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Dear Peer, Engi, Have you looked at the symmetry mates in Coot? If you look at the packing of your molecules and it makes sense to place an additional molecule in the gap in order to create crystal contacts then you should do so. I would place the whole molecule there and start with an occupancy of say 20% for the whole chain and refine and check the B factors if they are more like the other molecules or higher/lower. If they are lower then you can increase the occupancy, if they are higher than you have to lower it. You will then probably see more undefined, bloby density which means you have additional conformations for loops or secondary structural elements or maybe the whole molecule is a bit twisted. Engi, at 1.8A you should be able to build alternative conformations, for the 2.3A of Peer this will be more difficult. My guess is that your molecule is there but a bit "squashed" and hence has slightly different conformations in each unit cell and you will struggle to place just one model. You will also probably struggle to build side chains. Did you check for anisotropy? I would suspect that the resolution is lower in one or even two directions in connection with a large un-ordered/missing molecule. Cheers M ________________________________ From: CCP4 bulletin board <[log in to unmask]> on behalf of Kay Diederichs <[log in to unmask]> Sent: 23 July 2019 08:41:51 To: ccp4bb Subject: Re: [ccp4bb] Density questionable? Hi Engi, I understand that you say that the 2nd molecule in your case is not real; it's just model bias? I would not come to this conclusion. B values of NCS-related chains being quite different is not that rare. In my experience, at 1.8A and with a reasonably refined model (Rfree less than 30%), continuous electron density (even if it is visualized at low sigma) matching the model does not arise from model bias,. From your description, I infer that the second molecule exists in your crystal; it's "just" poorly ordered (or at least worse than the first) - otherwise ARP/wARP wouldn't build part of it. That ARP/wARP does not build the rest does not mean that a human cannot or should not build it, nor that it should not be built; the automatic programs typically only build the good parts. The statistics of the MR solution should tell whether the 2nd molecule was found due to a significant signal (LLG increase in phaser; good contrast in molrep), or because the user was "asking" the program to position two molecules. What is R/Rfree with/without the 2nd molecule? (Peer says that in his case the difference is 0.3% which I'd say is an insignificant difference) Can the crystal lattice be continued in all 3 dimensions without the 2nd molecule? best, Kay On Tue, 23 Jul 2019 06:22:31 +0200, Engi Hassaan <[log in to unmask]> wrote: >We faced a similar situation recently where our molecular replacement >suggested 2 molecules in the asymmetric unit but the B factors of the >second chain were twice as large as the first protien chain. At a >resolution of 1.8 Å2 you could see the electron density for both chains. We >performed ARP/wARP and realized only part of this second chain is resolved >in our structure. It was model bias. > >Best, >Engi > > >Engi Hassaan >PhD Student >Research Group of Prof. Dr. G. Klebe >Marburg, Germany > > >On Mon, Jul 22, 2019, 19:34 Bernhard Rupp <[log in to unmask]> wrote: > >> > Could there be two versions of each model: a "robustly-observed" and a >> "most-likely" version? >> >> We tried/suggested something in this spirit once. Not sure how it was >> received.... >> http://journals.iucr.org/d/issues/2016/12/00/rr5136/index.html >> >> Best, BR >> +++++++++++++++++++++++++++++++++++++++++++++++++ >> Jacob Pearson Keller >> Research Scientist / Looger Lab >> HHMI Janelia Research Campus >> 19700 Helix Dr, Ashburn, VA 20147 >> Desk: (571)209-4000 x3159 >> Cell: (301)592-7004 >> +++++++++++++++++++++++++++++++++++++++++++++++++ >> >> The content of this email is confidential and intended for the recipient >> specified in message only. It is strictly forbidden to share any part of >> this message with any third party, without a written consent of the sender. >> If you received this message by mistake, please reply to this message and >> follow with its deletion, so that we can ensure such a mistake does not >> occur in the future. >> >> -----Original Message----- >> From: CCP4 bulletin board <[log in to unmask]> On Behalf Of Peer Mittl >> Sent: Monday, July 22, 2019 6:05 AM >> To: [log in to unmask] >> Subject: [ccp4bb] Density questionable? >> >> Dear Colleagues, >> >> We are working on a structure where the density for a whole protein chain >> (>200 aa) is questionable, since the B-factors exceed 200 Å2 (2.3 Ang >> resolution). However, the initial difference density map and the feature >> enhanced map (normal 2fo-fc map to a minor extend) support the presence of >> this chain. Putting the chain seems equally wrong as not putting it. >> Putting >> it reduces Rfree by 0.3%. As a conservative researcher I feel tempted to >> deposit the structure without this highly mobile/weakly occupied chain, but >> other researchers may say "he has missed something". Handling this chain >> like a weakly occupied water is probably wrong, but what is the >> optimal/correct way? Is there a general opinion on how the escape this >> dilemma? >> >> All the best, >> Peer >> >> ######################################################################## >> >> 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=DwID-g&c=LU6cRtx0xgB8s29tIz9Olw&r >> >> =eLCg9eJ4Rs_LnxfUWsp7FSxhIEcZYmTSU4Uyq1bRYPI&m=rOm2j6iwxpg727UzObri1TbWnpwIi >> ZjKQvFr6ZVB9DY&s=Fk1bBPINkOI2P2wVJmv4cG2X8T6P0PwFqvAiyyTVnBk&e= >> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwID-g&c=LU6cRtx0xgB8s29tIz9Olw&r=eLCg9eJ4Rs_LnxfUWsp7FSxhIEcZYmTSU4Uyq1bRYPI&m=rOm2j6iwxpg727UzObri1TbWnpwIiZjKQvFr6ZVB9DY&s=Fk1bBPINkOI2P2wVJmv4cG2X8T6P0PwFqvAiyyTVnBk&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 >> > >######################################################################## > >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 -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. 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