Dear Hubing,
since your crystal is smaller than the beam, the shape of your spots
will be the shape of your crystal as viewed from the spot position on
the detector. This means that if your crystal has a rod shape, spots at
certain detector positions will have a rod shape. If your crystal has
ears, this may explain the ears you see in your diffraction pattern.
Different spot shapes at different detector regions are normally not a
problem since most processing programs use different local profiles for
different regions of the detector. Your high Rfree is not caused by the
different spot shapes, but must have other causes which may be
anisotropy of your data, ice rings, disorder of your protein etc.
If you do not like different spot shapes, you must collimate your beam
to be smaller than your crystal, but again, this is not the cause of
your high Rfree (and frustration)!
Good luck with your refinement!
Herman
________________________________
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On
Behalf Of Hubing Lou
Sent: Thursday, November 25, 2010 6:10 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] unusual diffraction spots
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, <elizabeth.d...@diamond.ac.uk>
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:ccp...@jiscmail.ac.uk]
On Behalf Of Hubing Lou
Sent: 24 November 2010 14:09
To: CCP4BB@JISCMAIL.AC.UK
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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