Re: [ccp4bb] Weird diffraction pattern
Dear all Thanks for all the input both on- and off- the list. We shall definitely look into these suggestions further and report again here in due course. Kind regards Sam On Tue, 9 Oct 2018 at 19:12, Sam Tang wrote: > Dear all > > Hello. We recently shot a crystal (a protein with small molecule as > ligand) at a synchrotron source and see a weird pattern. ( > https://drive.google.com/file/d/11bEtTJzKaAB5ZybezgN1cqBrckSRg2OV/view?usp=sharing > ) > > Crystal was grown in Citric acid and ammonium sulfate, cryoprotected with > glycerol. > > At first we thought it was a protein crystal contaminated with salt but on > second thought, the lowest resolution spot was at around 7 A, which doesn't > make sense for a protein. So we would like to solicit your experience and > perhaps someone may have encountered similar pattern before? > > Many thanks. > > Kind regards > > Sam > > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] AW: [EXTERNAL] [ccp4bb] Weird diffraction pattern
To me, it looks like some intergrown salt crystal. HS Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Sam Tang Gesendet: Dienstag, 9. Oktober 2018 13:13 An: CCP4BB@JISCMAIL.AC.UK Betreff: [EXTERNAL] [ccp4bb] Weird diffraction pattern Dear all Hello. We recently shot a crystal (a protein with small molecule as ligand) at a synchrotron source and see a weird pattern. (https://drive.google.com/file/d/11bEtTJzKaAB5ZybezgN1cqBrckSRg2OV/view?usp=sharing<https://urldefense.proofpoint.com/v2/url?u=https-3A__drive.google.com_file_d_11bEtTJzKaAB5ZybezgN1cqBrckSRg2OV_view-3Fusp-3Dsharing=DwMFaQ=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=ExvzTpwCzYQcP3Idr9vHnPcGFh0UXDAKsEB7jClqsF0=4dUd1KLQYV5G8_jUYdS8kORXsEkS-0kwKqiGRj5dvDg=>) Crystal was grown in Citric acid and ammonium sulfate, cryoprotected with glycerol. At first we thought it was a protein crystal contaminated with salt but on second thought, the lowest resolution spot was at around 7 A, which doesn't make sense for a protein. So we would like to solicit your experience and perhaps someone may have encountered similar pattern before? Many thanks. Kind regards Sam To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1=DwMFaQ=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=ExvzTpwCzYQcP3Idr9vHnPcGFh0UXDAKsEB7jClqsF0=Ud5SL2yONTX62SIhpLS7sPJxpTJogstplKUe7cQgN_g=> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Weird diffraction pattern
Hello again It could be a powder pattern aligned along one axis? The cell I gave (actually orthorhombic) is one of the crystal forms of one of the components in your mixture – glycerol. You may have another form of glycerol. It is worth checking. Colin PS I think glycerol diffraction has been raised previously on CCP4bb From: Sam Tang Sent: 09 October 2018 15:02 To: Nave, Colin (DLSLtd,RAL,LSCI) Cc: ccp4bb Subject: Re: [ccp4bb] Weird diffraction pattern Hello Colin Although the unit cell dimensions from mosflm should be largely unreliable in this case, the software actually returned a P2 space group with a=24.6, b=7.5, c=69.5 where b is so short that it resembles a small molecule crystal. Regards Sam Sam On Tue, 9 Oct 2018 at 20:53, colin.n...@diamond.ac.uk<mailto:colin.n...@diamond.ac.uk> mailto:colin.n...@diamond.ac.uk>> wrote: Sam Would this unit cell index some of the spots? a = 7.00 ± 0.04 A, b = 9.96 ± 0.05 A, c = 6.29 ± 0.04 A. Colin From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> On Behalf Of Sam Tang Sent: 09 October 2018 12:13 To: ccp4bb mailto:ccp4bb@jiscmail.ac.uk>> Subject: [ccp4bb] Weird diffraction pattern Dear all Hello. We recently shot a crystal (a protein with small molecule as ligand) at a synchrotron source and see a weird pattern. (https://drive.google.com/file/d/11bEtTJzKaAB5ZybezgN1cqBrckSRg2OV/view?usp=sharing) Crystal was grown in Citric acid and ammonium sulfate, cryoprotected with glycerol. At first we thought it was a protein crystal contaminated with salt but on second thought, the lowest resolution spot was at around 7 A, which doesn't make sense for a protein. So we would like to solicit your experience and perhaps someone may have encountered similar pattern before? Many thanks. Kind regards Sam To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 -- 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 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Weird diffraction pattern
Hello Colin Although the unit cell dimensions from mosflm should be largely unreliable in this case, the software actually returned a P2 space group with a=24.6, b=7.5, c=69.5 where b is so short that it resembles a small molecule crystal. Regards Sam Sam On Tue, 9 Oct 2018 at 20:53, colin.n...@diamond.ac.uk < colin.n...@diamond.ac.uk> wrote: > Sam > > Would this unit cell index some of the spots? > > a = 7.00 ± 0.04 A, b = 9.96 ± 0.05 A, c = 6.29 ± 0.04 A. > > Colin > > > > *From:* CCP4 bulletin board *On Behalf Of *Sam > Tang > *Sent:* 09 October 2018 12:13 > *To:* ccp4bb > *Subject:* [ccp4bb] Weird diffraction pattern > > > > Dear all > > > > Hello. We recently shot a crystal (a protein with small molecule as > ligand) at a synchrotron source and see a weird pattern. ( > https://drive.google.com/file/d/11bEtTJzKaAB5ZybezgN1cqBrckSRg2OV/view?usp=sharing > ) > > > > Crystal was grown in Citric acid and ammonium sulfate, cryoprotected with > glycerol. > > > > At first we thought it was a protein crystal contaminated with salt but on > second thought, the lowest resolution spot was at around 7 A, which doesn't > make sense for a protein. So we would like to solicit your experience and > perhaps someone may have encountered similar pattern before? > > > > Many thanks. > > > > Kind regards > > > Sam > > > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > > > > -- > > 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 > > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Weird diffraction pattern
Sam Would this unit cell index some of the spots? a = 7.00 ± 0.04 A, b = 9.96 ± 0.05 A, c = 6.29 ± 0.04 A. Colin From: CCP4 bulletin board On Behalf Of Sam Tang Sent: 09 October 2018 12:13 To: ccp4bb Subject: [ccp4bb] Weird diffraction pattern Dear all Hello. We recently shot a crystal (a protein with small molecule as ligand) at a synchrotron source and see a weird pattern. (https://drive.google.com/file/d/11bEtTJzKaAB5ZybezgN1cqBrckSRg2OV/view?usp=sharing) Crystal was grown in Citric acid and ammonium sulfate, cryoprotected with glycerol. At first we thought it was a protein crystal contaminated with salt but on second thought, the lowest resolution spot was at around 7 A, which doesn't make sense for a protein. So we would like to solicit your experience and perhaps someone may have encountered similar pattern before? Many thanks. Kind regards Sam To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 -- 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 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] weird diffraction pattern
Hello Tang, 1) For MR, you might want to try a range of homologs, or even a stack of overlapping homologs. A normal modes server like elNemo might also help if it can predict the "bend" your molecule undergoes upon binding. A long shot perhaps, but stranger things have happened. You also might be able to find the DNA by molecular replacement. 2) radiation damage increases with photons/area, not time. So no matter what your degrees/image you want the total shuttter-open time at the end of the data set to be below the damage limit of interest. A little web app I made once might help: http://bl831.als.lbl.gov/xtallife.html . These days, there is no reason not to know how long your crystal will last before you push "collect", and it is definitely worth knowing. -James Holton MAD Scientist On 7/28/2017 12:21 AM, Tang Chenjun wrote: Hi, Thanks to all who gave me suggestions concerning the weird diffraction pattern and I really appreciate it that Kay Diederichs help me processing my data set and answer my questions. Although the data set can be processed using HKL3000, XDS without problems, the Rwork/Rfree values are still above 0.5 after molecular replacement. There can be several reasons. 1) The structure change a lot after binding DNA, so it is not possible to find a solution using molecular replacement. 2) Strong radiation damage and 1.0 degree image widths prevent good integration results. It may be better to use 0.1 degree image widths. 3) Streaky spots appearing in certain directions because of anisotropy or lattice translocation disorder, or one very large unit cell dimension lying along the X-ray beam may also have an affect on data processing. Now I am optimizing the crystals to address these problems. Best wishes and thanks again for your help, Chenjun Tang
Re: [ccp4bb] weird diffraction pattern
Hi Chenjun Tang, I have not followed the original discussion, so my apologies if I am repeating the device already given to you. Crystallization optimization is always a good idea - nothing beats good quality crystals. In addition, you should try collecting few images, especially in the "bad" orientation of the crystal, at ambient temperature, from the samples that have NOT been cryo-protected yet. You need to figure out if the cryo-protection and/or cryo-cooling are damaging your crystals. Best, Nukri Ruslan Sanishvili (Nukri), Ph.D. Macromolecular Crystallographer GM/CA@APS X-ray Science Division, ANL 9700 S. Cass Ave. Lemont, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 rsanishv...@anl.gov From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Tang Chenjun <0910010...@cau.edu.cn> Sent: Friday, July 28, 2017 2:21 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] weird diffraction pattern Hi, Thanks to all who gave me suggestions concerning the weird diffraction pattern and I really appreciate it that Kay Diederichs help me processing my data set and answer my questions. Although the data set can be processed using HKL3000, XDS without problems, the Rwork/Rfree values are still above 0.5 after molecular replacement. There can be several reasons. 1) The structure change a lot after binding DNA, so it is not possible to find a solution using molecular replacement. 2) Strong radiation damage and 1.0 degree image widths prevent good integration results. It may be better to use 0.1 degree image widths. 3) Streaky spots appearing in certain directions because of anisotropy or lattice translocation disorder, or one very large unit cell dimension lying along the X-ray beam may also have an affect on data processing. Now I am optimizing the crystals to address these problems. Best wishes and thanks again for your help, Chenjun Tang
Re: [ccp4bb] weird diffraction pattern
Hi, Thanks to all who gave me suggestions concerning the weird diffraction pattern and I really appreciate it that Kay Diederichs help me processing my data set and answer my questions. Although the data set can be processed using HKL3000, XDS without problems, the Rwork/Rfree values are still above 0.5 after molecular replacement. There can be several reasons. 1) The structure change a lot after binding DNA, so it is not possible to find a solution using molecular replacement. 2) Strong radiation damage and 1.0 degree image widths prevent good integration results. It may be better to use 0.1 degree image widths. 3) Streaky spots appearing in certain directions because of anisotropy or lattice translocation disorder, or one very large unit cell dimension lying along the X-ray beam may also have an affect on data processing. Now I am optimizing the crystals to address these problems. Best wishes and thanks again for your help, Chenjun Tang
Re: [ccp4bb] weird diffraction pattern
Dear Gerd, I wasn't really giving much attention to the poke between the ribs ;-) - for me the more serious matter was to see the merits of pixel detectors over CCDs made light of, as if they didn't really make much difference. If some people get carried away in the way you describe, well, it doesn't hurt anyone; but if other people have very small, weakly diffracting crystals and they are told that they will do as well on a beamline with a CCD as on one with a Pilatus or an Eiger, then that will hurt someone. The original topic was whether certain images presented without any information about their angular width and recorded on a CCD were sufficient to diagnose a diffraction quality problem. As they showed evidene of at least one long axis, distinguishing streakiness from plain angular overlap caused by too great an image width seemed the most natural first step. The resolution I was talking about was resolving spots within images and neighbouring images, either thanks to a detector with a small PSF or thanks to a very fine image width. CCD detectors are poor in the first option because of their extended PSF, so emphasis has always been put on the importance of using the second one when pushed. Pixel array detectors allow both options to be used simultaneously, which is especially valuable in the investigation of crystals like Tang's. Hence, again, my advice in the strict context of the initial thread. With best wishes, Gerard. -- On Thu, Jul 13, 2017 at 02:39:58PM -0500, Gerd Rosenbaum wrote: > Dear Gerard, > >my "sound like a sales person" was meant as poking a little fun - nothing > serious, of course. > > I and our users like our not-so-new-anymore Pilatus3 6M. It's a great > detector in many ways. But, there is a lot of hype that this detector solves > all-problem, for instance fine slicing that is claimed to be only possible > with a pixel array detector. People get carried away and use 0.01 degree > slices even as the mosaicity of their sample is, say, 0.3 degree. Slicing > beyond 1/3 of the mosaicity will gain you very little - only more frames, > more processing time. > > This discourse is already drifting away from the original topic of the > thread so I will comment on the other arguments you made like resolution in > a private e-mail. > > Best regards, > > Gerd > > On 13.07.2017 14:00, Gerard Bricogne wrote: > >Dear Gerd, > > > > I can assure you that I have no shares in Dectris nor any > >commecial connections with them. What I do have is a lot of still > >vivid memories of CCD images, with their wooly point-spread function > >that was affected by fine-grained spatial variability as well as by > >irredicible inaccuracies in the geometric corrections required to try > >and undo the distortions introduced by the fiber-optic taper. By > >comparison the pixel-array detectors have a very regular structure, so > >that slight deviations from exact registering of the modules can be > >calibrated with high accuracy, making it possible to get very small > >residuals between calculated and observed spot positions. That, I > >certainly never saw with CCD images. > > > > I do think that asking for the image width was a highly pertinent > >question in this case, that had not been asked. As a specialist you > >might know how to use a CCD to good effect in fine-slicing mode, but > >it is amazing how many people there are still out there who are told > >to use 0.5 or even 1.0 degree image widths. > > > > Compensating the poor PSF of a CCD by fine slicing in the angular > >dimension is a tall order. With a Pilatus at 350mm from the crystal, > >the angular separation between 174-micron pixels is 0.5 milliradian. > >To achieve that separation in the angular (rotation) dimension, the > >equivalent image width would have to be 0.03 degree. For an EIGER the > >numbers become 75 microns, hence 0.21 milliradian i.e. 0.012 degree. > > > > Hence my advice, untainted by any commercial agenda :-) . > > With best wishes, > > Gerard. > > > >-- > >On Thu, Jul 13, 2017 at 01:25:08PM -0500, Gerd Rosenbaum wrote: > >>Dear Gerard, > >> > >>you sound like a sales person for Dectris. Fine slicing is perfectly fine > >>with CCD detectors - it takes a bit longer because of the step scan instead > >>of continuous scan. The read noise issue is often overstated compared to the > >>sample induced scatter background. If for fine slicing at 0.05 degree or > >>less the diffraction peaks go too close to the read noise make a longer > >>exposure - signal goes up, ratio signal to sample-induced-BG less, as for > >>any fine slicing, same read noise. > >> > >>It would be helpful to analyze the dense spot packing along layer lines if > >>we knew the wavelength and the sample-to-detector distance (assuming this is > >>a 300 mm detector) and the rotation width - as you pointed out. That would > >>help to distinguish between multiple crystals (my
Re: [ccp4bb] weird diffraction pattern
Dear Gerard, my "sound like a sales person" was meant as poking a little fun - nothing serious, of course. I and our users like our not-so-new-anymore Pilatus3 6M. It's a great detector in many ways. But, there is a lot of hype that this detector solves all-problem, for instance fine slicing that is claimed to be only possible with a pixel array detector. People get carried away and use 0.01 degree slices even as the mosaicity of their sample is, say, 0.3 degree. Slicing beyond 1/3 of the mosaicity will gain you very little - only more frames, more processing time. This discourse is already drifting away from the original topic of the thread so I will comment on the other arguments you made like resolution in a private e-mail. Best regards, Gerd On 13.07.2017 14:00, Gerard Bricogne wrote: Dear Gerd, I can assure you that I have no shares in Dectris nor any commecial connections with them. What I do have is a lot of still vivid memories of CCD images, with their wooly point-spread function that was affected by fine-grained spatial variability as well as by irredicible inaccuracies in the geometric corrections required to try and undo the distortions introduced by the fiber-optic taper. By comparison the pixel-array detectors have a very regular structure, so that slight deviations from exact registering of the modules can be calibrated with high accuracy, making it possible to get very small residuals between calculated and observed spot positions. That, I certainly never saw with CCD images. I do think that asking for the image width was a highly pertinent question in this case, that had not been asked. As a specialist you might know how to use a CCD to good effect in fine-slicing mode, but it is amazing how many people there are still out there who are told to use 0.5 or even 1.0 degree image widths. Compensating the poor PSF of a CCD by fine slicing in the angular dimension is a tall order. With a Pilatus at 350mm from the crystal, the angular separation between 174-micron pixels is 0.5 milliradian. To achieve that separation in the angular (rotation) dimension, the equivalent image width would have to be 0.03 degree. For an EIGER the numbers become 75 microns, hence 0.21 milliradian i.e. 0.012 degree. Hence my advice, untainted by any commercial agenda :-) . With best wishes, Gerard. -- On Thu, Jul 13, 2017 at 01:25:08PM -0500, Gerd Rosenbaum wrote: Dear Gerard, you sound like a sales person for Dectris. Fine slicing is perfectly fine with CCD detectors - it takes a bit longer because of the step scan instead of continuous scan. The read noise issue is often overstated compared to the sample induced scatter background. If for fine slicing at 0.05 degree or less the diffraction peaks go too close to the read noise make a longer exposure - signal goes up, ratio signal to sample-induced-BG less, as for any fine slicing, same read noise. It would be helpful to analyze the dense spot packing along layer lines if we knew the wavelength and the sample-to-detector distance (assuming this is a 300 mm detector) and the rotation width - as you pointed out. That would help to distinguish between multiple crystals (my guess) and lattice translocation disorder. Fine slicing is definitely needed to figure out what the diffraction pattern at 120 degree could tell you in terms of strong anisotropy . Best regard. Gerd On 13.07.2017 08:20, Gerard Bricogne wrote: Dear Tang, I noticed that your diffraction images seem to have been recorded on a 3x3 CCD detector. With this type of detector, fine slicing is often discouraged (because of the readout noise), and yet with the two long cell axes you have, any form of thick (or only semi-fine) slicing would result in spot overlaps. What, then, was your image width? Would you have access to a beamline with a Pilatus detector so that you could collect fine-sliced data? I would tend to agree with Herman that your crystals might be cursed with lattice translocation disorder (LTD), but you might as well try and put every chance of surviving this on your side by making sure that you collect fine-sliced data. LTD plus thick slicing would give you random data along the streaky direction. Use an image width of at most 0.1 degree (0.05 would be better) on a Pilatus, and use XDS to process your images. Good luck! Gerard -- On Thu, Jul 13, 2017 at 01:21:02PM +0100, Tang Chenjun wrote: Hi David, Thanks for your comments. Although the spots become streaky in certain directions, I have processed the data in HKL3000 and imosflm, which suggested the C2221 space group (66.59, 246.95 and 210.17). The Rmerge(0.14), completeness(94.8%), redundancy(4.6) are OK. When I tried to run Balbes with the solved native structure, the molecular replacement solution was poor. So I ran Balbes with the split domains of the native structure. Although the solutions were also
Re: [ccp4bb] weird diffraction pattern
Thanks for spelling it out! Would that advice still hold if the mosaicity of the crystal is 0.7 degrees? (I know, I should go read the paper., but . . .) eab On 07/13/2017 03:00 PM, Gerard Bricogne wrote: Dear Gerd, I can assure you that I have no shares in Dectris nor any commecial connections with them. What I do have is a lot of still vivid memories of CCD images, with their wooly point-spread function that was affected by fine-grained spatial variability as well as by irredicible inaccuracies in the geometric corrections required to try and undo the distortions introduced by the fiber-optic taper. By comparison the pixel-array detectors have a very regular structure, so that slight deviations from exact registering of the modules can be calibrated with high accuracy, making it possible to get very small residuals between calculated and observed spot positions. That, I certainly never saw with CCD images. I do think that asking for the image width was a highly pertinent question in this case, that had not been asked. As a specialist you might know how to use a CCD to good effect in fine-slicing mode, but it is amazing how many people there are still out there who are told to use 0.5 or even 1.0 degree image widths. Compensating the poor PSF of a CCD by fine slicing in the angular dimension is a tall order. With a Pilatus at 350mm from the crystal, the angular separation between 174-micron pixels is 0.5 milliradian. To achieve that separation in the angular (rotation) dimension, the equivalent image width would have to be 0.03 degree. For an EIGER the numbers become 75 microns, hence 0.21 milliradian i.e. 0.012 degree. Hence my advice, untainted by any commercial agenda :-) . With best wishes, Gerard. -- On Thu, Jul 13, 2017 at 01:25:08PM -0500, Gerd Rosenbaum wrote: Dear Gerard, you sound like a sales person for Dectris. Fine slicing is perfectly fine with CCD detectors - it takes a bit longer because of the step scan instead of continuous scan. The read noise issue is often overstated compared to the sample induced scatter background. If for fine slicing at 0.05 degree or less the diffraction peaks go too close to the read noise make a longer exposure - signal goes up, ratio signal to sample-induced-BG less, as for any fine slicing, same read noise. It would be helpful to analyze the dense spot packing along layer lines if we knew the wavelength and the sample-to-detector distance (assuming this is a 300 mm detector) and the rotation width - as you pointed out. That would help to distinguish between multiple crystals (my guess) and lattice translocation disorder. Fine slicing is definitely needed to figure out what the diffraction pattern at 120 degree could tell you in terms of strong anisotropy . Best regard. Gerd On 13.07.2017 08:20, Gerard Bricogne wrote: Dear Tang, I noticed that your diffraction images seem to have been recorded on a 3x3 CCD detector. With this type of detector, fine slicing is often discouraged (because of the readout noise), and yet with the two long cell axes you have, any form of thick (or only semi-fine) slicing would result in spot overlaps. What, then, was your image width? Would you have access to a beamline with a Pilatus detector so that you could collect fine-sliced data? I would tend to agree with Herman that your crystals might be cursed with lattice translocation disorder (LTD), but you might as well try and put every chance of surviving this on your side by making sure that you collect fine-sliced data. LTD plus thick slicing would give you random data along the streaky direction. Use an image width of at most 0.1 degree (0.05 would be better) on a Pilatus, and use XDS to process your images. Good luck! Gerard -- On Thu, Jul 13, 2017 at 01:21:02PM +0100, Tang Chenjun wrote: Hi David, Thanks for your comments. Although the spots become streaky in certain directions, I have processed the data in HKL3000 and imosflm, which suggested the C2221 space group (66.59, 246.95 and 210.17). The Rmerge(0.14), completeness(94.8%), redundancy(4.6) are OK. When I tried to run Balbes with the solved native structure, the molecular replacement solution was poor. So I ran Balbes with the split domains of the native structure. Although the solutions were also poor, I found the MR score of one solution above 35. On the basis of this solution, I tried to run Buccaneer and the Rfree could be 0.46. Unfortunately, there are four molecules in the asymmetric unit and it is to hard for me to reduce the Rfree further. All best, Chenjun Tang
Re: [ccp4bb] weird diffraction pattern
Dear Gerd, I can assure you that I have no shares in Dectris nor any commecial connections with them. What I do have is a lot of still vivid memories of CCD images, with their wooly point-spread function that was affected by fine-grained spatial variability as well as by irredicible inaccuracies in the geometric corrections required to try and undo the distortions introduced by the fiber-optic taper. By comparison the pixel-array detectors have a very regular structure, so that slight deviations from exact registering of the modules can be calibrated with high accuracy, making it possible to get very small residuals between calculated and observed spot positions. That, I certainly never saw with CCD images. I do think that asking for the image width was a highly pertinent question in this case, that had not been asked. As a specialist you might know how to use a CCD to good effect in fine-slicing mode, but it is amazing how many people there are still out there who are told to use 0.5 or even 1.0 degree image widths. Compensating the poor PSF of a CCD by fine slicing in the angular dimension is a tall order. With a Pilatus at 350mm from the crystal, the angular separation between 174-micron pixels is 0.5 milliradian. To achieve that separation in the angular (rotation) dimension, the equivalent image width would have to be 0.03 degree. For an EIGER the numbers become 75 microns, hence 0.21 milliradian i.e. 0.012 degree. Hence my advice, untainted by any commercial agenda :-) . With best wishes, Gerard. -- On Thu, Jul 13, 2017 at 01:25:08PM -0500, Gerd Rosenbaum wrote: > Dear Gerard, > > you sound like a sales person for Dectris. Fine slicing is perfectly fine > with CCD detectors - it takes a bit longer because of the step scan instead > of continuous scan. The read noise issue is often overstated compared to the > sample induced scatter background. If for fine slicing at 0.05 degree or > less the diffraction peaks go too close to the read noise make a longer > exposure - signal goes up, ratio signal to sample-induced-BG less, as for > any fine slicing, same read noise. > > It would be helpful to analyze the dense spot packing along layer lines if > we knew the wavelength and the sample-to-detector distance (assuming this is > a 300 mm detector) and the rotation width - as you pointed out. That would > help to distinguish between multiple crystals (my guess) and lattice > translocation disorder. Fine slicing is definitely needed to figure out what > the diffraction pattern at 120 degree could tell you in terms of strong > anisotropy . > > Best regard. > > Gerd > > On 13.07.2017 08:20, Gerard Bricogne wrote: > >Dear Tang, > > > > I noticed that your diffraction images seem to have been recorded > >on a 3x3 CCD detector. With this type of detector, fine slicing is > >often discouraged (because of the readout noise), and yet with the two > >long cell axes you have, any form of thick (or only semi-fine) slicing > >would result in spot overlaps. > > > > What, then, was your image width? Would you have access to a > >beamline with a Pilatus detector so that you could collect fine-sliced > >data? > > > > I would tend to agree with Herman that your crystals might be > >cursed with lattice translocation disorder (LTD), but you might as > >well try and put every chance of surviving this on your side by making > >sure that you collect fine-sliced data. LTD plus thick slicing would > >give you random data along the streaky direction. Use an image width > >of at most 0.1 degree (0.05 would be better) on a Pilatus, and use XDS > >to process your images. > > > > > > Good luck! > >Gerard > > > >-- > >On Thu, Jul 13, 2017 at 01:21:02PM +0100, Tang Chenjun wrote: > >>Hi David, > >>Thanks for your comments. Although the spots become streaky in certain > >>directions, I have processed the data in HKL3000 and imosflm, which > >>suggested the C2221 space group (66.59, 246.95 and 210.17). The > >>Rmerge(0.14), completeness(94.8%), redundancy(4.6) are OK. When I tried to > >>run Balbes with the solved native structure, the molecular replacement > >>solution was poor. So I ran Balbes with the split domains of the native > >>structure. Although the solutions were also poor, I found the MR score of > >>one solution above 35. On the basis of this solution, I tried to run > >>Buccaneer and the Rfree could be 0.46. Unfortunately, there are four > >>molecules in the asymmetric unit and it is to hard for me to reduce the > >>Rfree further. > >> > >>All best, > >> > >>Chenjun Tang
Re: [ccp4bb] weird diffraction pattern
Dear Gerard, you sound like a sales person for Dectris. Fine slicing is perfectly fine with CCD detectors - it takes a bit longer because of the step scan instead of continuous scan. The read noise issue is often overstated compared to the sample induced scatter background. If for fine slicing at 0.05 degree or less the diffraction peaks go too close to the read noise make a longer exposure - signal goes up, ratio signal to sample-induced-BG less, as for any fine slicing, same read noise. It would be helpful to analyze the dense spot packing along layer lines if we knew the wavelength and the sample-to-detector distance (assuming this is a 300 mm detector) and the rotation width - as you pointed out. That would help to distinguish between multiple crystals (my guess) and lattice translocation disorder. Fine slicing is definitely needed to figure out what the diffraction pattern at 120 degree could tell you in terms of strong anisotropy . Best regard. Gerd On 13.07.2017 08:20, Gerard Bricogne wrote: Dear Tang, I noticed that your diffraction images seem to have been recorded on a 3x3 CCD detector. With this type of detector, fine slicing is often discouraged (because of the readout noise), and yet with the two long cell axes you have, any form of thick (or only semi-fine) slicing would result in spot overlaps. What, then, was your image width? Would you have access to a beamline with a Pilatus detector so that you could collect fine-sliced data? I would tend to agree with Herman that your crystals might be cursed with lattice translocation disorder (LTD), but you might as well try and put every chance of surviving this on your side by making sure that you collect fine-sliced data. LTD plus thick slicing would give you random data along the streaky direction. Use an image width of at most 0.1 degree (0.05 would be better) on a Pilatus, and use XDS to process your images. Good luck! Gerard -- On Thu, Jul 13, 2017 at 01:21:02PM +0100, Tang Chenjun wrote: Hi David, Thanks for your comments. Although the spots become streaky in certain directions, I have processed the data in HKL3000 and imosflm, which suggested the C2221 space group (66.59, 246.95 and 210.17). The Rmerge(0.14), completeness(94.8%), redundancy(4.6) are OK. When I tried to run Balbes with the solved native structure, the molecular replacement solution was poor. So I ran Balbes with the split domains of the native structure. Although the solutions were also poor, I found the MR score of one solution above 35. On the basis of this solution, I tried to run Buccaneer and the Rfree could be 0.46. Unfortunately, there are four molecules in the asymmetric unit and it is to hard for me to reduce the Rfree further. All best, Chenjun Tang
Re: [ccp4bb] weird diffraction pattern
Hi Chenjun, Few suggestions from my side. Process the data with XDS and look into acentric intensity distribution (it indicates any twinning possibility). Run XTRIAGE and SFCHECK to understand any twinning or pseudo translation possibilities. Twinning can confuse the program and suggest you smaller unit cell with higher symmetry. Your images indicate longer cell axis. If you need more help, please send me an email. Thank you Rajesh ---x With regards Rajesh K. Harijan, Ph.D. Schramm Laboratory Albert Einstein College of Medicine 1300 Morris Park Ave., Bronx, NY 10461 Tel: 718.430.2777 | Fax: 718.430.8565 On Thu, Jul 13, 2017 at 3:56 AM, 唐晨骏 <0910010...@cau.edu.cn> wrote: > hello everyone, > I would like to seek your opinion on my crystal hits. I am working on a > helicase > > of which the native structure is solved and the all solution statistics are > > fine. I am trying to crystallize and solve the structure of the > protein/ssDNA > > complex. I recently got some hits from commercial screens using sitting > drop > > vapor diffusion. After crystallization optimization, these crystals > diffract > > weakly but to 3.2 Angstroms for the longer exposure time. However, when the > > crystals rotate between 120 degrees to 180 degrees, the spots become > streaky > > (attached), no matter the crystals are hexagonal or flaky. I have tried to > > determine the structure by molecular replacement method, but the > Rwork/Rfree > > values are huge (above 0.5) and can’t be reduced further. I suspect the > > obtained crystals quality and resulting processed statistics is the reason > for > > the observed high Rwork/Rfree values. Are there any suggestions? > > All comments will be appreciated! > > Best, > Chenjun Tang > > >
Re: [ccp4bb] weird diffraction pattern
- which may well be caused by your cryo-protection or flash-cooling procedure. I'd try to collect a few images at room temperature to see how good the crystals can be and if this procedure can be improved. To prevent overlaps, it may help to find a way to collect the data with the crystal rotating around the most problematic cell axis, which tends to be the shortest in the crystal. Bent loops might be helpful. Mark J van Raaij Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC calle Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://wwwuser <http://www.cnb.csic.es/~mjvanraaij>.cnb.csic.es/~mjvanraaij <http://www.cnb.csic.es/~mjvanraaij> > On 13 Jul 2017, at 11:13, Keller, Jacob <kell...@janelia.hhmi.org> wrote: > > You've got multiple lattices--try seeding approaches mentioned in a > recent/current thread. > > JPK > > -Original Message- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ??? > Sent: Thursday, July 13, 2017 3:56 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] weird diffraction pattern > > hello everyone, > I would like to seek your opinion on my crystal hits. I am working on a > helicase > > of which the native structure is solved and the all solution statistics are > > fine. I am trying to crystallize and solve the structure of the protein/ssDNA > > complex. I recently got some hits from commercial screens using sitting drop > > vapor diffusion. After crystallization optimization, these crystals diffract > > weakly but to 3.2 Angstroms for the longer exposure time. However, when the > > crystals rotate between 120 degrees to 180 degrees, the spots become streaky > > (attached), no matter the crystals are hexagonal or flaky. I have tried to > > determine the structure by molecular replacement method, but the Rwork/Rfree > > values are huge (above 0.5) and can’t be reduced further. I suspect the > > obtained crystals quality and resulting processed statistics is the reason > for > > the observed high Rwork/Rfree values. Are there any suggestions? > > All comments will be appreciated! > > Best, > Chenjun Tang > >
Re: [ccp4bb] weird diffraction pattern
Dear Tang, I noticed that your diffraction images seem to have been recorded on a 3x3 CCD detector. With this type of detector, fine slicing is often discouraged (because of the readout noise), and yet with the two long cell axes you have, any form of thick (or only semi-fine) slicing would result in spot overlaps. What, then, was your image width? Would you have access to a beamline with a Pilatus detector so that you could collect fine-sliced data? I would tend to agree with Herman that your crystals might be cursed with lattice translocation disorder (LTD), but you might as well try and put every chance of surviving this on your side by making sure that you collect fine-sliced data. LTD plus thick slicing would give you random data along the streaky direction. Use an image width of at most 0.1 degree (0.05 would be better) on a Pilatus, and use XDS to process your images. Good luck! Gerard -- On Thu, Jul 13, 2017 at 01:21:02PM +0100, Tang Chenjun wrote: > Hi David, > Thanks for your comments. Although the spots become streaky in certain > directions, I have processed the data in HKL3000 and imosflm, which suggested > the C2221 space group (66.59, 246.95 and 210.17). The Rmerge(0.14), > completeness(94.8%), redundancy(4.6) are OK. When I tried to run Balbes with > the solved native structure, the molecular replacement solution was poor. So > I ran Balbes with the split domains of the native structure. Although the > solutions were also poor, I found the MR score of one solution above 35. On > the basis of this solution, I tried to run Buccaneer and the Rfree could be > 0.46. Unfortunately, there are four molecules in the asymmetric unit and it > is to hard for me to reduce the Rfree further. > > All best, > > Chenjun Tang -- === * * * Gerard Bricogne g...@globalphasing.com * * * * Global Phasing Ltd. * * Sheraton House, Castle Park Tel: +44-(0)1223-353033 * * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 * * * ===
[ccp4bb] AW: [ccp4bb] weird diffraction pattern
Hi Chenjun Tang, From the images you sent, it looks like your crystal suffers from lattice translocation disorder. See e.g. http://onlinelibrary.wiley.com/doi/10.1107/S0907444909025153/epdf Calculating a native Patterson and looking for strange peaks may give some hints what is going on. Depending on the nature of the disorder, you may or may not correct for it. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Tang Chenjun Gesendet: Donnerstag, 13. Juli 2017 14:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] weird diffraction pattern Hi David, Thanks for your comments. Although the spots become streaky in certain directions, I have processed the data in HKL3000 and imosflm, which suggested the C2221 space group (66.59, 246.95 and 210.17). The Rmerge(0.14), completeness(94.8%), redundancy(4.6) are OK. When I tried to run Balbes with the solved native structure, the molecular replacement solution was poor. So I ran Balbes with the split domains of the native structure. Although the solutions were also poor, I found the MR score of one solution above 35. On the basis of this solution, I tried to run Buccaneer and the Rfree could be 0.46. Unfortunately, there are four molecules in the asymmetric unit and it is to hard for me to reduce the Rfree further. All best, Chenjun Tang
Re: [ccp4bb] weird diffraction pattern
Hi Jacob, I have tried seeding approaches but it didn't help. All best, Chenjun Tang
Re: [ccp4bb] weird diffraction pattern
Hi David, Thanks for your comments. Although the spots become streaky in certain directions, I have processed the data in HKL3000 and imosflm, which suggested the C2221 space group (66.59, 246.95 and 210.17). The Rmerge(0.14), completeness(94.8%), redundancy(4.6) are OK. When I tried to run Balbes with the solved native structure, the molecular replacement solution was poor. So I ran Balbes with the split domains of the native structure. Although the solutions were also poor, I found the MR score of one solution above 35. On the basis of this solution, I tried to run Buccaneer and the Rfree could be 0.46. Unfortunately, there are four molecules in the asymmetric unit and it is to hard for me to reduce the Rfree further. All best, Chenjun Tang
Re: [ccp4bb] weird diffraction pattern
You've got multiple lattices--try seeding approaches mentioned in a recent/current thread. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ??? Sent: Thursday, July 13, 2017 3:56 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] weird diffraction pattern hello everyone, I would like to seek your opinion on my crystal hits. I am working on a helicase of which the native structure is solved and the all solution statistics are fine. I am trying to crystallize and solve the structure of the protein/ssDNA complex. I recently got some hits from commercial screens using sitting drop vapor diffusion. After crystallization optimization, these crystals diffract weakly but to 3.2 Angstroms for the longer exposure time. However, when the crystals rotate between 120 degrees to 180 degrees, the spots become streaky (attached), no matter the crystals are hexagonal or flaky. I have tried to determine the structure by molecular replacement method, but the Rwork/Rfree values are huge (above 0.5) and can’t be reduced further. I suspect the obtained crystals quality and resulting processed statistics is the reason for the observed high Rwork/Rfree values. Are there any suggestions? All comments will be appreciated! Best, Chenjun Tang
Re: [ccp4bb] weird diffraction pattern
Hi, I'd need to see more processing stats to figure out your data issues. Streaky spots in certain directions can be indicative of anisotropy and/or lattice disorders. However, if your Rfree is above 0.5, it is likely that your molecular replacement solution is poor/bad/wrong. HTH, Dave -- Dr David C Briggs Hohenester Lab Department of Life Sciences Imperial College London UK http://about.me/david_briggs From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of 唐晨骏 <0910010...@cau.edu.cn> Sent: Thursday, July 13, 2017 8:56:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] weird diffraction pattern hello everyone, I would like to seek your opinion on my crystal hits. I am working on a helicase of which the native structure is solved and the all solution statistics are fine. I am trying to crystallize and solve the structure of the protein/ssDNA complex. I recently got some hits from commercial screens using sitting drop vapor diffusion. After crystallization optimization, these crystals diffract weakly but to 3.2 Angstroms for the longer exposure time. However, when the crystals rotate between 120 degrees to 180 degrees, the spots become streaky (attached), no matter the crystals are hexagonal or flaky. I have tried to determine the structure by molecular replacement method, but the Rwork/Rfree values are huge (above 0.5) and can’t be reduced further. I suspect the obtained crystals quality and resulting processed statistics is the reason for the observed high Rwork/Rfree values. Are there any suggestions? All comments will be appreciated! Best, Chenjun Tang
[ccp4bb] weird diffraction pattern
hello everyone, I would like to seek your opinion on my crystal hits. I am working on a helicase of which the native structure is solved and the all solution statistics are fine. I am trying to crystallize and solve the structure of the protein/ssDNA complex. I recently got some hits from commercial screens using sitting drop vapor diffusion. After crystallization optimization, these crystals diffract weakly but to 3.2 Angstroms for the longer exposure time. However, when the crystals rotate between 120 degrees to 180 degrees, the spots become streaky (attached), no matter the crystals are hexagonal or flaky. I have tried to determine the structure by molecular replacement method, but the Rwork/Rfree values are huge (above 0.5) and can’t be reduced further. I suspect the obtained crystals quality and resulting processed statistics is the reason for the observed high Rwork/Rfree values. Are there any suggestions? All comments will be appreciated! Best, Chenjun Tang
