From: Careina Edgooms
Date: 23 November 2011 07:54
To: CCP4BB@jiscmail.ac.uk
----------
From: Nat Echols
It could mean that the sidechain is correctly placed, but poorly
ordered, and the B-factor restraints are preventing the B-factors for
those atoms from going as high as they probably should. I don't think
this is terribly uncommon. I've seen it before, and each time I tried
truncating the residue(s) to alanine, I got positive difference
density back after refinement. If you see the same thing, my
inclination would be to leave it alone and not worry about it as long
as everything else looks sensible.
The negative density around Met S could be radiation damage - also not
uncommon, since these and carboxyl groups tend to get hit the hardest
(unless you have heavier atoms).
-Nat
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From: Ian Tickle
On 23 November 2011 15:40, Nat Echols wrote:
> The negative density around Met S could be radiation damage.
But you wouldn't expect to see -ve density in the 2Fo-Fc map from
radiation damage right? The Fo-Fc map for sure.
Cheers
-- Ian
----------
From: Nat Echols
I assumed that's what the original poster meant.
(and I apologize for the redundant comments, GMail groups messages by
subject, so every time someone changes the subject line, it becomes a
new thread, which I usually miss. That said, I thought after reading
Garib's Refmac paper published earlier this year that it was now using
distance-based B-factor restraints, instead of bond connectivity - is
this correct or did I misunderstand?)
-Nat
----------
From: James Holton
Sounds like rad dam to me. See Burmeister, W. (2000)."Structural changes in a cryo-cooled protein crystal owing to radiation damage", Acta Cryst. D 56, 328-341. The first sign of a Met loosing its S-CH3 group will be a negative difference peak on the S.
-James Holton
MAD Scientist
----------
From: Garib N Murshudov
----------
From: Ian Tickle
I assumed that since this topic came up fairly recently, in fact 3
weeks ago (see thread starting from
http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg23628.html), it
wasn't just a re-run of the same question.
Perhaps the original poster could clarify whether we are talking about
unexplained -ve density in the 2Fo-Fc map or in the Fo-Fc map?
Cheers
-- Ian
Date: 23 November 2011 07:54
To: CCP4BB@jiscmail.ac.uk
Good morning CCP4 members
I have a question about a 2F0-Fc difference map that I calculated with Refmac. In some instances it gives me negative (red) density around part of a side chain and no positive density in sight. Furthermore the entire residue fits well into the blue density of the complete map, including the part with negative density.
I am struggling to interpret this. Does the fact that it fits the blue density mean that the side chain is in the correct place or does the red blob on part of the side chain (eg on the sulphur in a Met residue) mean that something funky is happening with this side chain?
Thanks for any assistance
Careina
----------
From: Nat Echols
It could mean that the sidechain is correctly placed, but poorly
ordered, and the B-factor restraints are preventing the B-factors for
those atoms from going as high as they probably should. I don't think
this is terribly uncommon. I've seen it before, and each time I tried
truncating the residue(s) to alanine, I got positive difference
density back after refinement. If you see the same thing, my
inclination would be to leave it alone and not worry about it as long
as everything else looks sensible.
The negative density around Met S could be radiation damage - also not
uncommon, since these and carboxyl groups tend to get hit the hardest
(unless you have heavier atoms).
-Nat
----------
From: Ian Tickle
On 23 November 2011 15:40, Nat Echols wrote:
> The negative density around Met S could be radiation damage.
But you wouldn't expect to see -ve density in the 2Fo-Fc map from
radiation damage right? The Fo-Fc map for sure.
Cheers
-- Ian
----------
From: Nat Echols
I assumed that's what the original poster meant.
(and I apologize for the redundant comments, GMail groups messages by
subject, so every time someone changes the subject line, it becomes a
new thread, which I usually miss. That said, I thought after reading
Garib's Refmac paper published earlier this year that it was now using
distance-based B-factor restraints, instead of bond connectivity - is
this correct or did I misunderstand?)
-Nat
----------
From: James Holton
Sounds like rad dam to me. See Burmeister, W. (2000)."Structural changes in a cryo-cooled protein crystal owing to radiation damage", Acta Cryst. D 56, 328-341. The first sign of a Met loosing its S-CH3 group will be a negative difference peak on the S.
-James Holton
MAD Scientist
----------
From: Garib N Murshudov
There may be several reasons.
1) Artefacts (some of them)
a) effect of mask: if there are large holes inside molecule and there should be no electron density (for example hydrophobic holes) then in older versions masks would put a constant density there and as a result difference map would have negative densities. This has been dealt with in the newer version
b) Effect of TLS refinement. If you are using TLS then you can try without TLS and compare densities. If it is effect of TLS then without TLS you should not see negative density. TLS model is a simple model assumes that all atoms in the group oscillates as a rigid group. Loops and other flexible parts may not obey this assumption
2) Genuine density
Alternative conformations (Met almost always are in more than one conformations). In these cases you may be able to see alternative conformation if you look at very low level of electron density. And you may be able to see if it is case by looking at the B values. If neigbouring atoms have large differences in B values then it may be because of alt conformation
Regards
Garib
Garib N Murshudov
----------
From: Ian Tickle
I assumed that since this topic came up fairly recently, in fact 3
weeks ago (see thread starting from
http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg23628.html), it
wasn't just a re-run of the same question.
Perhaps the original poster could clarify whether we are talking about
unexplained -ve density in the 2Fo-Fc map or in the Fo-Fc map?
Cheers
-- Ian
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