Re: [ccp4bb] Negative density

[Gerard Bricogne]

Dear Renu,

 Bernhard is probably on the right track. It is likely that your crystal
diffracted to substantially higher resolution than 1.7A, and that its
diffraction was sharply truncated by the edges of a detector placed too far.
If so, the sharp truncation in reciprocal space of the Fourier coefficients
going into your map calculation will cause "series termination" ripples in
your map in real space, that may well be the cause of what you see. 

 Remember that the average electron density in the unit cell is zero, so
that all the positive density visible as the chemically interpretable
features of the map is necessarily offset by negative density elsewhere,
although usually not sharply peaked.

 Can you share one of your diffraction images? If not, you could process
your dataset with autoPROC [1]: the STARANISO pictures that are included in
the summary.html file would immediately show you whether it is the case that
a strong diffraction pattern was truncated by the detector edges. 


 With best wishes,

  Gerard

[1] https://www.globalphasing.com/autoproc/

--
On Tue, Feb 22, 2022 at 11:56:50AM -0800, Bernhard Rupp wrote:
> Hmm…compliment, pretty darn good density map for 1.7 A….maybe look at the 
> resolution cut-off. 
> 
> If it is at a significant level, it could be truncation ripples?
> 
> Otherwise I second the noise opinion if it is already a well completed model.
> 
>  
> 
> Cheers, BR
> 
>  
> 
>  
> 
> From: CCP4 bulletin board  On Behalf Of S
> Sent: Monday, February 21, 2022 21:34
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Negative density
> 
>  
> 
> Hi All,
> 
>  
> 
> I am getting this negative density in the centre of the ring. Could you 
> please help me with this?
> 
>  
> 
>  
> 
> Resolution: 1.7A
> 
> 2FoFc - 1.5
> 
> FoFc - 3.0
> 
>  
> 
> Thanks in advance.
> 
> Regards,
> 
> Renu
> 
>  
> 
>   _  
> 
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Re: [ccp4bb] Negative density

[Bernhard Rupp]

Hmm…compliment, pretty darn good density map for 1.7 A….maybe look at the 
resolution cut-off. 

If it is at a significant level, it could be truncation ripples?

Otherwise I second the noise opinion if it is already a well completed model.

 

Cheers, BR

 

 

From: CCP4 bulletin board  On Behalf Of S
Sent: Monday, February 21, 2022 21:34
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Negative density

 

Hi All,

 

I am getting this negative density in the centre of the ring. Could you please 
help me with this?

 

 

Resolution: 1.7A

2FoFc - 1.5

FoFc - 3.0

 

Thanks in advance.

Regards,

Renu

 

  _  

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Re: [ccp4bb] Negative density

[Mark J. van Raaij]

or perhaps something in the non-visible part of the ligand pulling electrons 
out of the rings? can this somehow be specified as ligand input?
on the other hand, I guess this is near the "end" of refinement, so the Fo-Fc 
difference map is probably very flat - meaning that 3 sigma corresponds to very 
few electrons per cubic Å and may be called "noise".

> On 22 Feb 2022, at 13:01, Jon Cooper 
> <488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> I thought it was tryptophan but the nitrogen and extra bond are in the wrong 
> places so it must be a ligand and, if so, have you tried refining the 
> occupancy? Cheers, Jon.C.
> 
> 
> Sent from ProtonMail mobile
> 
> 
> 
>  Original Message 
> On 22 Feb 2022, 05:34, S < shine.star1...@gmail.com> wrote:
> 
> Hi All,
> 
> I am getting this negative density in the centre of the ring. Could you 
> please help me with this?
> 
> 
> Resolution: 1.7A
> 2FoFc - 1.5
> FoFc - 3.0
> 
> Thanks in advance.
> Regards,
> Renu
> 
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Negative density

[Jon Cooper]

I thought it was tryptophan but the nitrogen and extra bond are in the wrong 
places so it must be a ligand and, if so, have you tried refining the 
occupancy? Cheers, Jon.C.

Sent from ProtonMail mobile

 Original Message 
On 22 Feb 2022, 05:34, S wrote:

> Hi All,
>
> I am getting this negative density in the centre of the ring. Could you 
> please help me with this?
>
> Resolution: 1.7A
> 2FoFc - 1.5
> FoFc - 3.0
>
> Thanks in advance.
> Regards,
> Renu
>
> ---
>
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Re: [ccp4bb] negative density around disulfide bond

[William G. Scott]

On Jun 1, 2014, at 10:08 PM, Eze Chivi ezech...@outlook.com.ar wrote:

 Hello, when I refine my structure, I see negative density around the 
 disulfide bond. I have 7 copies per ASU, and I can see this density in many 
 of them. In some cases, I see positive density also (negative in the center 
 of the straight line linking S atoms, and positive in both sides). What can I 
 try to solve it? Is it due to radiation damage? Alternative conformation 
 (partial oxidation)? Incorrect disulfide geometry parameters? My resolution 
 is 2.1 A, R/Rfree are around 0.220/0.243, and similar results with refmac5, 
 phenix and PDBREDO. Please find two example pictures in attachment.
 Thanks for your help!
 
 
 Ezequiel
 disulfide.jpgdisulfide2.jpg

Dear Ezequiel:

I apologize if I missed some crucial fact, but the simplest naive explanation 
would be the disulfide bonds became reduced somewhere along the line, since 
that would push the non-bonded sulfur atoms apart, relative to where you have 
them. You should be able to tell with a denaturing gel (run in the absence of 
DTT or equivalent).  I’ve found a bit of Cu(II)-1,10-phenanthroline can help 
keep the disulfides oxidized. Try re-refining without the disulfide linkages, 
and see if the difference densities go away.

Bill

Re: [ccp4bb] negative density around disulfide bond

[Ed Pozharski]

Try refining without disulfide bond - from the way density looks it might work. 
 Whether this is what happens in vivo is a different question entirely. 


Sent on a Sprint Samsung Galaxy S® III

div Original message /divdivFrom: Eze Chivi 
ezech...@outlook.com.ar /divdivDate:06/02/2014  1:08 AM  (GMT-05:00) 
/divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] negative 
density around disulfide bond /divdiv
/divHello, when I refine my structure, I see negative density around the 
disulfide bond. I have 7 copies per ASU, and I can see this density in many of 
them. In some cases, I see positive density also (negative in the center of the 
straight line linking S atoms, and positive in both sides). What can I try to 
solve it? Is it due to radiation damage? Alternative conformation (partial 
oxidation)? Incorrect disulfide geometry parameters? My resolution is 2.1 A, 
R/Rfree are around 0.220/0.243, and similar results with refmac5, phenix and 
PDBREDO. Please find two example pictures in attachment.
Thanks for your help!


Ezequiel

Re: [ccp4bb] negative density around disulfide bond

[Eleanor Dodson]

I have seen similar features fairly often when the data collection has been
pushed to the limit.
The theory is that it is due to radiation damage - you could check by only
merging say  the first 50% of your data, then seeing if the di-sulphide is
intact in those maps. ( wouldnt re-refine much - just set CB-SG occs to
0.00 - recalculate SFs and check the maps)
Eleanor




On 2 June 2014 07:44, Marjolein Thunnissen 
marjolein.thunnis...@biochemistry.lu.se wrote:

  Hi,

  I would guess radiation damage, see Weik et al., 2000, PNAS 97, 623-628
 or for a more recent discussion and thorough overview Sutton et al., 2013,
 Acta Cryst D69, 2381-2394.

  best regards

  Marjolein


  On 02 Jun 2014, at 07:08, Eze Chivi ezech...@outlook.com.ar wrote:

   Hello, when I refine my structure, I see negative density around the
 disulfide bond. I have 7 copies per ASU, and I can see this density in many
 of them. In some cases, I see positive density also (negative in the center
 of the straight line linking S atoms, and positive in both sides). What can
 I try to solve it? Is it due to radiation damage? Alternative conformation
 (partial oxidation)? Incorrect disulfide geometry parameters? My resolution
 is 2.1 A, R/Rfree are around 0.220/0.243, and similar results with refmac5,
 phenix and PDBREDO. Please find two example pictures in attachment.
 Thanks for your help!


 Ezequiel
  disulfide.jpgdisulfide2.jpg






 *Marjolein Thunnissen*
  Science Coordinator MX

 MAX IV Laboratory
 Lund University
 P.O. Box 118, SE-221 00 Lund, Sweden
 Visiting address: Ole Römers väg 1, 223 63 Lund
  Telephone: +46 766 32 04 17
  www.maxlab.lu.se

Re: [ccp4bb] negative density around disulfide bond

[Pavel Afonine]

Ezequiel,

since you mentioned you tried Phenix too:

in Phenix you can remove a particular disulfide bond by using a parameter,
for example:

disulfide_bond_exclusions_selection_string=(chain A and resseq 1 and name
SG) or (chain B and resseq 10 and name SG)

This works in the command line and GUI. Please let me know (off-list) if
you any help with this.

Pavel


On Sun, Jun 1, 2014 at 10:08 PM, Eze Chivi ezech...@outlook.com.ar wrote:

  Hello, when I refine my structure, I see negative density around the
 disulfide bond. I have 7 copies per ASU, and I can see this density in many
 of them. In some cases, I see positive density also (negative in the center
 of the straight line linking S atoms, and positive in both sides). What can
 I try to solve it? Is it due to radiation damage? Alternative conformation
 (partial oxidation)? Incorrect disulfide geometry parameters? My resolution
 is 2.1 A, R/Rfree are around 0.220/0.243, and similar results with refmac5,
 phenix and PDBREDO. Please find two example pictures in attachment.
 Thanks for your help!


 Ezequiel

Re: [ccp4bb] negative density around disulfide bond

[Reza Khayat]

What does an omit map look like, if you omit both cysteines?

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org


 Original message 
Date: Mon, 2 Jun 2014 09:31:28 -0700
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf 
of Pavel Afonine pafon...@gmail.com)
Subject: Re: [ccp4bb] negative density around disulfide bond  
To: CCP4BB@JISCMAIL.AC.UK

   Ezequiel,
   since you mentioned you tried Phenix too:
   in Phenix you can remove a particular disulfide bond
   by using a parameter, for**example:

   disulfide_bond_exclusions_selection_string=(chain A
   and resseq 1 and name SG) or (chain B and resseq 10
   and name SG)

   This works in the command line and GUI. Please let
   me know (off-list) if you any help with this.

   Pavel

   On Sun, Jun 1, 2014 at 10:08 PM, Eze Chivi
   ezech...@outlook.com.ar wrote:

 Hello, when I refine my structure, I see negative
 density around the disulfide bond. I have 7 copies
 per ASU, and I can see this density in many of
 them. In some cases, I see positive density also
 (negative in the center of the straight line
 linking S atoms, and positive in both sides). What
 can I try to solve it? Is it due to radiation
 damage? Alternative conformation (partial
 oxidation)? Incorrect disulfide geometry
 parameters? My resolution is 2.1 A, R/Rfree are
 around 0.220/0.243, and similar results with
 refmac5, phenix and PDBREDO. Please find two
 example pictures in attachment.
 Thanks for your help!

 Ezequiel

Re: [ccp4bb] negative density at some places in the side chain of residues

[Eleanor Dodson]

What I would do - not net ideal!

1) the ASP looks in the wrong place - shift it into green density and one
OE is probably a water..
2) MET notoriously hard to model - I suspect there often are multiple
conformations.. And of course at some wave lengths the S contribution
should be down weighted by f'  - the default is to use the S scattering for
CuKa wavelength..

3) I think you need to flip the GLY O and then refit the adjacent residues
4) SER is often in 2 conformations - I suspect that is the case here..
Eleanor

On 26 April 2012 22:03, Antony Oliver antony.oli...@sussex.ac.uk wrote:

 Alaksa,

 1) What rmsd / sigma are you contouring your density at ? i.e. are you
 down in the noise or are you at a reasonable value for your Fo-Fc map?

 2) It looks like some of your side-chains appear to have more than one
 conformation - it's fairly easy in Coot to position and model both.

 Tony.

 ---
 Mobile Account
 ---

 On 26 Apr 2012, at 21:55, Alaksa xtal.cc...@gmail.com wrote:

 
  Dear all
  I am refining the crystal structure of a protein (Rfree and Rvalue are
 25 and 20 A respectively). However, I am getting the negative density at
 some places in the side chain of residues. All side chains are properly
 fitting into the blue density, however red density blobs are also present
 at the same place along with blue density . At some other place this red
 density is also present in the main chain along with blue density (see the
 attached snaps). If I have mutate the residues to alanine then density
 becomes blue, but when change into the original residue, after refmac5
 again it is showing red blob. Also if I rotate the chain to place it in
 green density, but after running refmac it attain original position having
 red blob. I am not using TLS.
  I am seeking some strategy so that the problem can be solved. Please
 suggest me the possible reasons and remedy. Also i am naive in
 crystallography.
 
  Thanks
  Alaksa
 
 
  coot2.png
  coot1.png
  coot3.png
  coot4.png

Re: [ccp4bb] negative density at some places in the side chain of residues

[Antony Oliver]

Alaksa,

1) What rmsd / sigma are you contouring your density at ? i.e. are you down in 
the noise or are you at a reasonable value for your Fo-Fc map?

2) It looks like some of your side-chains appear to have more than one 
conformation - it's fairly easy in Coot to position and model both. 

Tony. 

---
Mobile Account
---

On 26 Apr 2012, at 21:55, Alaksa xtal.cc...@gmail.com wrote:

 
 Dear all
 I am refining the crystal structure of a protein (Rfree and Rvalue are 25 
 and 20 A respectively). However, I am getting the negative density at some 
 places in the side chain of residues. All side chains are properly fitting 
 into the blue density, however red density blobs are also present at the same 
 place along with blue density . At some other place this red density is also 
 present in the main chain along with blue density (see the attached snaps). 
 If I have mutate the residues to alanine then density becomes blue, but when 
 change into the original residue, after refmac5 again it is showing red blob. 
 Also if I rotate the chain to place it in green density, but after running 
 refmac it attain original position having red blob. I am not using TLS.
 I am seeking some strategy so that the problem can be solved. Please suggest 
 me the possible reasons and remedy. Also i am naive in crystallography.
 
 Thanks
 Alaksa
 
 
 coot2.png
 coot1.png
 coot3.png
 coot4.png

Re: [ccp4bb] negative density in difference map [SEC=UNCLASSIFIED]

[DUFF, Anthony]

Delete (set occupancies to zero) the side chain back to CA.  Do a few rounds of 
refinement and calculate Fo-Fc and examine.



It is possible that the side chain is disordered, or ordered in multiple 
conformations.  Compare the density for alternate confonformers against the 
density for CA.



Alternatively, your side chain B-factor restaints might be too tight.



Anthony






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Careina Edgooms 
[careinaedgo...@yahoo.com]
Sent: Wednesday, 23 November 2011 6:54 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] negative density in difference map

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

Re: [ccp4bb] negative density in difference map [SEC=UNCLASSIFIED]

[Eleanor Dodson]

I wish I could answer this!
One possibility is that the side-chain B values are too tightly 
restrained - Ian Tickle recommends releasing these somewhat..


Here are the default refmac values.
THERMAL FACTORS
  Weight= 1.00
 Main chain bond (1-2 neighbour)  1.5A**2
 Main chain angle (1-3 neighbour) 2.0A**2
 Side chain bond  3.0A**2
 Side chain angle 4.5A**2

and you could change them say to:


Under GUI - check geometric parameters and alter the Bfactor values

data line becomes:  temp 1.0 1.5 2.0  4.0 6.0

The trouble is that resyraints for ARG or MET say should be looser than 
those for SER or VAL.


But I often finish up with some inexplicable red blobs - sometimes 
floating in space..

Eleanor


On 11/23/2011 08:39 AM, DUFF, Anthony wrote:



Delete (set occupancies to zero) the side chain back to CA.  Do a few rounds of 
refinement and calculate Fo-Fc and examine.







It is possible that the side chain is disordered, or ordered in multiple 
conformations.  Compare the density for alternate confonformers against the 
density for CA.



Alternatively, your side chain B-factor restaints might be too tight.



Anthony






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Careina Edgooms 
[careinaedgo...@yahoo.com]
Sent: Wednesday, 23 November 2011 6:54 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] negative density in difference map

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

Re: [ccp4bb] negative density in difference map [SEC=UNCLASSIFIED]

[Ian Tickle]

Hi Careina

Since my name came up I felt compelled to comment, though I don't have
a definitive answer.  Over-restraining of B factors is certainly a
possibility and could well explain otherwise inexplicable difference
density.  IMO B factors are generally over-restrained.  They are a bit
like the R factors: tight restraints keep the B factors low and people
(referees in particular!) seem to feel happier when they see low
values, even though that is not necessarily optimal (this mind-set is
of course known as wishful thinking - see Wikipedia article).

Personally I never restrain 1-3 B factors, and only use the 1-2
restraints, and even then I relax those from the default values.  The
1-3 B restraints are clearly not independent of the 1-2 ones, and
restraints should ideally be independent of each other, otherwise you
are applying them more than once,  IMO the overall B factor restraint
weight should be optimised in the same way that the X-ray weight
should be, by minimising Rfree or -LLfree.  X-plor, CNS (and I would
guess phenix.refine) have had this option for many years
(optimize_rweight.inp in CNS), and it seems an excellent thing to do,

Disorder is also an obvious possibility as Anthony said, and I would
follow his sensible advice.

Cheers

-- Ian

Cheers

-- Ian

On 23 November 2011 10:58, Eleanor Dodson c...@ysbl.york.ac.uk wrote:
 I wish I could answer this!
 One possibility is that the side-chain B values are too tightly restrained -
 Ian Tickle recommends releasing these somewhat..

 Here are the default refmac values.
 THERMAL FACTORS
          Weight= 1.00
     Main chain bond (1-2 neighbour)          1.5A**2
     Main chain angle (1-3 neighbour)         2.0A**2
     Side chain bond                          3.0A**2
     Side chain angle                         4.5A**2

 and you could change them say to:


 Under GUI - check geometric parameters and alter the Bfactor values

 data line becomes:  temp 1.0     1.5 2.0  4.0 6.0

 The trouble is that resyraints for ARG or MET say should be looser than
 those for SER or VAL.

 But I often finish up with some inexplicable red blobs - sometimes floating
 in space..
 Eleanor


 On 11/23/2011 08:39 AM, DUFF, Anthony wrote:


 Delete (set occupancies to zero) the side chain back to CA.  Do a few
 rounds of refinement and calculate Fo-Fc and examine.





 It is possible that the side chain is disordered, or ordered in multiple
 conformations.  Compare the density for alternate confonformers against the
 density for CA.



 Alternatively, your side chain B-factor restaints might be too tight.



 Anthony





 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Careina
 Edgooms [careinaedgo...@yahoo.com]
 Sent: Wednesday, 23 November 2011 6:54 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] negative density in difference map

 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

Re: [ccp4bb] negative density in difference map

[Nat Echols]

On Tue, Nov 22, 2011 at 11:54 PM, Careina Edgooms
careinaedgo...@yahoo.com wrote:
 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?

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

Re: [ccp4bb] negative density in difference map

[Ian Tickle]

On 23 November 2011 07:54, Careina Edgooms careinaedgo...@yahoo.com wrote:
 I have a question about a 2F0-Fc difference map that I calculated with Refmac.

On 23 November 2011 15:40, Nat Echols nathaniel.ech...@gmail.com 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

Re: [ccp4bb] negative density in difference map

[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

On 11/22/2011 11:54 PM, Careina Edgooms wrote:

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

Re: [ccp4bb] negative density in difference map

[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

On 23 Nov 2011, at 07:54, Careina Edgooms wrote:

 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

Garib N Murshudov 
Structural Studies Division
MRC Laboratory of Molecular Biology
Hills Road 
Cambridge 
CB2 0QH UK
Email: ga...@mrc-lmb.cam.ac.uk 
Web http://www.mrc-lmb.cam.ac.uk

Re: [ccp4bb] negative density in difference map

[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

On 23 November 2011 16:03, Nat Echols nathaniel.ech...@gmail.com wrote:
 On Wed, Nov 23, 2011 at 7:57 AM, Ian Tickle ianj...@gmail.com wrote:
 On 23 November 2011 07:54, Careina Edgooms careinaedgo...@yahoo.com wrote:
 I have a question about a 2F0-Fc difference map that I calculated with 
 Refmac.

 On 23 November 2011 15:40, Nat Echols nathaniel.ech...@gmail.com 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.

 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

[ccp4bb] negative density in difference map

[Careina Edgooms]

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

Re: [ccp4bb] negative density peaks where there is no model.

[Edward A. Berry]

Yes, I've seen this behavior. I attribute it to lack of the 0,0,0 and ultra-low
resolution reflections. Remember that the average density in a map (with 
difference
coefficients or whatever) is zero. That means that if the model lacks a few 
atoms,
but has no atoms that don't belong, so that an absolute difference map would go
from zero to some positive value, The calculated map would go from some negative
value to a lower positive value. Thus a point where there is no atom and should
be no atom , with a value of 0-0 = 0, may be the lowest point in the map and 
come
out negative in the AC-coupled map.

That still wouldn't account for *significant* negative peaks, since the positive
density peaks are few and the near-zero points are many, the baseline will not
drop much to float the peaks. However if low-resolution data is missing, the
density profile is being put through a hi-frequency filter, so the whole 
baseline
is not shifted down equally, but rather the baseline in the region of the peaks
sinks under the weight of the peaks. So, out of all the points in the absolute
map that were near zero, a few points in the vicinity of the highest positive
peaks may end up lower than all the rest and appear significant.

Also, if you are judging significant by the old 3-sigma rule, remember that as you 
improve your model the difference map tends to zero everywhere, and the absolute

value associated with 3 sigma gets smaller and smaller. Much better to look at
difference maps in terms of absolute density (e-/A^3; the default now in O), or
compare the sigma value of the negative peaks with that of the positive peak
produced by omitting a well-ordered water. Then they may not seem so 
significant!

This explanation is not completely satisfactory, so if anyone has a better one
I'm eager to hear it.

Ed

Waight, Andrew wrote:

Hello everyone I have a question for the experts.

 I am in the final stages of refining my model placing waters and whatnot. However when I refine my model against the pure scalepack output, I see some rather signifigant negative difference density peaks (3sigma) in a marginally important region of the protein. The strange thing is that there is no model built into this region. How can there be negative difference density when there is no model built there? It is obviously not a weighting problem because the structure factors are directly from the raw reflection list. Has anyone else ecperienced this phenomenon and what if any actions would you suggest. I have attached also a screenshot. Thanks for your advice everyone I have learned so much from reading this BB everyday. 


  
Drew


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Re: [ccp4bb] negative density peaks where there is no model.

[Joe Cockburn]

Hi Andrew,
If there is no *protein* model built into this region of the map, then it
will be modelled by the bulk solvent correction - therefore, the negative
peaks are telling you that the mean electron density there is lower than
in the bulk solvent. Probably.
Joe


 Hello everyone I have a question for the experts.

  I am in the final stages of refining my model
 placing waters and whatnot. However when I refine
 my model against the pure scalepack output, I see
 some rather signifigant negative difference
 density peaks (3sigma) in a marginally important
 region of the protein. The strange thing is that
 there is no model built into this region. How can
 there be negative difference density when there
 is no model built there? It is obviously not a
 weighting problem because the structure factors
 are directly from the raw reflection list. Has
 anyone else ecperienced this phenomenon and what
 if any actions would you suggest. I have attached
 also a screenshot. Thanks for your advice
 everyone I have learned so much from reading this
 BB everyday.

   
 Drew

 
 This email message, including any attachments, is for the sole use of the
 intended recipient(s) and may contain information that is proprietary,
 confidential, and exempt from disclosure under applicable law. Any
 unauthorized review, use, disclosure, or distribution is prohibited. If
 you have received this email in error please notify the sender by return
 email and delete the original message. Please note, the recipient should
 check this email and any attachments for the presence of viruses. The
 organization accepts no liability for any damage caused by any virus
 transmitted by this email.
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Re: [ccp4bb] Negative density around C of COO-

[Nave, C (Colin)]

 Hi
Just for completeness (seeing as we are discussing scattering factors
and radiation damage) there is an increased susceptibility to primary
damage for sulphur due to the increased absorption which follows f'',
tabulated for example in
http://henke.lbl.gov/optical_constants/asf.html. The x-ray absorption of
the sample is related to these.

f''  for nitrogen is 0.034 at 6keV and 0.007 12keV 
f'' for sulphur is 0.936 at 6keV and 0.258 12keV 

Much greater absorption for sulphur and much more likely to be damaged
by direct absorption of a photon.

However this direct primary damage is not the main factor. Each absorbed
photon produces a high energy photoelectron (ejected from the absorbing
atom). This photoelectron gradually loses energy as it travels through
the sample, creating several hundred sites of secondary damage. These
damaged sites are the main effect, rather divorced from the initial
absorption site. Weak bonds are more likely to be disrupted due to these
inelastic interactions with the photo-electron. Even then, one will not
see an effect (by x-ray diffraction) unless the atom has some freedom
(at approx 100K) to move within its local environment.  This is more
likely to happen on the outside of the protein rather than in the
interior. 

Cheers
   Colin


-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Ian Tickle
Sent: 06 May 2008 11:26
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Negative density around C of COO-

Hi, I think you'll find that if you work out the contribution from f' to
the density, in most cases it's not significant relative to the RMSD.
This will be particularly true for S which of course has a low value of
f' for all commonly used wavelengths.  This must be true otherwise we
would observe these effects routinely.  The explanation must involve
some effect which is peculiar to the crystal being studied, as opposed
to being peculiar to the wavelength, and radiation damage seems the most
likely explanation (and has been unequivocally demonstrated in a number
of cases).

-- Ian 

 -Original Message-
 From: [EMAIL PROTECTED]
 [mailto:[EMAIL PROTECTED] On Behalf Of Lijun Liu
 Sent: 05 May 2008 21:37
 To: James Holton
 Cc: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Negative density around C of COO-
 
 I believe some, may not be all, negative density may be due to the 
 choice of wavelength.  C,O and N are not so sensitive to normally used

 wavelength (1-1.54A), but S does.  This is more true when you have 
 higher resolution and better quality data.  You can estimate this by 
 f'.
 
 By the way, although I am not a real supporter of radicals from 
 damage, I believe that a photo-induced (here X-ray) chemical reaction

 is much different from a heat-induced one.
 Low temperature is not a problem.  But this not for diffusion, as it 
 is ~100% heat-related.  Please point out if I am wrong.
 
   This assignment of free radicals to damage is often made 
 (flippantly) in the literature, but I feel a strong need to point out 
 that there is NO EVIDENCE of a free radical diffusion mechanism for 
 radiation damage below ~130K.
 
 
 Lijun Liu, PhD
 Institute of Molecular Biology
 HHMI  Department of Physics
 University of Oregon
 Eugene, OR 97403
 541-346-4080
 
 
 


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Re: [ccp4bb] Negative density around C of COO-

[James Holton]

Indeed there are radicals, but they are not free.

Perhaps a better terminology for them would be fixed radicals.  
Species such as the disulphide radical have been well established by 
spectrophotometric and other evidence (M. Weik et. al. and others).  It 
is quite remarkable actually how long-lived different radical species 
can be a ~100K.  The most extensive literature on radical states at low 
temperature is the ESR field.  Much of it is quite old, but still 
accessible in textbooks, such as Box (1977).


Still, I think my best answer to your first question (Bart) is that 
cryo-cooled samples are solids, and solid-state chemistry is different 
from liquid-state chemistry.  This fact is further supported by 
observations of the effects of so-called radical scavengers under 
cryo.  Some of these still work in the cold, others do not, and recently 
it has been reported (In a talk at RD5) that some scavengers can be 
effective against damage to one part of the protein, but not others, 
even for chemically identical sites.  Indeed, chemically identical sites 
in the same protein are often seen to decay at wildly different rates 
with or without scavengers.  Since two disulphide bonds have exactly the 
same likelihood of interacting directly with an x-ray photon (called the 
x-ray absorption cross section), these different decay rates cannot be 
explained this way.  It can also not be explained by solvent 
accessibility as first pointed out in Burmeister (2000). 

So, basically, the model that liquid-phase chemistry is still going on 
under cryo is inconsistent with almost every observation made in 
cryo-cooled samples.  The kinetics in particular make no sense at all 
(first pointed out by R. Thorne, and also detailed in Holton, JSR 
2007).  The only thing solid-state and liquid-state radiation damage 
have in common is that some of the chemical consequences (such as 
disulphide breakage) are similar.


The jumping radicals you describe actually fall very nicely into the 
quasiparticle category I was talking about.  That is: migration of a 
chemical state with no mass transport.  Believe it or not, the problem 
right now is that there are so many quasiparticle mechanisms that can do 
this we still aren't sure which one is involved.  The only thing that is 
fairly certain is that molecular diffusion is not. 

Most of what we know about solid-state chemistry comes from the 
semiconductor field, where the reactions are VERY well understood.  
Unfortunately, that literature is rather opaque to biologists who aren't 
working at a synchrotron. ;)  The most well-studied quasiparticles in 
silicon are the so-called electrons and holes.  The electrons 
aren't really electrons, but rather a charged excited state of a silicon 
atom in  the lattice.  I will admit that the term diffusion is used in 
this field.  This is largely because these jumping quasiparticle 
entities moving about a silicon crystal lattice actually obey the ideal 
gas law!  However, since these are quasiparticles and not real 
particles, using the term diffusion gives biochemists the wrong 
impression of the mechanism.


I know the primary and secondary damage formalism, but I think the 
problem these days is that I, for one, am fairly convinced that noone is 
ever going to be able to observe primary damage.  The effects we 
classify as secondary damage outnumber these reactions by thousands to 
one.  Colin Nave just described this in his posting, so I won't repeat 
it here, but the primary consequence of any ionizing radiation is to put 
a whole bunch of atoms into chemically excited states.  How this 
chemical energy dissipates depends on the structure of the material, and 
this makes it hard to predict if you don't know the structure.


-James


Bart Hazes wrote:

Hi James,

We used to talk about primary and secondary radiation damage. The 
former operates at room temperature where free radicals were said to 
be formed in solution and diffuse around to damage proteins. Under 
cryoconditions this no longer happens, leading to greatly improved 
crystal life time, but we still have primary radiation damage, with 
the photons directly hitting the protein.


It was my understanding that this was still considered to form a free 
radical at the affected atom without there being any diffusion 
involved. Sulfur atoms would be more sensitive as they have a larger 
X-ray cross-section or because they may act as free-radical sinks 
where free radicals generated nearby strip an electron from the 
sulfur, thereby satisfying their own electronic configuration and 
converting the sulfur into a radical state. For instance, in 
ribonucleotide reductase a tyrosine free radical is formed 
spontaneously (using oxygen and an dinuclear iron site) and jumps 
over 20 Angstrom from one subunit to another to form a thiyl free 
radical in the active site. It then jumps back to the tyrosine upon 
completion of the catalytic cycle. Although we don't know how it 
jumps, 

[ccp4bb] Negative density around C of COO-

[Narayanan Ramasubbu]

Dear all:
I am noticing that in some of my structures, at 1.5 A resolution, 1) 
there is some negative density around the C of the carboxyl groups.
2) I also notice the negative density around S in a disulfide region. It 
is as though the disulfide is broken.


Could some body enlighten me on this feature?

Re: [ccp4bb] Negative density around C of COO-

[Kevin P Madauss]

I would suspect radiation damage for the S density .  I have not seen 
radiation damage of only COO carbons, but then again, I do not routinely 
get such good diffraction. 





Kevin P Madauss  V-161a
US Structural Biology   GlaxoSmithKline 
Office:  919.483.3759  5 Moore Drive 
Cell:  919.924.6436  RTP, NC 27709
 

Re: [ccp4bb] Negative density around C of COO-

[Roger Rowlett]

Narayanan Ramasubbu wrote:

Dear all:
I am noticing that in some of my structures, at 1.5 A resolution, 1)
there is some negative density around the C of the carboxyl groups.
2) I also notice the negative density around S in a disulfide region. It
is as though the disulfide is broken.

Could some body enlighten me on this feature?
  
Synchrotron radiation can cause free radical mediated damage to 
proteins. The most frequently observed consequences are decarboxlation 
of Asp and Glu, and the breakage of disulfide bonds. See, for example, 
Wiek et al. (2000) PNAS 97, 623-628, and Leiros et al. (2001) Acta 
Cryst. D 57, 488-497.


Cheers,


--

Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [EMAIL PROTECTED]

Re: [ccp4bb] Negative density around C of COO-

[James Holton]

I don't mean to single anyone out, but the assignment of free radicals 
as the species mediating radiation damage at cryo temperatures is a pet 
peeve of mine.  Free radicals have been shown to mediate damage at room 
temperature (and there is a VERY large body of literature on this), but 
there are a great many good reasons to think that free radicals do NOT 
play a role in radiation damage under cryo.


This assignment of free radicals to damage is often made (flippantly) 
in the literature, but I feel a strong need to point out that there is 
NO EVIDENCE of a free radical diffusion mechanism for radiation damage 
below ~130K.  To the contrary there is a great deal of evidence that 
water, buffers and protein crystals below ~130 K are in a state of 
matter known as a solid, and molecules (such as free radicals) do not 
diffuse through solids (except on geological timescales).  If you are 
worried that the x-ray beam is heating your crystal to 130 K, then have 
a look at Snell et. al. JSR 14 109-15 (2007).  They showed quite 
convincingly that this just can't happen for anything but the most 
exotic situations.


There is evidence, however, of energy transfer taking place between 
different regions of the crystal, but energy transfer does not require 
molecular diffusion or any other kind of mass transport.  In fact, 
solid-state chemistry is generally mediated by cascading 
neighbor-to-neighbor reactions that do not involve diffusion in the 
traditional sense.  Electricity is an example of this kind of chemistry, 
and these reactions are a LOT faster than diffusion.  The closest 
analogy to diffusion is that the propagating reaction can be seen as a 
species of sorts that is moving around inside the sample.  Entities 
like this are formally called quasiparticles.  Some quasiparticles are 
charged, but others are not.  If you don't know what a quasiparticle is, 
you can look them up in wikipedia. 

Some have tried to rescue the free radical statements about radiation 
damage by claiming that individual electrons are radicals.  I guess 
this must come from the pressure of such a large body of free-radical 
literature at room temperature.  However, IMHO this is about as useful 
as declaring that every chemical reaction is a free radical reaction 
(since they involve the movement of electrons).   I think it best that 
we try to call the chemistry what it is and try to stamp out rumors that 
mechanisms are known when in reality they are not.


Just my little rant.

-James Holton
MAD Scientist

Re: [ccp4bb] Negative density around C of COO-

[Lijun Liu]

I believe some, may not be all, negative density may be due to the  
choice of wavelength.  C,O and N are not so sensitive to normally  
used wavelength (1-1.54A), but S does.  This is more true when you  
have higher resolution and better quality data.  You can estimate  
this by f'.


By the way, although I am not a real supporter of radicals from  
damage, I believe that a photo-induced (here X-ray) chemical  
reaction is much different from a heat-induced one.  Low temperature  
is not a problem.  But this not for diffusion, as it is ~100% heat- 
related.  Please point out if I am wrong.
This assignment of free radicals to damage is often made  
(flippantly) in the literature, but I feel a strong need to point  
out that there is NO EVIDENCE of a free radical diffusion mechanism  
for radiation damage below ~130K.


Lijun Liu, PhD
Institute of Molecular Biology
HHMI  Department of Physics
University of Oregon
Eugene, OR 97403
541-346-4080

Re: [ccp4bb] Negative density around C of COO-

[Bart Hazes]

Hi James,

We used to talk about primary and secondary radiation damage. The former 
operates at room temperature where free radicals were said to be formed 
in solution and diffuse around to damage proteins. Under cryoconditions 
this no longer happens, leading to greatly improved crystal life time, 
but we still have primary radiation damage, with the photons directly 
hitting the protein.


It was my understanding that this was still considered to form a free 
radical at the affected atom without there being any diffusion involved. 
Sulfur atoms would be more sensitive as they have a larger X-ray 
cross-section or because they may act as free-radical sinks where free 
radicals generated nearby strip an electron from the sulfur, thereby 
satisfying their own electronic configuration and converting the sulfur 
into a radical state. For instance, in ribonucleotide reductase a 
tyrosine free radical is formed spontaneously (using oxygen and an 
dinuclear iron site) and jumps over 20 Angstrom from one subunit to 
another to form a thiyl free radical in the active site. It then jumps 
back to the tyrosine upon completion of the catalytic cycle. Although we 
don't know how it jumps, certainly not by diffusion, there is general 
agreement that it does happen.


People have also observed broken disulfides in cryocrystal structures 
with the sulfurs at a distance that is too long for a disulfide but too 
short for a normal non-bonded sulfur-sulfur interaction. I seem to 
remember that this distance was suggested to indicate the presence of a 
thiyl free radical. I'm no chemist of physicist so can't evaluate if 
that claim is reasonable but if it is then that would be direct evidence 
to support the involvement of a free radical state in radiation damage.


So I guess my questions/comments are
- what are the great many good reasons to think that free radicals do 
NOT play a role in radiation damage under cryo.
- although diffusion does not happen below 130K, radicals do appear to 
teleport, at least over short distances.


Bart

James Holton wrote:
I don't mean to single anyone out, but the assignment of free radicals 
as the species mediating radiation damage at cryo temperatures is a pet 
peeve of mine.  Free radicals have been shown to mediate damage at room 
temperature (and there is a VERY large body of literature on this), but 
there are a great many good reasons to think that free radicals do NOT 
play a role in radiation damage under cryo.


This assignment of free radicals to damage is often made (flippantly) 
in the literature, but I feel a strong need to point out that there is 
NO EVIDENCE of a free radical diffusion mechanism for radiation damage 
below ~130K.  To the contrary there is a great deal of evidence that 
water, buffers and protein crystals below ~130 K are in a state of 
matter known as a solid, and molecules (such as free radicals) do not 
diffuse through solids (except on geological timescales).  If you are 
worried that the x-ray beam is heating your crystal to 130 K, then have 
a look at Snell et. al. JSR 14 109-15 (2007).  They showed quite 
convincingly that this just can't happen for anything but the most 
exotic situations.


There is evidence, however, of energy transfer taking place between 
different regions of the crystal, but energy transfer does not require 
molecular diffusion or any other kind of mass transport.  In fact, 
solid-state chemistry is generally mediated by cascading 
neighbor-to-neighbor reactions that do not involve diffusion in the 
traditional sense.  Electricity is an example of this kind of chemistry, 
and these reactions are a LOT faster than diffusion.  The closest 
analogy to diffusion is that the propagating reaction can be seen as a 
species of sorts that is moving around inside the sample.  Entities 
like this are formally called quasiparticles.  Some quasiparticles are 
charged, but others are not.  If you don't know what a quasiparticle is, 
you can look them up in wikipedia.
Some have tried to rescue the free radical statements about radiation 
damage by claiming that individual electrons are radicals.  I guess 
this must come from the pressure of such a large body of free-radical 
literature at room temperature.  However, IMHO this is about as useful 
as declaring that every chemical reaction is a free radical reaction 
(since they involve the movement of electrons).   I think it best that 
we try to call the chemistry what it is and try to stamp out rumors that 
mechanisms are known when in reality they are not.


Just my little rant.

-James Holton
MAD Scientist





--

==

Bart Hazes (Assistant Professor)
Dept. of Medical Microbiology  Immunology
University of Alberta
1-15 Medical Sciences Building
Edmonton, Alberta
Canada, T6G 2H7
phone:  1-780-492-0042
fax:1-780-492-7521

==