Dear Hubing, Thankyou for the extra details. The MARCCD to my experience does not show 'bleeding' from pixels at strong spots. Such effects on a CCD anyway are in a line not like the 'ears' you have here. It also does not look like diffuse scattering. So, since it seems to be an effect visible on very strong spots, I wonder if there is some texture to your crystal ie in effect a very small degree of fragmentation on this particular sample. Best wishes, John On Thu, Nov 25, 2010 at 5:09 AM, Hubing Lou <[email protected]> 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, <[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 >> >> >> > -- Professor John R Helliwell DSc
