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Dear Markus,
You could also try the "inse" keyword, which will tell refmac two mutually exclude two atoms occupying the same position but we occupancy for example of 0.6 and 0.4, stopping them from repelling each other.
Here is an example in the case of an Fe site which was contaminated by Mn in some of the molecules in the crystal:
"external distance first residue 401 inse A chain A atom MN second residue 401 chain A atom FE value 0.0 sigma 0.01 type 1"
I guess you could use it on your sugar atoms ...
Best,
D
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of [log in to unmask]
Sent: 19 April 2013 08:47
To: ccp4bb
Subject: Re: [ccp4bb] Alternate sugar conformations in refmac 5.5.0110
Dear Markus,
I just did a test: I generated in Coot an alternative conformation for an ASN with attached NAG, wrote out the coordinates and manually adapted the LINK record, read it back in in Coot and hit the Refmac button (version 5.7.0029). Except for some green difference density because I fitted an alternate conformation where there was none, everything went fine, there was no repulsion. I used the following link records in PDB standard (I believe it is long overdue that Refmac gives up his own idiosyncratic syntax, but that is another story).
LINK ND2AASN B 199 C1 ANAG B 501
LINK ND2BASN B 199 C1 BNAG B 501
No link type is specified, but Coot/Refmac figure out themselves the correct link.
I think Thomas Lueteke made a valid point. If you fit the alternative conformation in a strained conformation, it will move away, maybe even out of density, to relieve this strain. Coot/Refmac handle sugars very well and just doing a real space refinement usually solves the problem in less than a second. Just specify one of the altnernative NAG molecules. Do not include the ASN and the other NAG. If that does not work, you could try to first refine the B-conformer alone, and then combine the two conformers.
Best regards,
Herman
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Markus Meier
Sent: Thursday, April 18, 2013 10:41 PM
To: [log in to unmask]
Subject: Re: [ccp4bb] Alternate sugar conformations in refmac 5.5.0110
Hi Robbie,
Oops, sorry, I used
CCP4 6.3: Refmac_5.7.0032 version 5.7.0032 : 05/09/12
(confirmed by the log file)
so I am using the current version and the problem still persists.
Thanks!
Markus
On 18/04/13 03:31 PM, Robbie Joosten wrote:
> Hi Markus,
>
> You could try changing your Refmac version. The version you are using
> is ancient. You may have an old version in your PATH next to the new one because your CCP4 seems up to date.
>
> AFAICT there is nothing wrong with the LINKR or the HETATM records
>
> Sent from my Windows Phone
> ----------------------------------------------------------------------
> --------------------------------------------------
> From: Markus Meier
> Sent: 2013-04-18 22:19
> To: [log in to unmask]
> Subject: [ccp4bb] Alternate sugar conformations in refmac 5.5.0110
>
> Dear all,
>
> I am trying to refine a beta-D-N-Acetyl glucosamine moiety (NAG)
> linked to an asparagine (ASN) in Refmac.
> (version CCP4 6.3: Refmac_5.5.0110 version 5.5.0110 : 08/05/10)
>
> The sugar has two alternate conformations that occupy the same
> position in the electron density but are rotated 180° relative to each
> other. Even though I have defined two alternate conformations for the
> asparagine side chain, the sugar and the beta-link, Refmac pushes the
> two conformations apart, out of the electron density (while still
> honouring the link to Asn). So it seems that Refmac applies repulsive forces between the alternate conformations.
>
> Did anyone else experience this and/or can suggest a fix?
> All help is very much appreciated!
>
> My definitions for the alternate conformations in the PDB file are below:
>
> LINKR C1 ANAG E 1 ND2AASN A 116 NAG-ASN
> LINKR C1 BNAG E 1 ND2BASN A 116 NAG-ASN
> HETATM 125 C1 ANAG E 1 30.978 40.626 -25.446 0.50 98.96 E C
> HETATM 126 C2 ANAG E 1 30.428 41.897 -26.138 0.50100.81 E C
> HETATM 127 N2 ANAG E 1 29.783 41.751 -27.447 0.50 94.26 E N
> HETATM 128 C7 ANAG E 1 28.807 42.561 -27.924 0.50 89.45 E C
> HETATM 129 O7 ANAG E 1 28.325 42.377 -29.035 0.50 83.16 E O
> HETATM 130 C8 ANAG E 1 28.252 43.720 -27.128 0.50 87.63 E C
> HETATM 131 C3 ANAG E 1 31.618 42.847 -26.221 0.50102.77 E C
> HETATM 132 O3 ANAG E 1 31.420 43.888 -27.159 0.50102.25 E O
> HETATM 133 C4 ANAG E 1 31.840 43.369 -24.802 0.50103.37 E C
> HETATM 134 O4 ANAG E 1 32.969 44.217 -24.782 0.50104.91 E O
> HETATM 135 C5 ANAG E 1 32.040 42.223 -23.785 0.50 94.99 E C
> HETATM 136 C6 ANAG E 1 31.564 42.633 -22.381 0.50 86.60 E C
> HETATM 137 O6 ANAG E 1 32.632 42.591 -21.462 0.50 77.76 E O
> HETATM 138 O5 ANAG E 1 31.458 40.954 -24.130 0.50 99.19 E O
> HETATM 139 C1 BNAG E 1 30.271 40.925 -24.108 0.50 98.96 E C
> HETATM 140 C2 BNAG E 1 31.415 41.878 -23.684 0.50100.81 E C
> HETATM 141 N2 BNAG E 1 32.048 41.664 -22.378 0.50 94.26 E N
> HETATM 142 C7 BNAG E 1 33.331 41.981 -22.079 0.50 89.45 E C
> HETATM 143 O7 BNAG E 1 33.784 41.778 -20.959 0.50 83.16 E O
> HETATM 144 C8 BNAG E 1 34.276 42.586 -23.092 0.50 87.63 E C
> HETATM 145 C3 BNAG E 1 30.816 43.277 -23.775 0.50102.77 E C
> HETATM 146 O3 BNAG E 1 31.569 44.240 -23.061 0.50102.25 E O
> HETATM 147 C4 BNAG E 1 30.718 43.597 -25.266 0.50103.37 E C
> HETATM 148 O4 BNAG E 1 30.115 44.862 -25.440 0.50104.91 E O
> HETATM 149 C5 BNAG E 1 29.907 42.532 -26.038 0.50 94.99 E C
> HETATM 150 C6 BNAG E 1 30.373 42.426 -27.500 0.50 86.60 E C
> HETATM 151 O6 BNAG E 1 29.320 42.743 -28.381 0.50 77.76 E O
> HETATM 152 O5 BNAG E 1 29.866 41.216 -25.458 0.50 99.19 E O
> ATOM 745 N ASN A 116 27.207 36.475 -25.453 1.00 62.15 A N
> ATOM 746 CA AASN A 116 28.475 37.144 -25.135 0.50 61.77 A C
> ATOM 747 CB AASN A 116 28.182 38.336 -24.223 0.50 69.02 A C
> ATOM 748 CG AASN A 116 29.037 39.555 -24.495 0.50 79.45 A C
> ATOM 749 OD1AASN A 116 28.656 40.644 -24.047 0.50 83.18 A O
> ATOM 750 ND2AASN A 116 30.178 39.419 -25.218 0.50 87.72 A N
> ATOM 751 C ASN A 116 29.353 36.143 -24.389 1.00 53.28 A C
> ATOM 752 O ASN A 116 29.778 36.380 -23.266 1.00 48.83 A O
> ATOM 753 CA BASN A 116 28.575 37.144 -25.135 0.50 61.77 A C
> ATOM 754 CB BASN A 116 28.282 38.336 -24.223 0.50 69.02 A C
> ATOM 755 CG BASN A 116 29.487 39.201 -23.920 0.50 79.45 A C
> ATOM 756 OD1BASN A 116 30.611 38.710 -24.084 0.50 83.18 A O
> ATOM 757 ND2BASN A 116 29.299 40.471 -23.478 0.50 87.72 A N
>
> The Refmac log output shows that link description was recognized correctly for each conformation:
> WARNING : residue: NAG 1 chain:EE
> atom: "O1 " is absent in coord_file
> WARNING : link(spec):NAG-ASN is found dist = 1.466 ideal_dist= 1.439
> ch:EE res: 1 NAG at:C1 A->AA res: 116 ASN at:ND2 A
> WARNING : link(spec):NAG-ASN is found dist = 1.244 ideal_dist= 1.439
> ch:EE res: 1 NAG at:C1 B->AA res: 116 ASN at:ND2 B
>
>
> Best regards,
> Markus
>
> --
> Markus Meier, Ph.D.
> University of Manitoba
> Department of Chemistry
> 144 Dysart Road
--
Markus Meier, Ph.D.
University of Manitoba
Department of Chemistry
144 Dysart Road
Winnipeg, MB, R3T 2N2, Canada
Phone: +1 204 474 7172
Fax: +1 204 474 7608
E-mail: [log in to unmask]
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