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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