc1BU6xjU4le8GxWW0NB5xhp-20uHVws&s=gY9 >>>>>> 1rA 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=Dbf9z >>>>>> osw >>>>>> cQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R >>>>>> 4H8 >>>>>> oHzZyRHMQu1AQ&m=EoLwt0JuWdDOc1BU6xjU4le8GxWW0NB5xhp-20uHVws&s=C6m >>>>>> 6fY 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=Dbf9zos >>>>> wcQ >>>>> -CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8 >>>>> oHz >>>>> ZyRHMQu1AQ&m=EoLwt0JuWdDOc1BU6xjU4le8GxWW0NB5xhp-20uHVws&s=C6m6fYE >>>>> OPl 79xkEzUw4GMTCpsV1JmEBJmKwXr5Gk4CQ&e= >>>> >>>> >>>> >>>> ################################################################### >>>> ### >>>> ## >>>> >>>> To unsubscribe from the CCP4BB list, click the following link: >>>> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.a >>>> c.u >>>> k_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwIBaQ&c=Dbf9zoswc >>>> Q-C >>>> RvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHz >>>> ZyR >>>> HMQu1AQ&m=EoLwt0JuWdDOc1BU6xjU4le8GxWW0NB5xhp-20uHVws&s=C6m6fYEOPl7 >>>> 9xk 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 >>> .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=DwIDaQ&c=Dbf9zoswcQ >> -CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHz >> ZyRHMQu1AQ&m=kipCxLuHZ7OC5aeoTWZt7hc-qlbyE1cjH2cpgs_78u0&s=6-a91Aeyf4 >> hm_0AFdV4c51zzaAkNzUmcjuwVnKq41sY&e= ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
