Dear Hubing,
please don't discard a structure just because the Rfree > 30%, or approve a structure just because the Rfree < 30%. Other quality parameters like relative absence of clashes and Ramachandran outliers are much more important. Perhaps even more important is whether the structure gives an important new insight - even a structure with mediocre overall quality parameters may yield a clear, defendable, interesting feature.
Of course, this is not to say one should not try to get better crystals and data where at all possible...but perhaps what you wanted to discover about your protein is already in your present structure.
Mark van Raaij


On 25 Nov 2010, at 06:09, Hubing Lou wrote:

> 
> 
> 
> To further clarify things, the data was collected at a synchrotron beamline with collimator size ~130*40(um*square), beam divergence ~0.3*0.1mRad. The detector type was MarCCD.
> The crystal was multiple-faced trigonal (space group P3121) the size was about 0.1*0.1*0.15mm. The exposure time was 2s for each image. 
> 
> I am currently refining the structure, however the Rfree stays above 30%. A close inspection shows at high resolution shell the spots become rod shaped. As I said we are preparing new constructs with N-terminal his-tag cleaved. But any other good suggestions out there might be helpful to avoid future frustration.
> 
> Thanks,
> Hubing
> 
> On Wed, Nov 24, 2010 at 10:26 PM, <[log in to unmask]> wrote:
> I think there may be two effects going on here:
> 
>  
> I think the “ears” on the round spots which also feature on the more rod shaped spots if you look closely could be related to a misalignment of the beamline optics.
> 
>  
> I think the change in spot shape from round to rod shaped is due to the crystal quality.
> 
>  
> Do the “ears” only feature on this image of this crystal or do they appear on other images? If the ear effect is a one off then that would tend to suggest it isn’t a beamline optic effect.
> 
>  
> Liz
> 
>  
> Dr. Liz Duke
> 
> Principal Beamline Scientist
> 
> Diamond Light Source
> 
> Harwell Science and Innovation Campus
> 
> Chilton
> 
> OX11 0DE
> 
> UK
> 
>  
> Tel. 01235 778057
> 
> Mob. 07920 138148
> 
>  
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Hubing Lou
> Sent: 24 November 2010 14:09
> To: [log in to unmask]
> Subject: [ccp4bb] unusual diffraction spots
> 
>  
> Dear CCP4BBer, 
> 
> I recently collected a dataset at synchrotron. The diffraction was quite anisotropic with one direction to 2.1Angstrom while the other is 3.0Ang. What unusual is in the diffraction image (see the attached file), clearly at low resolution there were some spots with tails ("two ears") and at the high resolution shell the spots turned to be rod-shaped.  Please, can anyone explain how this could be? Is this related to the anisotropy? The protein was N-terminal his6-tagged, we  are currently preparing new samples with the His-tag removed. But any other suggestions are also very welcomed.
> 
> Regards,
> 
> Hubing
> 
> 
>  
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