Dear all Herman might be correct in this case but spot shapes can be affected by imperfactions in the crystal rather than the crystal shape. Some types of imperfections (e.g. strain) manifest themselves more for higher resolution data. They are still there for the low resolution data, but buried by the overall instrument (detector, beamline) resolution. An interesting thing to do is to examine spot shapes at different detector distances making sure the beam divergence doesn't dominate. . In this case one might find that the low resolution spots also have this behavior (ears etc.). Of course it is probably too late to do this. Regards Colin ________________________________
From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of [email protected] Sent: 25 November 2010 08:28 To: [email protected] Subject: Re: [ccp4bb] unusual diffraction spots 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:[email protected]] On Behalf Of Hubing Lou Sent: Thursday, November 25, 2010 6:10 AM To: [email protected] 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, <[email protected]> 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:[email protected]] On Behalf Of Hubing Lou Sent: 24 November 2010 14:09 To: [email protected] 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 -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
