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

[ccp4bb] AW: [ccp4bb] Contouring 2Fo-Fc map, large blobs in Fo-Fc

[Herman . Schreuder]

Hi Roger,

First, sigma is a relative measure. If sigma is very low, e.g. since your map 
contains 80% solvent, 3 sigma may correspond to the same absolute value e.g. in 
electrons/Å3 as 1 sigma in a standard map, so I would not be worried about that.

However, an Rfree of 0.45 and a large off-origin Patterson peak indicates that 
you have to place another copy of your search molecule. This brings a few 
questions:

-Is the real space group P1, or is there higher symmetry? In the latter 
case you have to move to the higher symmetry space group.

-Did you ask phaser to search for 2 (or more) molecules? If the answer 
is no, that would be the first thing to do.

-Otherwise, I would translate the molecule already found according to 
the off-origin Patterson vector and look how this fits into the electron 
density maps.

Good luck!
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Roger 
Shek
Gesendet: Mittwoch, 26. April 2017 03:23
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Contouring 2Fo-Fc map, large blobs in Fo-Fc

Hi everyone,

We have a structure that phaser found a solution for in P1, even though there 
is a large off-origin Patterson peak indicating pseudo NCS. However, the 
2mFo-Fc map needs to be contoured up to >3 sigma to be reasonable, going lower 
makes it look like what I normally see at <1 sigma. If you look at the Fo-Fc 
map, there are large areas of positive and negative density. The 2mFo-Fc map 
looks very convincing, you can see the tyrosines, phenyl rings, etc...Also 
phaser gave LLG of >400 and a TFZ of ~14. Rfree is ~0.45. Does anyone know 
what's going on?

Thanks,
Roger

--
Roger Shek

Stony Brook University
Graduate Student in Biochemistry and Structural Biology (PhD)
Stony Brook Integrative Structural Biology 
Organization
Cell: (808) 386-3879