Re: [ccp4bb] large number in ASU

[Eleanor Dodson]

Have you looked at a self rotation function? Is there a clear 3-fold axis
perhaps?
Eleanor

On 27 April 2017 at 10:45, Mark J van Raaij  wrote:

> Does the self-rotation function suggest presence of NCS axes? If so, this
> may help you figure out the symmetry inside the a.u..
> If you haven't done so already, try diffracting a crystal at room
> temperature, to make sure cryo-protection and freezing did not affect the
> diffraction.
> In any case, at 3.65Å I would recommend not to spend too much of your time
> on this dataset.
> Just set your computer(s) to try MR with different parameters, numbers of
> copies to search for etc. Instead of 40 copies at 50% solvent, you may have
> down to 20 copies with 75% solvent (or maybe even less). Or more than 40
> with less solvent (perhaps less likely). Even then, judging whether an MR
> solution is really correct might still not be that easy.
> And in the meantime spend your own time to try to get derivatives and
> better-diffracting crystals. At better than 2.5Å resolution it will all be
> much easier, for instance auto-building with your input sequence will allow
> much better identification of correct MR solutions.
> And if you are lucky, a heavy atom soaking experiment might actually
> improve the crystal.
> If you haven't already, I'd also try to get some complementary information
> on whether your protein forms stable complexes. EM, SAXS and AUC come to
> mind.
>
> Good luck!
>
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> tel. (+34) 91 585 4616 <+34%20915%2085%2046%2016>
> http://wwwuser.cnb.csic.es/~mjvanraaij
>
> On 26 Apr 2017, at 21:34, Jademilson Santos 
> wrote:
>
> Greetings all,
>
> I am having trouble with a data set and would like to know if somebody can
> help. I'm working with a protein of approximately 50 kDa, which I have
> successfully crystallized. The crystals diffracted at a resolution of 3,65
> angstroms and upon initial processing using XDS i obtained the following
> information:
>
> space group: P21
> ISA = 33.3
> cell unit: a=285.2, b=135.9, c= 287.5, α=90, β=117.5, γ=90
>
> Matthews coefficient indicates that there are 40 molecules in the
> asymmetric unit
>
> I am currently running the program Phaser (Phenix) to determine the phase
> via molecular replacement with a model that has 49% homology and query
> coverage of 94% and the program is taking extremly long to finish. In this
> case in which there is an extremly high number of molecules in the
> asymmetric unit, is this actually possible? Does someone know how to work
> with these values and is there a specific strategy which i must follow?
>
> Regards
>
>
>
> *Jademilson Celestino dos Santos*
>
> *Laboratory of Applied Structural Biology*
>
>
> *Department of MicrobiologyInstitute of Biomedical SciencesUniversity of
> São Paulo- USP*
>
>
>

Re: [ccp4bb] large number in ASU

[Andrew Leslie]

Dear Jademilson,

At a CCP4 APS workshop a few years ago, one of the 
students solved a molecular replacement problem where I think there were close 
to 40 copies in the asu. I have to say that many people were surprised by this, 
but I think the protein was smaller and they had a very good search model - I 
can’t remember the resolution of the data.  The job certainly ran for a long 
time (overnight I think), but it does show that in some circumstances this can 
work. Possibly Randy Ready can remember the details better than I do. However 
in your case you may need to resort to experimental phasing.

Good luck!

Andrew


> On 26 Apr 2017, at 20:34, Jademilson Santos  
> wrote:
> 
> Greetings all,
> 
> I am having trouble with a data set and would like to know if somebody can 
> help. I'm working with a protein of approximately 50 kDa, which I have 
> successfully crystallized. The crystals diffracted at a resolution of 3,65 
> angstroms and upon initial processing using XDS i obtained the following 
> information: 
> 
> space group: P21
> ISA = 33.3
> cell unit: a=285.2, b=135.9, c= 287.5, α=90, β=117.5, γ=90
> 
> Matthews coefficient indicates that there are 40 molecules in the asymmetric 
> unit 
> 
> I am currently running the program Phaser (Phenix) to determine the phase via 
> molecular replacement with a model that has 49% homology and query coverage 
> of 94% and the program is taking extremly long to finish. In this case in 
> which there is an extremly high number of molecules in the asymmetric unit, 
> is this actually possible? Does someone know how to work with these values 
> and is there a specific strategy which i must follow?
> 
> Regards
> 
> Jademilson Celestino dos Santos
> 
> Laboratory of Applied Structural Biology
> Department of Microbiology
> Institute of Biomedical Sciences
> University of São Paulo- USP

Re: [ccp4bb] large number in ASU

[CRAIG A BINGMAN]

Congratulations!

I think you are now looking for additional crystallographic and 
non-crystallographic symmetry, because finding 40 particles in arbitrary 
positions and orientations is going to be brutal.

I wouldn’t take the cell and point group assignment from XDS at face value.  
Rather I think you should put XDS_ASCII.HKL through phenix.xtriage and 
POINTLESS in the CCP4 suite of programs.  Both can be invoked from the command 
line.

pointless HKLIN XDS_ASCII.HKL
phenix.xtriage XDS_ASCII.HKL

That handles the crystallographic symmetry part.  Let us know how it turns out.

It is also possible that there is extensive NCS, and if the space group 
assignment holds, I’d ask again about that.  As Jacob Keller’s response 
suggests, most people are going to suggest that you lock down crystallographic 
symmetry before looking at NCS.

Craig



On Apr 26, 2017, at 2:34 PM, Jademilson Santos 
> wrote:

Greetings all,

I am having trouble with a data set and would like to know if somebody can 
help. I'm working with a protein of approximately 50 kDa, which I have 
successfully crystallized. The crystals diffracted at a resolution of 3,65 
angstroms and upon initial processing using XDS i obtained the following 
information:

space group: P21
ISA = 33.3
cell unit: a=285.2, b=135.9, c= 287.5, α=90, β=117.5, γ=90

Matthews coefficient indicates that there are 40 molecules in the asymmetric 
unit

I am currently running the program Phaser (Phenix) to determine the phase via 
molecular replacement with a model that has 49% homology and query coverage of 
94% and the program is taking extremly long to finish. In this case in which 
there is an extremly high number of molecules in the asymmetric unit, is this 
actually possible? Does someone know how to work with these values and is there 
a specific strategy which i must follow?

Regards

Jademilson Celestino dos Santos

Laboratory of Applied Structural Biology
Department of Microbiology
Institute of Biomedical Sciences
University of São Paulo- USP

Re: [ccp4bb] large number in ASU

[Keller, Jacob]

Use Zanuda to see whether the space group is actually a higher one—looks like a 
and c axes are pretty similar, and beta might be 120, suggesting a threefold. 
Otherwise it’s a pretty large beta. I wonder what the largest beta ever seen in 
the pdb is?

JPK


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Jademilson Santos
Sent: Wednesday, April 26, 2017 3:35 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] large number in ASU

Greetings all,

I am having trouble with a data set and would like to know if somebody can 
help. I'm working with a protein of approximately 50 kDa, which I have 
successfully crystallized. The crystals diffracted at a resolution of 3,65 
angstroms and upon initial processing using XDS i obtained the following 
information:

space group: P21
ISA = 33.3
cell unit: a=285.2, b=135.9, c= 287.5, α=90, β=117.5, γ=90
Matthews coefficient indicates that there are 40 molecules in the asymmetric 
unit

I am currently running the program Phaser (Phenix) to determine the phase via 
molecular replacement with a model that has 49% homology and query coverage of 
94% and the program is taking extremly long to finish. In this case in which 
there is an extremly high number of molecules in the asymmetric unit, is this 
actually possible? Does someone know how to work with these values and is there 
a specific strategy which i must follow?

Regards

Jademilson Celestino dos Santos
Laboratory of Applied Structural Biology
Department of Microbiology
Institute of Biomedical Sciences
University of São Paulo- USP