Re: [ccp4bb] Cys modification - deciding between CSS and SMC

[Briggs, David C]

Hi Mohamed,

Did you try CSO or CSD?

I've seen oxidation of Cys residues in the active site of Cysteine-dependent 
Phosphatases before.

In addition, oxidation is more easily explainable than CSS and SMC, especially 
if reducing agents were omitted from final purification steps.

HTH,

Dave

--
Dr David C Briggs
Hohenester Lab
Department of Life Sciences
Imperial College London
UK
http://about.me/david_briggs



From: Mohamed Noor
Sent: Sunday, 21 May, 13:00
Subject: [ccp4bb] Cys modification - deciding between CSS and SMC
To: ccp4bb@jiscmail.ac.uk


I am working on a model at a resolution of 2.1 A. The active site Cys in both 
copies have a positive density towards the S end of the residue and these blobs 
are there in FEM and Polder maps. When I replace these residues with either CSS 
or SMC, I get the following statistics. What is the best way of deciding which 
one is correct? CSS: R/Rfree - 0.2016/0.2299 Local CC (chain A/B) - 0.950/0.937 
Bfactor (chain A N/CA/C/O/CB/SG/SD) - 42.8/48.6/46.7/42.8/57.3/66.2/98.8 
Bfactor (chain B N/CA/C/O/CB/SG/SD) -42.0/44.5/47.2/45.7/57/60.3/95.3 No 
residual positive density left. SMC: R/Rfree - 0.2087/0.2299 Local CC (chain 
A/B) - 0.903/0.886 Bfactor (chain A N/CA/C/O/CB/SG/CS) - 
43.9/47.9/46.5/42.1/51.8/64.4/66.5 Bfactor (chain A N/CA/C/O/CB/SG/CS) - 
43.4/45.0/47.3/45.4/51.8/59.4/66.1 Crescent shaped positive density left 
towards the CS atom end. Although CSS seems more plausible, the high B factors 
of SD make me wonder if they are correct. Thanks. Mohamed

[ccp4bb]

[Qingfeng Chen]

Thanks for the suggestion about using NCS constraint to apply change on one
subunit to other subunits. That should make life much easier.

About the problem of phenix realspace refinement, I figured it out. I used
GUI and when I checked the log file, the "ramachandran_restraints" was
turned off. It is odd since there does not seem to have any buttons to turn
"ramachandran_restraints" off in GUI. Then I tried command line, the
Ramachandron statistics look normal now ("ramachandran_restraints" is on in
log file).

Thanks.

On Fri, May 19, 2017 at 11:01 AM, Pavel Afonine  wrote:

>
>
> Yes, that is what I have been doing. Build one subunit and assemble into
>> tetramer before realspace refinement (with "ncs" constraints). I used
>> tetramer for refinement because I want the distances between the inter
>> subunits interaction partners to be considered. The problem is, whenever I
>> want to make some manual adjustments of the model afterwards, I have to
>> break it into monomer and repeat the whole process again. I assume if the
>> map is in P4 spacegroup, I only need to modify one subunit of this protein
>> and changes will be automatically applied to the other subunits. Am I
>> right?
>>
>
> So you have 4 copies and NCS group consisting of one master (or reference)
> copy and three related by NCS symmetry. Then you make changes in master
> copy and when you run refinement with NCS constraints the changes will be
> propagated onto the related copies by applying NCS constraints. This is how
> this works in phenix.real_space_refine. So no need to make identical
> changes in all 4 copies or go to P4.
>
>
>
>> Another question is, when I tried to refine my model using phenix
>> realspace refinement (energy minimization and adp), the statistics
>> generally become better (map CC, rotamer outlier etc), however, the
>> ramachandron statistics become worse, with increase number of outliers
>> (from below 1% to around 5%) and allowed (from several percent to 10-20%).
>> Do you know what could be the cause? I know I am asking the right person...
>>
>
> This is not expected (about worsening Ramachandran plot outliers). I'm
> sure we can solve this problem if you send me model and map files (and
> resolution) off list (if files too large to send by email please use
> Dropbox or similar file sharing tools).
>
> Pavel
>
>

Re: [ccp4bb] crystallization screen for protein-protein complex

[Edward Snell]

Dear Hena,

You already have excellent advice but I would also look at the PEG smear 
approach developed by Frank von Delft’s group – Chaikuad et al., Acta Cryst D. 
7, 1627-39 (2015). It’s commercially marketed by Molecular Dimensions and has 
features which may be amenable to complex systems. Caveat emptor, we don’t have 
any data on any of the academic samples that have been submitted to our 
crystallization center to vouch for its success but we would suggest taking a 
look.

If you are interested in testing a lot of conditions in an efficient way the 
high-throughput crystallization screening center at our institute uses a lot of 
the commercially available screens 
(http://hwi.buffalo.edu/crystallization-cocktails/) and covers a fairly 
extensive area of chemical space.  That can save a lot of time and effort. 
Details are at http://getacrystal.org.

Good luck,

Eddie


Edward Snell Ph.D.
President and CEO Hauptman-Woodward Medical Research Institute
Assistant Prof. Department of Structural Biology, University at Buffalo
700 Ellicott Street, Buffalo, NY 14203-1102
hwi.buffalo.edu
Phone: (716) 898 8631 Fax: (716) 898 8660
Skype:  eddie.snell Email: esn...@hwi.buffalo.edu
[cid:image003.png@01D2D241.8F507E50]
Heisenberg was probably here!

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hena Dutta
Sent: Saturday, May 20, 2017 5:15 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] crystallization screen for protein-protein complex

Dear Members,
I am trying to crystallize a protein-protein complex. Both are soluble proteins 
and form complex as observed in FPLC either co-purifying or separately 
purifying and then mixing with equi-molar ratio.
Looking for suggested screens to try for crystallization.
So far tried with Qiagen - ProComplex Suite, Classics Suite and Classics II 
Suite
No success yet.
Is there any guidance which screen to try?
Looking for your help...
Regards,
Hena.

[ccp4bb] Cys modification - deciding between CSS and SMC

[Mohamed Noor]

I am working on a model at a resolution of 2.1 A. The active site Cys in both 
copies have a positive density towards the S end of the residue and these blobs 
are there in FEM and Polder maps. When I replace these residues with either CSS 
or SMC, I get the following statistics. What is the best way of deciding which 
one is correct?

CSS:

R/Rfree - 0.2016/0.2299
Local CC (chain A/B) - 0.950/0.937
Bfactor (chain A N/CA/C/O/CB/SG/SD) - 42.8/48.6/46.7/42.8/57.3/66.2/98.8
Bfactor (chain B N/CA/C/O/CB/SG/SD) -42.0/44.5/47.2/45.7/57/60.3/95.3

No residual positive density left.

SMC:

R/Rfree - 0.2087/0.2299
Local CC (chain A/B) - 0.903/0.886
Bfactor (chain A N/CA/C/O/CB/SG/CS) - 43.9/47.9/46.5/42.1/51.8/64.4/66.5
Bfactor (chain A N/CA/C/O/CB/SG/CS) - 43.4/45.0/47.3/45.4/51.8/59.4/66.1

Crescent shaped positive density left towards the CS atom end.

Although CSS seems more plausible, the high B factors of SD make me wonder if 
they are correct.

Thanks.
Mohamed

Re: [ccp4bb] crystallization screen for protein-protein complex

[Patrick Shaw Stewart]

Hena

There was a very interesting paper by Peter Sun and coworker from 2002.
They pointed out that there is a very strong bias towards crystallizing
protein-protein complexes with PEG rather than salt as the main precipitant.

Patrick

___

Radaev and Sun.  Crystallization of protein-protein complexes. J. Appl.
Cryst. (2002). 35, 674-676





On 20 May 2017 at 22:14, Hena Dutta  wrote:
>
> Dear Members,
> I am trying to crystallize a protein-protein complex. Both are soluble
proteins and form complex as observed in FPLC either co-purifying or
separately purifying and then mixing with equi-molar ratio.
> Looking for suggested screens to try for crystallization.
>
> So far tried with Qiagen - ProComplex Suite, Classics Suite and Classics
II Suite
> No success yet.
>
> Is there any guidance which screen to try?
>
> Looking for your help...
> Regards,
> Hena.




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