Re: [ccp4bb] negative density around disulfide bond

[Sampson, Jared]

Hi Eze -

I’m glad to see you solved your problem.

On Jun 2, 2014, at 9:07 PM, Eze Chivi 
mailto:ezech...@outlook.com.ar>> wrote:

Thanks to everyone for the useful information.
I refined my structure in the reduced form (using the method suggested by 
Pavel). Negative density disappeared, and also R/Rfree improved a little bit. I 
attach two example pictures.
In parallel, I mutated Cys residues to Ala and re-refined again adding the 
simulated annealing option in phenix (Is this the way to make an omit map?).

In the future, instead of mutating to Ala, you could try using the 
delete-residue-sidechain function, which prunes the side chain of the active 
residue back to CB.  The function is bound to capital letter K (as “Kill 
Sidechain”) in Paul’s key bindings: 
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Pauls-key-bindings-for-coot.
  That way, the sequence will remain correct, and until you add the atoms back 
into your model, you will be able to remind yourself about them via Extensions 
> Modeling > Residues with Missing Atoms.

Cheers,
Jared

Positive density appeared in the place of S atoms (another picture attached, 
the oxidized model is shown).
It appears that radiation damage caused full reduction...
Thank you again!
Regards
Ezequiel
PS: This protein was crystalized before by my group (and other group) in 
oxidized form (several times), it is a single point mutant. Data were collected 
at SLS using a Pilatus Detector.






--
Jared Sampson
Xiangpeng Kong Lab
NYU Langone Medical Center
http://kong.med.nyu.edu/

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.
=

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  (on behalf 
of Pavel Afonine )
>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
>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

[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  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

[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  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
>  
>
>
>
>
>
>
> *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

[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

 Original message From: Eze Chivi 
 Date:06/02/2014  1:08 AM  (GMT-05:00) 
To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] negative 
density around disulfide bond 
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

[Marjolein Thunnissen]

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 
mailto: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


[cid:3A2194AC-734C-45EB-B5A7-CE7FC0F0F1BC]




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

[William G. Scott]

On Jun 1, 2014, at 10:08 PM, Eze Chivi  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
> 

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