From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Koromyslova, Anna
Sent: Tuesday, July 11, 2017 3:37 PM
To: [log in to unmask]
Subject: Re: [ccp4bb] Problem with a cell content

Eleanor

On 11 July 2017 at 19:12, Koromyslova, Anna <[log in to unmask]> wrote:

Dear Eleanor,

 

NCS translation vector = 0 0 -0.5 in the final sp P6222, and in P622 or C121. All had the same solution.

The protein tends to form a homodimer and if I use P1 space group molecular replacement can find several dimers, there is no translational ncs found during MR, but still solvent cell content was similarly high.

Within a protein monomer there are no similar domains.

 

Best regards,

Anna

 

 

 

From: CCP4 bulletin board <[log in to unmask]> on behalf of Eleanor Dodson <[log in to unmask]>
Reply-To: Eleanor Dodson <[log in to unmask]>
Date: Tuesday, July 11, 2017 at 7:29 PM
To: "[log in to unmask]" <[log in to unmask]>
Subject: Re: [ccp4bb] Problem with a cell content

 

This is very strange . If you have a large non- crystallographic translation vector you would expect either to have two molecules in the asymmetric unit or your one molecule must have two very similar domains? 

What is the n-c translation vector? 

 

Could you have assigned too high symmetry ? SG maybe P 62? That could explain the packing clashes Z scores of 27 are pretty good.

 

Eleanor

 

 

On 11 July 2017 at 18:05, Oganesyan, Vaheh <[log in to unmask]> wrote:

Похоже, что вы уже решили структуру. Почему вас беспокоит плотность ваших кристаллов? Кристаллы бывают разные.

 

First of all Fab by itself is already almost 50 kDa, so complex with antigen should be more than 50 kDa. Because you already solved the structure calculate the molecular mass based on your pdb file and rerun Matthews with correct mass. New numbers may be quite a bit different. Good indication of relatively low crystal density and consequently loose packing is the resolution of your data set. If you did not throw away data beyond 2.9A I’d suggest use them all. The reflections are too valuable to throw away. If data beyond some resolution is weak then they will have low contribution to the structure. Best if you calculate electron density maps at different resolutions at the end of refinement, compare them and use resolution that makes difference.

 

 

 

Regards,

 

Vaheh

8-5851

 

From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Koromyslova, Anna
Sent: Tuesday, July 11, 2017 12:32 PM
To: [log in to unmask]
Subject: [ccp4bb] Problem with a cell content

 

Dear CCP4 members,

 

I am working on a structure of a protein in complex with an antibody fragment (approx. 50kDa together). Molecular replacement with closely related proteins always comes up with one complex in the asymmetric unit, although MW of protein to which Matthews applies is 125kDa and corresponds to two complexes.

Phaser gives two warnings:

Large non-origin Patterson peak indicates that translational NCS is present.

Solutions with Z-scores greater than 27.2 (the threshold indicating a definite solution) were rejected for failing packing test

 

I couldn’t get a solution with two subunits although I have tried multiple combinations including only conserved parts of both proteins and different space groups including P1. Phenix Autobuild also yielded only one complex.

 

So, the question is whether I can use that structure as is despite very high solvent content (80%) or should I try smth else. I would be very grateful for any suggestions.

 

When the solution with a single complex is refined the statistics are the following:

 

R-work                  0.2129

R-free                  0.2459

Matthews Coefficient: 6.22

Percentage Solvent: 80.22

Resolution range (Å)  48.34  - 2.9 (2.98  - 2.9)

Space group                         P 62 2 2

Unit cell  167.45 167.45 143.538 90 90 120

Multiplicity                      19.1 (18.3)

Completeness (%)                    99.44 (94.39)

Mean I/sigma(I)                     24.59 (2.71)

Wilson B-factor                            64.28

R-merge                   0.1256 (1.186)

R-meas                           0.1291

CC1/2                     0.999 (0.85)

CC*                        1 (0.959)

 

Thank you very much for your help,

 

Anna

 

 

Dr. Anna Koromyslova, Postdoctoral researcher

German Cancer Research Center (DKFZ), F150

Im Neuenheimer Feld 242

D-69120 Heidelberg

Germany

 

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