Re: [ccp4bb] changing resolution bins statistics in CORRECT.LP
Dear Sebastiano, sorry, it is not possible. Indeed, only XCSALE allows you to define the resolution ranges yourself. HTH, Kay On Tue, 15 Jan 2013 16:22:14 +0100, Sebastiano Pasqualato sebastiano.pasqual...@gmail.com wrote: Hi all, I was wondering if XDS allows to change the number of resolution bins appearing in the table: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION of CORRECT.LP. Please, note that I am not referring to the table output by XSCALE, in which you can change the resolution bins with the keyword RESOLUTION_SHELLS=, but rather the table output by the CORRECT job of XDS. Thanks in advance, ciao, Sebastiano
[ccp4bb] protein degradation in crystal
Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several samll bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa
[ccp4bb] REMINDER: Joint CeBEM/CCP4 Macromolecular Crystallography School announcement 2013 in Uruguay
Dear Colleagues, We are pleased to announce the *second Macromolecular Crystallography School 2013 *at the Institut Pasteur de Montevideo (Uruguay). All details can be found at www.pasteur.edu.uy/mx2013 http://www.pasteur.edu.uy/mx2013 *Title:* Macromolecular Crystallography School 2013: From data processing to structure refinement and beyond *Dates:*April 9th-17th, 2013. *Site:*Institut Pasteur de Montevideo (Montevideo), Uruguay *The workshop content:* Conceived to provide theoretical background and hands-on abilities in the use of computational tools to exploit X ray diffraction data. Through lectures, tutorials and hands-on trouble-shooting, the students will be trained in state-of-the-art macromolecular crystallography. Particular emphasis will be given to data processing, phasing/structure determination and model refinement/validation. The workshop will feature authors and experts of many modern crystallographic software packages. This workshop represents the continuation within the series started in 2010 (http://www.pasteur.edu.uy/mxcourse) on Macromolecular Crystallography. In this opportunity it is being co-organized by the Center for Structural Biology of the Mercosur CeBEM (www.cebem.org.ar http://www.cebem.org.ar), jointly with the Collaborative Computational Project Number 4 (CCP4 -- UK www.ccp4.ac.uk http://www.ccp4.ac.uk). Support from the Institut Pasteur International Network, the International Union of Crystallography and Institut Pasteur de Montevideo is also greatly acknowledged. *Applicants:* Graduate students, postdoctoral researchers and young scientists are encouraged to apply. Only 20 applicants will be selected for participation. Participants of the workshop are strongly encouraged to bring their own problem data sets so they can be used during the workshop's hands-on sessions. There is no registration fee. Support for accommodation, per diem and local transportation will be provided to all participants from abroad. Support to cover travel expenses will be considered on a case-by-case basis. Specific requests should be well-grounded as we will only be able to select a limited number. *Application:* Application *deadline is February 10, 2013*. Application form, the program, contact info and other details can be found at www.pasteur.edu.uy/mx2013 http://www.pasteur.edu.uy/mx2013 Please address further inquiries to mx2...@pasteur.edu.uy mailto:mx2...@pasteur.edu.uy Ronan Keegan and Alejandro Buschiazzo -- Alejandro Buschiazzo, PhD Research Scientist Unit of Protein Crystallography Institut Pasteur de Montevideo Mataojo 2020 Montevideo 11400 URUGUAY Phone: +598 25220910 int. 120 Fax: +598 25224185 http://www.pasteur.edu.uy/pxf
Re: [ccp4bb] changing resolution bins statistics in CORRECT.LP
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Sebastiano, you could use xprep to get the statistics in user defined resolution shells. Out of curiosity: Would you mind sharing why you want to do this and why you don't want to use the XSCALE statistics instead? The statistics are probably more meaningful after scaling, I guess. Best, Tim On 01/15/2013 04:22 PM, Sebastiano Pasqualato wrote: Hi all, I was wondering if XDS allows to change the number of resolution bins appearing in the table: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION of CORRECT.LP. Please, note that I am not referring to the table output by XSCALE, in which you can change the resolution bins with the keyword RESOLUTION_SHELLS=, but rather the table output by the CORRECT job of XDS. Thanks in advance, ciao, Sebastiano - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQ9pk/UxlJ7aRr7hoRAqreAJ9/aKSHyXWahBc96/3x0U5iriZk4gCg2fWC GVLgXUEN+10M9IpCDRENGF0= =SK0P -END PGP SIGNATURE-
Re: [ccp4bb] protein degradation in crystal
Dear Lisa It is not uncommon to see breakdown products when you run crystals on gel. Espesially if they are older crystals, sometimes you even see higher molecular bands, these are probably due to intra molecular cross links formed over time. If you are worried about stability, try to increase the crystallization speed, we have one example where we see a clear difference in both crystal quality and even space group depending on when we fish the crystals. The crystals appear within 5 min, the best quality data sets come from crystals we fish after only 30-60 min. You may also have a little protease contamination of course, to prevent this add protease inhibitor, or DTT, or EDTA to you protein before you set it up. cheers Preben On 1/16/13 12:14 PM, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several samll bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 http://www.jpmorth.dk
Re: [ccp4bb] advices
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Mike, difficult to give specific advice without knowing the crystallisation conditions. - - at the beginning in the unmixed drop your protein is soluble - - after equilibration with the mother liquor there is a crystal. Think about what is happening within the crystallisation chamber and find the spot where you don't have crystals. It must exist. I would first lower the precipitant concentration. You may instead want to lower the precipitation concentration difference by adding some of the precipitant to the protein solution (making sure the protein stays soluble). Good luck, Tim On 01/16/2013 02:30 AM, Mike John wrote: Hello, all, My crystal grows very fast even though the protein conc. is now 1.5 mg/ml. The shape of the crystal likes a ruler plate with one side very thin. The crystal quality has diffraction to about 3.5A in a home source of 1.2 KW sealed tube Angilent equipment. But the data can not be indexed due to one direction has poor diffraction and the crystal quality. Seeking advices on improving the crystal quality. Thank you very much Mike - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQ9puXUxlJ7aRr7hoRAhDKAJ9dW9KufS3w5pdrgKqB34LBBMKKIgCfZs5f 9gss5fNzJi+CrSAmX1kr2L0= =Z5BF -END PGP SIGNATURE-
Re: [ccp4bb] crystal dehydration
Judith, If you can, try monitoring the diffraction during the dehydration experiment. There are plenty of ways of doing this. Perhaps the simplest is to shoot your crystals in-situ after swapping the precipitant for a more concentrated one. The is also the Free Mounting System. David Hargreaves Associate Principal Scientist _ AstraZeneca Discovery Sciences, Structure Biophysics Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.com Please consider the environment before printing this e-mail -- AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking Compliance with our Code of Conduct and Policies. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Judith A Ronau Sent: 15 January 2013 14:47 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystal dehydration Greetings! I have recently been attempting crystal dehydration experiments to improve diffraction following the procedures from the ERSF in which crystals are exposed to increased concentrations of precipitant. I would like to know if anyone knew of any alternative methods for dehydration of protein crystals. Thanks! Best, Judith
Re: [ccp4bb] changing resolution bins statistics in CORRECT.LP
Thanks to Kay and Tim or the feedback. The reason I wanted to get statistics from the CORRECT step of XDS is that I have refined a structure using the mtz output by the GO.COM automatic reduction routine of SLS beamline PXIII, which does not involve a scaling step (I discovered recently). I was willing to have the integration statistics of the reflection file I used in the refinement's high resolution bin. I will definitely give xprep a try. Another question that raised by looking deeper into their automatic procedure (thanks Meitian for the help) is that when integrating with XDS CORRECT keeping the FRIEDEL'S_LAW=TRUE or =FALSE I get a different number of reflections in the final mtz. In my case, if I run the same XDS.INP script and change only the FRIEDEL'S_LAW flag, I obtain: =TRUE: 11551 reflections * Resolution Range : 0.000430.11138 ( 48.225 - 2.996 A ) * Sort Order : 1 2 3 0 0 * Space group = 'P 3 2 1' (number 150) OVERALL FILE STATISTICS for resolution range 0.000 - 0.111 === Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 29 0 100.00 13.7 13.7 48.22 3.00 H H 2 NONE 0 16 0 100.00 4.7 4.7 48.22 3.00 H K 3 NONE -32 32 0 100.00 0.5 12.2 48.22 3.00 H L 4 NONE1.4 292.0 0 100.0019.4019.40 48.22 3.00 F FP 5 NONE0.1 4.3 0 100.00 0.70 0.70 48.22 3.00 Q SIGFP 6 NONE0.0 1.0 0 100.00 0.95 0.95 48.22 3.00 I FreeRflag No. of reflections used in FILE STATISTICS11551 =FALSE: 11643 reflections * Cell Dimensions : (obsolete - refer to dataset cell dimensions above) 100.5450 100.5450 96.4500 90. 90. 120. * Resolution Range : 0.000430.11138 ( 48.225 - 2.996 A ) * Sort Order : 1 2 3 0 0 * Space group = 'P 3 2 1' (number 150) OVERALL FILE STATISTICS for resolution range 0.000 - 0.111 === Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 29 0 100.00 13.7 13.7 48.22 3.00 H H 2 NONE 0 16 0 100.00 4.7 4.7 48.22 3.00 H K 3 NONE -32 32 0 100.00 0.5 12.2 48.22 3.00 H L 4 NONE1.4 291.9 0 100.0019.4019.40 48.22 3.00 F FP 5 NONE0.1 4.3 0 100.00 0.67 0.67 48.22 3.00 Q SIGFP 6 NONE -13.613.269 99.41-0.01 0.69 48.22 3.00 D DANO 7 NONE0.0 5.769 99.41 1.13 1.13 48.22 3.00 Q SIGDANO 8 NONE 0 2 0 100.00 0.0 0.0 48.22 3.00 Y ISYM 9 NONE0.0 1.0 0 100.00 0.95 0.95 48.22 3.00 I FreeRflag No. of reflections used in FILE STATISTICS11643 Aren't they supposed to be the exact same number? Thanks, ciao, s On Jan 16, 2013, at 1:12 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Sebastiano, you could use xprep to get the statistics in user defined resolution shells. Out of curiosity: Would you mind sharing why you want to do this and why you don't want to use the XSCALE statistics instead? The statistics are probably more meaningful after scaling, I guess. Best, Tim On 01/15/2013 04:22 PM, Sebastiano Pasqualato wrote: Hi all, I was wondering if XDS allows to change the number of resolution bins appearing in the table: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION of CORRECT.LP. Please, note that I am not referring to the table output by XSCALE, in which you can change the resolution bins with the keyword RESOLUTION_SHELLS=, but rather the table output by the CORRECT job of XDS. Thanks in advance, ciao, Sebastiano - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQ9pk/UxlJ7aRr7hoRAqreAJ9/aKSHyXWahBc96/3x0U5iriZk4gCg2fWC GVLgXUEN+10M9IpCDRENGF0= =SK0P -END PGP SIGNATURE- -- Sebastiano Pasqualato, PhD Crystallography Unit Department of Experimental Oncology European Institute of Oncology IFOM-IEO Campus via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5167 fax +39 02 9437 5990 please note the change in email address! sebastiano.pasqual...@ieo.eu
[ccp4bb] side question re crystal dehydration
Dear all, From Leonid's reply earlier you can see a problem some of us have been having for a while now, when looking for literature regarding dehydration. Most of you that perform dehydration either don't consider it happening or don't report it in great detail in your publications. This is only understandable because it isn't the focus of your work and it only helps you get to where you want to get to. I'm trying to get an up to date picture of what is out there but I haven't got the time or eyes to go through everyone's methods to pick the couple of lines that describe your particular method. I really want to find out what is being done to be able to give people better advice. So: Could people out there that think that in their particular projects dehydration/hydration had an effect send me a ref. or a short description? (can be done outside the BB to not spam everyone) I will duly acknowledge everyone!! By dehydration I mean: 1 Soaking with increasing concentration of precipitants or salts 2 By equilibrating against a new precipitant or salt (by vapour diffusion or dialysis) 3 By letting the drops dry (controlled or uncontrolled) 4 by using an FMS/HC1/MicroRT or any other gadget 5 By some other magical trick you may have Thank you all for your help, Regards Juan Juan Sanchez-Weatherby, PhD Beamline Scientist - I02 Macromolecular Crystallography Group Diamond Light Source Ltd Diamond House DR1.64 Harwell Science and Innovation Campus RAL, Chilton, Didcot Oxfordshire OX11 0DE United Kingdom Tel: +44 (0)1235 778661 Mob:+44 (0)7795 641259 Fax:+44 (0)1235 778052 juan.sanchez-weathe...@diamond.ac.uk http://www.diamond.ac.uk -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Leonid Sazanov Sent: 15 January 2013 19:32 To: ccp4bb Subject: Re: [ccp4bb] crystal dehydration In case if dehydration needs to be done slowly and under tight control of all parameters, one possibility is to use micro-dialysis buttons. We used it for a large membrane protein complex and diffraction improved from ~7 to 2.7 A. The crystal is fished out and put into mother liquor solution in the button, sealed with dialysis membrane and the button is then placed into about 5 mls of mother liquor with slightly higher PEG concentration. Then you just exchange outside buffer every day or so for solutions containing higher concentrations of PEG. We went from ~9 to 30 % PEG4000 in about a week. You can easily observe crystal under microscope and if it cracks - you went too far/too quickly with PEG and need to use a bit less next time. Also, this method allows you to control all other components of the dehydrating solution - we needed to decrease salt concentration at the same time as increasing PEG. You can also introduce/increase cryo-protectant concentration at the same time. With these crystals, otherwise excellent dehydration machines already mentioned did not work, possibly because the process had to be really slow. The reference is here: http://www.ncbi.nlm.nih.gov/pubmed/21822288 Best wishes.
Re: [ccp4bb] changing resolution bins statistics in CORRECT.LP
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Sebastiano, if the output of GO.COM produces an mtz-file you can use for phasing or refinement, I am very confident there is a scaling step involved. This might also be an explanation for the discrepancy you point out: With FRIEDEL'S_LAW=TRUE the statistics which affects the rejection of outliers would have more reflections within each group of symmetry related reflections and hence a greater spread which might lead to the rejection of some of the classes. But this is a mere guess. Cheers, Tim On 01/16/2013 02:03 PM, Sebastiano Pasqualato wrote: Thanks to Kay and Tim or the feedback. The reason I wanted to get statistics from the CORRECT step of XDS is that I have refined a structure using the mtz output by the GO.COM automatic reduction routine of SLS beamline PXIII, which does not involve a scaling step (I discovered recently). I was willing to have the integration statistics of the reflection file I used in the refinement's high resolution bin. I will definitely give xprep a try. Another question that raised by looking deeper into their automatic procedure (thanks Meitian for the help) is that when integrating with XDS CORRECT keeping the FRIEDEL'S_LAW=TRUE or =FALSE I get a different number of reflections in the final mtz. In my case, if I run the same XDS.INP script and change only the FRIEDEL'S_LAW flag, I obtain: =TRUE: 11551 reflections * Resolution Range : 0.000430.11138 ( 48.225 - 2.996 A ) * Sort Order : 1 2 3 0 0 * Space group = 'P 3 2 1' (number 150) OVERALL FILE STATISTICS for resolution range 0.000 - 0.111 === Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 29 0 100.00 13.7 13.7 48.22 3.00 H H 2 NONE 0 16 0 100.00 4.7 4.7 48.22 3.00 H K 3 NONE -32 32 0 100.00 0.5 12.2 48.22 3.00 H L 4 NONE1.4 292.0 0 100.0019.40 19.40 48.22 3.00 F FP 5 NONE0.1 4.3 0 100.00 0.70 0.70 48.22 3.00 Q SIGFP 6 NONE0.0 1.0 0 100.00 0.95 0.95 48.22 3.00 I FreeRflag No. of reflections used in FILE STATISTICS11551 =FALSE: 11643 reflections * Cell Dimensions : (obsolete - refer to dataset cell dimensions above) 100.5450 100.5450 96.4500 90. 90. 120. * Resolution Range : 0.000430.11138 ( 48.225 - 2.996 A ) * Sort Order : 1 2 3 0 0 * Space group = 'P 3 2 1' (number 150) OVERALL FILE STATISTICS for resolution range 0.000 - 0.111 === Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 29 0 100.00 13.7 13.7 48.22 3.00 H H 2 NONE 0 16 0 100.00 4.7 4.7 48.22 3.00 H K 3 NONE -32 32 0 100.00 0.5 12.2 48.22 3.00 H L 4 NONE1.4 291.9 0 100.0019.40 19.40 48.22 3.00 F FP 5 NONE0.1 4.3 0 100.00 0.67 0.67 48.22 3.00 Q SIGFP 6 NONE -13.613.269 99.41-0.01 0.69 48.22 3.00 D DANO 7 NONE0.0 5.769 99.41 1.13 1.13 48.22 3.00 Q SIGDANO 8 NONE 0 2 0 100.00 0.0 0.0 48.22 3.00 Y ISYM 9 NONE0.0 1.0 0 100.00 0.95 0.95 48.22 3.00 I FreeRflag No. of reflections used in FILE STATISTICS11643 Aren't they supposed to be the exact same number? Thanks, ciao, s On Jan 16, 2013, at 1:12 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear Sebastiano, you could use xprep to get the statistics in user defined resolution shells. Out of curiosity: Would you mind sharing why you want to do this and why you don't want to use the XSCALE statistics instead? The statistics are probably more meaningful after scaling, I guess. Best, Tim On 01/15/2013 04:22 PM, Sebastiano Pasqualato wrote: Hi all, I was wondering if XDS allows to change the number of resolution bins appearing in the table: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION of CORRECT.LP. Please, note that I am not referring to the table output by XSCALE, in which you can change the resolution bins with the keyword RESOLUTION_SHELLS=, but rather the table output by the CORRECT job of XDS. Thanks in advance, ciao, Sebastiano - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/
Re: [ccp4bb] changing resolution bins statistics in CORRECT.LP
The XDS CORRECT step will also (by default) do scaling - if I understand correctly the same scaling as in XSCALE, but for a single sweep and with no zero-dose. If what you want is merging statistics, I find it helpful to write out the data unmerged and then use pointless -c and aimless to merge the data (with scales constant) which provides a very nice summary. Best wishes, Graeme On 16 January 2013 13:19, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Sebastiano, if the output of GO.COM produces an mtz-file you can use for phasing or refinement, I am very confident there is a scaling step involved. This might also be an explanation for the discrepancy you point out: With FRIEDEL'S_LAW=TRUE the statistics which affects the rejection of outliers would have more reflections within each group of symmetry related reflections and hence a greater spread which might lead to the rejection of some of the classes. But this is a mere guess. Cheers, Tim On 01/16/2013 02:03 PM, Sebastiano Pasqualato wrote: Thanks to Kay and Tim or the feedback. The reason I wanted to get statistics from the CORRECT step of XDS is that I have refined a structure using the mtz output by the GO.COM automatic reduction routine of SLS beamline PXIII, which does not involve a scaling step (I discovered recently). I was willing to have the integration statistics of the reflection file I used in the refinement's high resolution bin. I will definitely give xprep a try. Another question that raised by looking deeper into their automatic procedure (thanks Meitian for the help) is that when integrating with XDS CORRECT keeping the FRIEDEL'S_LAW=TRUE or =FALSE I get a different number of reflections in the final mtz. In my case, if I run the same XDS.INP script and change only the FRIEDEL'S_LAW flag, I obtain: =TRUE: 11551 reflections * Resolution Range : 0.000430.11138 ( 48.225 - 2.996 A ) * Sort Order : 1 2 3 0 0 * Space group = 'P 3 2 1' (number 150) OVERALL FILE STATISTICS for resolution range 0.000 - 0.111 === Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 29 0 100.00 13.7 13.7 48.22 3.00 H H 2 NONE 0 16 0 100.00 4.7 4.7 48.22 3.00 H K 3 NONE -32 32 0 100.00 0.5 12.2 48.22 3.00 H L 4 NONE1.4 292.0 0 100.0019.40 19.40 48.22 3.00 F FP 5 NONE0.1 4.3 0 100.00 0.70 0.70 48.22 3.00 Q SIGFP 6 NONE0.0 1.0 0 100.00 0.95 0.95 48.22 3.00 I FreeRflag No. of reflections used in FILE STATISTICS11551 =FALSE: 11643 reflections * Cell Dimensions : (obsolete - refer to dataset cell dimensions above) 100.5450 100.5450 96.4500 90. 90. 120. * Resolution Range : 0.000430.11138 ( 48.225 - 2.996 A ) * Sort Order : 1 2 3 0 0 * Space group = 'P 3 2 1' (number 150) OVERALL FILE STATISTICS for resolution range 0.000 - 0.111 === Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 29 0 100.00 13.7 13.7 48.22 3.00 H H 2 NONE 0 16 0 100.00 4.7 4.7 48.22 3.00 H K 3 NONE -32 32 0 100.00 0.5 12.2 48.22 3.00 H L 4 NONE1.4 291.9 0 100.0019.40 19.40 48.22 3.00 F FP 5 NONE0.1 4.3 0 100.00 0.67 0.67 48.22 3.00 Q SIGFP 6 NONE -13.613.269 99.41-0.01 0.69 48.22 3.00 D DANO 7 NONE0.0 5.769 99.41 1.13 1.13 48.22 3.00 Q SIGDANO 8 NONE 0 2 0 100.00 0.0 0.0 48.22 3.00 Y ISYM 9 NONE0.0 1.0 0 100.00 0.95 0.95 48.22 3.00 I FreeRflag No. of reflections used in FILE STATISTICS11643 Aren't they supposed to be the exact same number? Thanks, ciao, s On Jan 16, 2013, at 1:12 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear Sebastiano, you could use xprep to get the statistics in user defined resolution shells. Out of curiosity: Would you mind sharing why you want to do this and why you don't want to use the XSCALE statistics instead? The statistics are probably more meaningful after scaling, I guess. Best, Tim On 01/15/2013 04:22 PM, Sebastiano Pasqualato wrote: Hi all, I was wondering if XDS allows to change the number of resolution bins appearing in the table: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION of CORRECT.LP. Please, note that I am not referring to the table output by XSCALE, in which you can change
Re: [ccp4bb] changing resolution bins statistics in CORRECT.LP
On Jan 16, 2013, at 2:19 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Sebastiano, if the output of GO.COM produces an mtz-file you can use for phasing or refinement, I am very confident there is a scaling step involved. Sorry, my bad. I meant there's no XSCALE nor scala that has been run. CORRECT does the scaling. This might also be an explanation for the discrepancy you point out: With FRIEDEL'S_LAW=TRUE the statistics which affects the rejection of outliers would have more reflections within each group of symmetry related reflections and hence a greater spread which might lead to the rejection of some of the classes. But this is a mere guess. The way I performed the two runs was by running CORRECT step of XDS followed by XDSCONV, f2mtz and cad. When working with the FRIEDEL'S_LAW flag on, CORRECT outputs F(+), F(-) but also merged F. I was expecting those were merged identically to the case in which FRIEDEL'S_LAW is off. But maybe your point is correct. Thanks, s Cheers, Tim On 01/16/2013 02:03 PM, Sebastiano Pasqualato wrote: Thanks to Kay and Tim or the feedback. The reason I wanted to get statistics from the CORRECT step of XDS is that I have refined a structure using the mtz output by the GO.COM automatic reduction routine of SLS beamline PXIII, which does not involve a scaling step (I discovered recently). I was willing to have the integration statistics of the reflection file I used in the refinement's high resolution bin. I will definitely give xprep a try. Another question that raised by looking deeper into their automatic procedure (thanks Meitian for the help) is that when integrating with XDS CORRECT keeping the FRIEDEL'S_LAW=TRUE or =FALSE I get a different number of reflections in the final mtz. In my case, if I run the same XDS.INP script and change only the FRIEDEL'S_LAW flag, I obtain: =TRUE: 11551 reflections * Resolution Range : 0.000430.11138 ( 48.225 - 2.996 A ) * Sort Order : 1 2 3 0 0 * Space group = 'P 3 2 1' (number 150) OVERALL FILE STATISTICS for resolution range 0.000 - 0.111 === Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 29 0 100.00 13.7 13.7 48.22 3.00 H H 2 NONE 0 16 0 100.00 4.7 4.7 48.22 3.00 H K 3 NONE -32 32 0 100.00 0.5 12.2 48.22 3.00 H L 4 NONE1.4 292.0 0 100.0019.40 19.40 48.22 3.00 F FP 5 NONE0.1 4.3 0 100.00 0.70 0.70 48.22 3.00 Q SIGFP 6 NONE0.0 1.0 0 100.00 0.95 0.95 48.22 3.00 I FreeRflag No. of reflections used in FILE STATISTICS11551 =FALSE: 11643 reflections * Cell Dimensions : (obsolete - refer to dataset cell dimensions above) 100.5450 100.5450 96.4500 90. 90. 120. * Resolution Range : 0.000430.11138 ( 48.225 - 2.996 A ) * Sort Order : 1 2 3 0 0 * Space group = 'P 3 2 1' (number 150) OVERALL FILE STATISTICS for resolution range 0.000 - 0.111 === Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 29 0 100.00 13.7 13.7 48.22 3.00 H H 2 NONE 0 16 0 100.00 4.7 4.7 48.22 3.00 H K 3 NONE -32 32 0 100.00 0.5 12.2 48.22 3.00 H L 4 NONE1.4 291.9 0 100.0019.40 19.40 48.22 3.00 F FP 5 NONE0.1 4.3 0 100.00 0.67 0.67 48.22 3.00 Q SIGFP 6 NONE -13.613.269 99.41-0.01 0.69 48.22 3.00 D DANO 7 NONE0.0 5.769 99.41 1.13 1.13 48.22 3.00 Q SIGDANO 8 NONE 0 2 0 100.00 0.0 0.0 48.22 3.00 Y ISYM 9 NONE0.0 1.0 0 100.00 0.95 0.95 48.22 3.00 I FreeRflag No. of reflections used in FILE STATISTICS11643 Aren't they supposed to be the exact same number? Thanks, ciao, s On Jan 16, 2013, at 1:12 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear Sebastiano, you could use xprep to get the statistics in user defined resolution shells. Out of curiosity: Would you mind sharing why you want to do this and why you don't want to use the XSCALE statistics instead? The statistics are probably more meaningful after scaling, I guess. Best, Tim On 01/15/2013 04:22 PM, Sebastiano Pasqualato wrote: Hi all, I was wondering if XDS allows to change the number of resolution bins appearing in the table: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION of
Re: [ccp4bb] protein degradation in crystal
Hi Lisa, Speed is definitely a big factor here. With a protein I work with I can get large crystals in myriad conditions that only diffract to about 4-5 Ang. What I ended up doing was taking these crystals and seeding entire screens. I found that not only would crystals appear sooner but it revealed novel crystallization conditions. These seeded crystals would appear within minutes as Preben described and diffracted to better than 2 Ang. Another thought would be to try limited proteolysis to see if you can identify a more stable construct. -John -- John Domsic Postdoctoral Fellow Gene Expression and Regulation Program The Wistar Institute Philadelphia, PA 19104 On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth j.p.mo...@ncmm.uio.nowrote: Dear Lisa It is not uncommon to see breakdown products when you run crystals on gel. Espesially if they are older crystals, sometimes you even see higher molecular bands, these are probably due to intra molecular cross links formed over time. If you are worried about stability, try to increase the crystallization speed, we have one example where we see a clear difference in both crystal quality and even space group depending on when we fish the crystals. The crystals appear within 5 min, the best quality data sets come from crystals we fish after only 30-60 min. You may also have a little protease contamination of course, to prevent this add protease inhibitor, or DTT, or EDTA to you protein before you set it up. cheers Preben On 1/16/13 12:14 PM, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several samll bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 http://www.jpmorth.dk
[ccp4bb] EMBO 2013 Practical Course - Exploiting Anomalous Scattering - ESRF - 10 - 14 June 2013
COURSE ANNOUNCEMENT EMBO 2013 Practical Course - Exploiting Anomalous Scattering in Macromolecular Structure Determination ESRF-EMBL, Grenoble, France, 10 - 14 June 2013 The EMBO 2013 Practical Course on Exploiting Anomalous Scattering in Macromolecular Structure Determination will be hosted by the ESRF in Grenoble, France from 10 to 14, June 2013. The course aims to impart the theoretical and practical basis for the 3-dimensional structure determination of bio-macromolecules using Anomalous Dispersion techniques (SAD MAD) to young scientists who intend to apply these methods in macromolecular crystallography. Through a series of lectures, software demonstrations, practicals on the ESRF beamlines and tutorials, participants will get insights into all aspects of the structure determination process including beamline instrumentation, data collection and processing, heavy atom substructure determination, phasing and model building. There will also be sessions focusing on automated structure solution procedures and newer methods. Additional sessions will include presentations of distinguished structures solved with these techniques. The number of participants is limited to 20 and the deadline for application is March 10th, 2013. Additional information, course programme and instructions to apply to the course can be found in the course webpages: http://events.embo.org/13-crystallography/ Confirmed invited speakers include: Andreas Bracher, Kay Diederichs, Rita Giordano, Adrian Goldman, Wolfgang Kabsch, Gordon Leonard, Airlie McCoy, Sean McSweeney, Christoph Müller-Dieckmann, Max Nanao, Alexander Popov, Harry Powell, Thomas Terwilliger, Andrew Thompson, Isabel Uson, Clemens Vonhrein -- ἀρετή --- Daniele de Sanctis, PhD Structural Biology Group ESRF, Grenoble, France Tel 33 (0)4 76 88 2869
Re: [ccp4bb] side question re crystal dehydration
Juan, Humidity variation is what vapour diffusion crystallization achieves. In your list of all possible dehydration methods you would end up classifying all vapour diffusion experiments as a case of dehydration. After nucleation, crystals continue to grow and the drop continues to become more concentrated in precipitant. I agree you are completely right! The concept of crystal hydration is very complicated: Crystals are grown in liquid water and often analysed in vitrified water with a solvent-hydrogen bond network that is different from that in the liquid state. What does this mean in terms of hydration? The use of cryo-protectant alter the solvent hydrogen bonding pattern. What does this mean in terms of hydration? VM, the Matthews coefficient, defined as the crystal volume per unit of protein molecular weight is a a measure of hydration? So if the VM is the same the hydration is the same? I agree that all the methods that you mention will affect hydration of the crystals, but the way that X-ray crystallography is carried out today cannot avoid it. Crystal dehydration must be defined as an explicit effort to use methodology designed to alter the hydration of crystals, preferably using a defined measured and controlled relative humidity value. As you start to consider badly defined systems, you will also have badly defined hydration. Enrico. On Wed, 16 Jan 2013 14:18:05 +0100, Juan Sanchez-Weatherby juan.sanchez-weathe...@diamond.ac.uk wrote: Dear all, From Leonid's reply earlier you can see a problem some of us have been having for a while now, when looking for literature regarding dehydration. Most of you that perform dehydration either don't consider it happening or don't report it in great detail in your publications. This is only understandable because it isn't the focus of your work and it only helps you get to where you want to get to. I'm trying to get an up to date picture of what is out there but I haven't got the time or eyes to go through everyone's methods to pick the couple of lines that describe your particular method. I really want to find out what is being done to be able to give people better advice. So: Could people out there that think that in their particular projects dehydration/hydration had an effect send me a ref. or a short description? (can be done outside the BB to not spam everyone) I will duly acknowledge everyone!! By dehydration I mean: 1 Soaking with increasing concentration of precipitants or salts 2 By equilibrating against a new precipitant or salt (by vapour diffusion or dialysis) 3 By letting the drops dry (controlled or uncontrolled) 4 by using an FMS/HC1/MicroRT or any other gadget 5 By some other magical trick you may have Thank you all for your help, Regards Juan Juan Sanchez-Weatherby, PhD Beamline Scientist - I02 Macromolecular Crystallography Group Diamond Light Source Ltd Diamond House DR1.64 Harwell Science and Innovation Campus RAL, Chilton, Didcot Oxfordshire OX11 0DE United Kingdom Tel: +44 (0)1235 778661 Mob:+44 (0)7795 641259 Fax:+44 (0)1235 778052 juan.sanchez-weathe...@diamond.ac.uk http://www.diamond.ac.uk -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Leonid Sazanov Sent: 15 January 2013 19:32 To: ccp4bb Subject: Re: [ccp4bb] crystal dehydration In case if dehydration needs to be done slowly and under tight control of all parameters, one possibility is to use micro-dialysis buttons. We used it for a large membrane protein complex and diffraction improved from ~7 to 2.7 A. The crystal is fished out and put into mother liquor solution in the button, sealed with dialysis membrane and the button is then placed into about 5 mls of mother liquor with slightly higher PEG concentration. Then you just exchange outside buffer every day or so for solutions containing higher concentrations of PEG. We went from ~9 to 30 % PEG4000 in about a week. You can easily observe crystal under microscope and if it cracks - you went too far/too quickly with PEG and need to use a bit less next time. Also, this method allows you to control all other components of the dehydrating solution - we needed to decrease salt concentration at the same time as increasing PEG. You can also introduce/increase cryo-protectant concentration at the same time. With these crystals, otherwise excellent dehydration machines already mentioned did not work, possibly because the process had to be really slow. The reference is here: http://www.ncbi.nlm.nih.gov/pubmed/21822288 Best wishes. -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/ibitecs/simopro/ltmb/cristallogenese
Re: [ccp4bb] crystal dehydration
Indeed! We have a short communication in Acta D that is under revision (no preprint yet, sorry!), going in the same direction than what Hagelueken et al have done (using saturated salt solutions in the reservoir and transferring your crystals in perfluoropolyether oil (see paper here that we have just seen: http://journals.iucr.org/d/issues/2012/10/00/bw5408/bw5408.pdf), but instead of transferring the crystals in the 96 wells, you grow your crystals first in 96-wells, you then pierce your tray with a thin needle and add your gradient of salt concentration in the reservoir, re-seal it, let it equilibrate and use in-situ plate screening facilities available in several European synchrotrons (@ Diamond, SLS, Bessy, ESRF-FIP beamline) or in-house facilities to get an x-ray feedback of your experiment. You would follow changes in your cell parameters (like with the HC1). The advantage of this is that hopefully you get an overall picture of whether dehydration has an effect on your samples within one experiment without handling your crystals (which could add another detrimental parameter to your samples). This is a work in progress at Diamond and we are happy to help/talk to anyone who would like to try it, just contact us: Alice Douangamath (alice.douangam...@diamond.ac.uk) Pierre Aller (pierre.al...@diamond.ac.uk) Juan Sanchez-Weatherby (juan.sanchez-weathe...@diamond.ac.uk) Jose Brandao-Neto (jose.brandao-n...@diamond.ac.uk) -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hargreaves, David Sent: 16 January 2013 12:37 To: ccp4bb Subject: Re: [ccp4bb] crystal dehydration Judith, If you can, try monitoring the diffraction during the dehydration experiment. There are plenty of ways of doing this. Perhaps the simplest is to shoot your crystals in-situ after swapping the precipitant for a more concentrated one. The is also the Free Mounting System. David Hargreaves Associate Principal Scientist _ AstraZeneca Discovery Sciences, Structure Biophysics Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.com Please consider the environment before printing this e-mail -- AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking Compliance with our Code of Conduct and Policies. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Judith A Ronau Sent: 15 January 2013 14:47 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystal dehydration Greetings! I have recently been attempting crystal dehydration experiments to improve diffraction following the procedures from the ERSF in which crystals are exposed to increased concentrations of precipitant. I would like to know if anyone knew of any alternative methods for dehydration of protein crystals. Thanks! Best, Judith
Re: [ccp4bb] protein degradation in crystal
Were the crystals which run different on gels from the same drop, or separate drops? Yes, it is possible that the protein is being cleaved in the crystal (self-cleavage?); but it may also be that it is being cleaved in the mother liquor, and that crystallization is enriching one form or another. Remember that crystallization is a useful purification technique. There should be enough protein in a crystal to get some mass spec data, which will tell you the size of the components, and which should be precise enough to tell you the cleavage site. This will tell you what type of protease you are dealing with. Then as possible remedies: You could include an appropriate protease inhibitor during purification to limit degradation. You could add a bit of protease to encourage proteolysis to go to completion. You want your sample to be homogeneous, so whether this is useful depends on whether the cleaved part is the interesting part. You could engineer the gene with a stop codon at or near the cleavage site. This is what we did with HO-1, with some success. DOI: 10.1002/pro.5560070820 You could engineer the cleavage site to eliminate cleavage. Cheers, On 01/16/13 06:14, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several small bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] protein degradation in crystal
Were the crystals which run different on gels from the same drop, or separate drops? Yes, it is possible that the protein is being cleaved in the crystal (self-cleavage?); but it may also be that it is being cleaved in the mother liquor, and that crystallization is enriching one form or another. Remember that crystallization is a useful purification technique. There should be enough protein in a crystal to get some mass spec data, which will tell you the size of the components, and which should be precise enough to tell you the cleavage site. This will tell you what type of protease you are dealing with. Then as possible remedies: You could include an appropriate protease inhibitor during purification to limit degradation. You could add a bit of protease to encourage proteolysis to go to completion. You want your sample to be homogeneous, so whether this is useful depends on whether the cleaved part is the interesting part. You could engineer the gene with a stop codon at or near the cleavage site. This is what we did with HO-1, with some success. DOI: 10.1002/pro.5560070820 You could engineer the cleavage site to eliminate cleavage. Cheers, On 01/16/13 06:14, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several small bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] protein degradation in crystal
Hi John, This is really an amazing wild west story: the man who crystallizes faster than his protease! I really must compliment you with how you successfully performed these experiments! Unfortunately, proteins usually do not crystallize that fast (at least not in my hands), so in these cases other methods have to be used. As has mentioned before, protease inhibitors are the way to go. Especially with autolysis, as one protease cuts another one, the speed of the reaction goes with the square of the protease concentration. Whereas in dilute solutions not much happens, as soon as you start to concentrate towards crystallization conditions, say 10 mg/ml, degradation suddenly goes very fast. There are 2 cases to consider: 1) the protein you want to crystallize is a protease and is destroying itself. In this case you need to cocrystallize with a potent and specific inhibitor. With serine proteases, Wolfram Bode was very successful by using chloromethylketone-containing peptides (e.g. PPACK). These compounds would make covalent links with both the active site serine and histidine, effectively killing any protease activity. 2) the protein you want to crystallize is not a protease and it is a contaminant which is causing the problems. In this case I would add a protease inhibitor coctail in an earlier step of the purification to block the protease before the final purification steps. I would also add some small broad protease inhibitor e.g. PMSF to the protein solution used for crystallization. Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of John Domsic Sent: Wednesday, January 16, 2013 2:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein degradation in crystal Hi Lisa, Speed is definitely a big factor here. With a protein I work with I can get large crystals in myriad conditions that only diffract to about 4-5 Ang. What I ended up doing was taking these crystals and seeding entire screens. I found that not only would crystals appear sooner but it revealed novel crystallization conditions. These seeded crystals would appear within minutes as Preben described and diffracted to better than 2 Ang. Another thought would be to try limited proteolysis to see if you can identify a more stable construct. -John -- John Domsic Postdoctoral Fellow Gene Expression and Regulation Program The Wistar Institute Philadelphia, PA 19104 On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth j.p.mo...@ncmm.uio.no wrote: Dear Lisa It is not uncommon to see breakdown products when you run crystals on gel. Espesially if they are older crystals, sometimes you even see higher molecular bands, these are probably due to intra molecular cross links formed over time. If you are worried about stability, try to increase the crystallization speed, we have one example where we see a clear difference in both crystal quality and even space group depending on when we fish the crystals. The crystals appear within 5 min, the best quality data sets come from crystals we fish after only 30-60 min. You may also have a little protease contamination of course, to prevent this add protease inhibitor, or DTT, or EDTA to you protein before you set it up. cheers Preben On 1/16/13 12:14 PM, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several samll bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 tel:%2B47%202284%200794 http://www.jpmorth.dk
Re: [ccp4bb] protein degradation in crystal
Just to add to Herman's suggestions, if you are trying to crystallise a protease then you could also try using the S195A variant rather than an inhibitor. This would certainly be the case if you ever want to co-crystallise in a substrate, as PPACK (or the like) would occupy the active site cleft and prevent formation of the protease-substrate complex. Tom ** Hi John, This is really an amazing wild west story: the man who crystallizes faster than his protease! I really must compliment you with how you successfully performed these experiments! Unfortunately, proteins usually do not crystallize that fast (at least not in my hands), so in these cases other methods have to be used. As has mentioned before, protease inhibitors are the way to go. Especially with autolysis, as one protease cuts another one, the speed of the reaction goes with the square of the protease concentration. Whereas in dilute solutions not much happens, as soon as you start to concentrate towards crystallization conditions, say 10 mg/ml, degradation suddenly goes very fast. There are 2 cases to consider: 1) the protein you want to crystallize is a protease and is destroying itself. In this case you need to cocrystallize with a potent and specific inhibitor. With serine proteases, Wolfram Bode was very successful by using chloromethylketone-containing peptides (e.g. PPACK). These compounds would make covalent links with both the active site serine and histidine, effectively killing any protease activity. 2) the protein you want to crystallize is not a protease and it is a contaminant which is causing the problems. In this case I would add a protease inhibitor coctail in an earlier step of the purification to block the protease before the final purification steps. I would also add some small broad protease inhibitor e.g. PMSF to the protein solution used for crystallization. Herman -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *John Domsic *Sent:* Wednesday, January 16, 2013 2:22 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] protein degradation in crystal Hi Lisa, Speed is definitely a big factor here. With a protein I work with I can get large crystals in myriad conditions that only diffract to about 4-5 Ang. What I ended up doing was taking these crystals and seeding entire screens. I found that not only would crystals appear sooner but it revealed novel crystallization conditions. These seeded crystals would appear within minutes as Preben described and diffracted to better than 2 Ang. Another thought would be to try limited proteolysis to see if you can identify a more stable construct. -John -- John Domsic Postdoctoral Fellow Gene Expression and Regulation Program The Wistar Institute Philadelphia, PA 19104 On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth j.p.mo...@ncmm.uio.nowrote: Dear Lisa It is not uncommon to see breakdown products when you run crystals on gel. Espesially if they are older crystals, sometimes you even see higher molecular bands, these are probably due to intra molecular cross links formed over time. If you are worried about stability, try to increase the crystallization speed, we have one example where we see a clear difference in both crystal quality and even space group depending on when we fish the crystals. The crystals appear within 5 min, the best quality data sets come from crystals we fish after only 30-60 min. You may also have a little protease contamination of course, to prevent this add protease inhibitor, or DTT, or EDTA to you protein before you set it up. cheers Preben On 1/16/13 12:14 PM, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several samll bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 %2B47%202284%200794 http://www.jpmorth.dk -- Skype: tom.murray.rust Twitter: tmurrayrust http://twitpic.com/photos/tmurrayrust +44 7970 480 601 (UK)
Re: [ccp4bb] ccp4 update
Dear Andreas Förster, You can use ccp4um (ccp4 update manage) to update CCP4 from command line. Best wishes, Q. Cai Qixu Cai Email: caiq...@gmail.com School of Life Sciences, Xiamen University, Fujian, China 2013/1/14 Andreas Förster docandr...@gmail.com Dear CCP4 maintainers, I've come to appreciate the CCP4 update functionality, which, in our multiuser network (RHEL 6.2), I used to invoke by calling $CCP4/bin/update. Update 012 removed that script with no immediately obvious replacement. Was that on purpose? Is there a way of updating CCP4 from the command line without calling CCP4i? Thanks Andreas (from update.log: [Thu Jan 3 2013 11:00:21] Ready to make changes --- applying update 6.3.0-012 --- update header read --- creating restore package, please wait ... --- done ... file '/csb/soft/Linux64/share/ccp4-**6.3.0/bin/update' removed ... file '/csb/soft/Linux64/share/ccp4-**6.3.0/lib_exec/update' removed ... file '/csb/soft/Linux64/share/ccp4-**6.3.0/lib_exec/_update' removed ... directory '/csb/soft/Linux64/share/ccp4-**6.3.0/lib_exec' removed some more blah blah) -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
Re: [ccp4bb] ccp4 update
Ah, I see. This was part of update 10. Thank you. Andreas On 16/01/2013 2:51, Qixu Cai wrote: Dear Andreas Förster, You can use ccp4um (ccp4 update manage) to update CCP4 from command line. Best wishes, Q. Cai -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
[ccp4bb] ccp4 update
Dear Andreas, Sorry for late response to your post and confusion with the updater. For technical reasons (Windows does not like names containing update :)), the updater was renamed into ccp4um (ccp4 update manager) and is found in $CCP4/bin. I hope this helps, Eugene Dear CCP4 maintainers, I've come to appreciate the CCP4 update functionality, which, in our multiuser network (RHEL 6.2), I used to invoke by calling $CCP4/bin/update. Update 012 removed that script with no immediately obvious replacement. Was that on purpose? Is there a way of updating CCP4 from the command line without calling CCP4i? Thanks Andreas (from update.log: [Thu Jan 3 2013 11:00:21] Ready to make changes --- applying update 6.3.0-012 --- update header read --- creating restore package, please wait ... --- done ... file '/csb/soft/Linux64/share/ccp4-6.3.0/bin/update' removed ... file '/csb/soft/Linux64/share/ccp4-6.3.0/lib_exec/update' removed ... file '/csb/soft/Linux64/share/ccp4-6.3.0/lib_exec/_update' removed ... directory '/csb/soft/Linux64/share/ccp4-6.3.0/lib_exec' removed some more blah blah) -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London -- Scanned by iCritical.
Re: [ccp4bb] changing resolution bins statistics in CORRECT.LP
Sebastiano, CORRECT does scale the data. XSCALE is only needed for two or more datasets, or if other requirements exist - you named one. The two varieties of FRIEDEL'S_LAW differ in their rejections! HTH, Kay Sebastiano Pasqualato sebastiano.pasqual...@gmail.com schrieb: Thanks to Kay and Tim or the feedback. The reason I wanted to get statistics from the CORRECT step of XDS is that I have refined a structure using the mtz output by the GO.COM automatic reduction routine of SLS beamline PXIII, which does not involve a scaling step (I discovered recently). I was willing to have the integration statistics of the reflection file I used in the refinement's high resolution bin. I will definitely give xprep a try. Another question that raised by looking deeper into their automatic procedure (thanks Meitian for the help) is that when integrating with XDS CORRECT keeping the FRIEDEL'S_LAW=TRUE or =FALSE I get a different number of reflections in the final mtz. In my case, if I run the same XDS.INP script and change only the FRIEDEL'S_LAW flag, I obtain: =TRUE: 11551 reflections * Resolution Range : 0.000430.11138 ( 48.225 - 2.996 A ) * Sort Order : 1 2 3 0 0 * Space group = 'P 3 2 1' (number 150) OVERALL FILE STATISTICS for resolution range 0.000 - 0.111 === Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 29 0 100.00 13.7 13.7 48.22 3.00 H H 2 NONE 0 16 0 100.00 4.7 4.7 48.22 3.00 H K 3 NONE -32 32 0 100.00 0.5 12.2 48.22 3.00 H L 4 NONE1.4 292.0 0 100.0019.4019.40 48.22 3.00 F FP 5 NONE0.1 4.3 0 100.00 0.70 0.70 48.22 3.00 Q SIGFP 6 NONE0.0 1.0 0 100.00 0.95 0.95 48.22 3.00 I FreeRflag No. of reflections used in FILE STATISTICS11551 =FALSE: 11643 reflections * Cell Dimensions : (obsolete - refer to dataset cell dimensions above) 100.5450 100.5450 96.4500 90. 90. 120. * Resolution Range : 0.000430.11138 ( 48.225 - 2.996 A ) * Sort Order : 1 2 3 0 0 * Space group = 'P 3 2 1' (number 150) OVERALL FILE STATISTICS for resolution range 0.000 - 0.111 === Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 29 0 100.00 13.7 13.7 48.22 3.00 H H 2 NONE 0 16 0 100.00 4.7 4.7 48.22 3.00 H K 3 NONE -32 32 0 100.00 0.5 12.2 48.22 3.00 H L 4 NONE1.4 291.9 0 100.0019.4019.40 48.22 3.00 F FP 5 NONE0.1 4.3 0 100.00 0.67 0.67 48.22 3.00 Q SIGFP 6 NONE -13.613.269 99.41-0.01 0.69 48.22 3.00 D DANO 7 NONE0.0 5.769 99.41 1.13 1.13 48.22 3.00 Q SIGDANO 8 NONE 0 2 0 100.00 0.0 0.0 48.22 3.00 Y ISYM 9 NONE0.0 1.0 0 100.00 0.95 0.95 48.22 3.00 I FreeRflag No. of reflections used in FILE STATISTICS11643 Aren't they supposed to be the exact same number? Thanks, ciao, s On Jan 16, 2013, at 1:12 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Sebastiano, you could use xprep to get the statistics in user defined resolution shells. Out of curiosity: Would you mind sharing why you want to do this and why you don't want to use the XSCALE statistics instead? The statistics are probably more meaningful after scaling, I guess. Best, Tim On 01/15/2013 04:22 PM, Sebastiano Pasqualato wrote: Hi all, I was wondering if XDS allows to change the number of resolution bins appearing in the table: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION of CORRECT.LP. Please, note that I am not referring to the table output by XSCALE, in which you can change the resolution bins with the keyword RESOLUTION_SHELLS=, but rather the table output by the CORRECT job of XDS. Thanks in advance, ciao, Sebastiano - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQ9pk/UxlJ7aRr7hoRAqreAJ9/aKSHyXWahBc96/3x0U5iriZk4gCg2fWC GVLgXUEN+10M9IpCDRENGF0= =SK0P -END PGP SIGNATURE- -- Sebastiano Pasqualato, PhD Crystallography Unit Department of Experimental Oncology European Institute of Oncology IFOM-IEO Campus via Adamello, 16 20139 - Milano Italy
[ccp4bb] CCP4 package and update managers: relocating installations
Dear CCP4, While we're talking about the package and update managers, I have a question. Is there any way to install the CCP4 suite in a non-standard location under OS X and still use the package manager and update facilities? With a large number of Macs it's much easier for me to have CCP4 living in a single, network-mounted directory than on each machine. Under Linux, the package manager and updater allow me to put things where I like. Under OS X the package manager wants to put things in /Applications and I can't edit that, despite it being in a GUI element that looks like an input field. Regards, Chris -- Dr Chris Richardson :: Sysadmin, structural biology, icr.ac.uk The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network.
Re: [ccp4bb] CCP4 package and update managers: relocating installations
Hello Chris, Is there any way to install the CCP4 suite in a non-standard location under OS X and still use the package manager and update facilities? With a large number of Macs it's much easier for me to have CCP4 living in a single, network-mounted directory than on each machine. I use Pacifist to extract the files into a single directory tree and then move that to our custom install location. You'll have to modify the shell set up scripts to reflect the new location. I usually grep through the install to see if there are any other hardcoded paths that need modification, but the CCP4 packaging QA is generally quite good, and we haven't had many problems. -ben -- | Ben Eisenbraun | SBGrid Consortium | http://sbgrid.org | | Harvard Medical School | http://hms.harvard.edu |
Re: [ccp4bb] CCP4 package and update managers: relocating installations
Dear Chris, Indeed, you cannot use Package Manager on Mac OSX for installing CCP4 in custom location, which is due to petty technicalities and pragmatism. However, CCP4 can be installed in non-standard locations with raw *.dmg's downloadable from our web site, which is supposed to be as easy as with the PM. In difference of Package Manager, the updater is immune to the location and can be used in non-standard setups. Does this help? Regards, Eugene On 16 Jan 2013, at 15:27, Chris Richardson wrote: Dear CCP4, While we're talking about the package and update managers, I have a question. Is there any way to install the CCP4 suite in a non-standard location under OS X and still use the package manager and update facilities? With a large number of Macs it's much easier for me to have CCP4 living in a single, network-mounted directory than on each machine. Under Linux, the package manager and updater allow me to put things where I like. Under OS X the package manager wants to put things in /Applications and I can't edit that, despite it being in a GUI element that looks like an input field. Regards, Chris -- Dr Chris Richardson :: Sysadmin, structural biology, icr.ac.uk The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network. -- Scanned by iCritical.
[ccp4bb] Call for Beamtime@EMBL Hamburg - Deadline 20th January 2013
Dear Colleagues, We announce a call for synchrotron beam time applications in biological small-angle scattering (SAXS) and macromolecular crystallography (MX). The beamtime will be available at the PETRA-III storage ring from *March, 1st, 2013 to September, 15th, 2013* (please note the end date change compared to the announcement in December 2012!). In biological SAXS, we offer access to an undulator beamline P12: * 200 x 100 micron2 beam with 1013 ph/sec * Automated data collection with a robotic sample changer * Online FPLC and biophysical characterization of the sample * Remote and Fedex access * PILATUS 2M pixel detector covering the resolution from 2000 to 10 Å * http://www.embl-hamburg.de/facilities/saxs/index.html Part of the beamtime on P12 will be available for soft condensed matter applications through a Collaborative Research Group model in partnership with the Helmholtz Zentrum Geesthacht. For MX, access to two undulator beamlines - P13 and P14 - is offered. P13: * 50 x 50 micron2 beam with 1013 ph/sec * Apertures of 10/50/100 micron diameter * Energy 4.5 to 15 keV / Wavelength 0.82 - 2.7 A * MD2 diffractometer * PILATUS 6M (25 Hz) with 80 core compute server for XDS-processing * http://www.embl-hamburg.de/facilities/mx/P13 P14: * 5 x 10 micron2 focused beam with up to 5 x 1012 ph/sec * Quick toggling (1 min) to unfocused flexible beam mode with 5-150 micron beam size. * Fixed energy 10 keV = 1.24 A * MD3 diffractometer with vertical spindle and kappa goniostat * PILATUS 6M (25 Hz) with 80 core compute server for XDS-processing * http://www.embl-hamburg.de/facilities/mx/P14 Electronic beam proposal forms and a detailed description of the beamlines are available at http://www.embl-hamburg.de/facilities/access_infrastructures/index.html *The deadline for submission of proposals is January, 20th, 2013. *An external Project Evaluation Committee will assess the proposals. Access to the EMBL Hamburg facilities will in part be supported by the European Commission, Research Infrastructure Action under the FP7 project BioStruct-X (http://www.biostruct-x.eu/). Users that are interested in assistance with crystallization, sample preparation and characterization (SPC, also in combination with SAXS measurements) are encouraged to apply to the Biostruct-X support at http://www.biostruct-x.eu/content/embl-hamburg-protein-production-and-htx For further information tel. +49 40-89902-111 s...@embl-hamburg.de (SAXS) m...@embl-hamburg.de (MX) s...@embl-hamburg.de (SPC) With best regards, EMBL Hamburg User Office
Re: [ccp4bb] side question re crystal dehydration
I think one may need to distinguish between three different kinds of dehydration experiment, because of the different forces they will exert on a crystal to shrink the unit cell, creating new stabilizing crystal contacts or perhaps causing contacts to fail in a chaotic manner, disordering the crystal: 1. dehydration while bathed with copious solution, by increasing concentration of glycerol or other small solute. 2. same, but the dehydrating solute is too large to enter (some) solvent channels. Large PEG molecules may do this(?). But PEG is often seen in crystal structures. 3. The crystal is fished out of solution before dehydration, with a thin layer of solution adhering to the surface. This is what is done with the FMS and I suppose other humidity-controlling systems, or more cheaply by fishing the crystal in a cryocap with a short pin and storing it screwed into an upright cryovial with 100 ul or so of defined humectant solution in the bottom. #1 would exert no direct forces on the crystal, but internal surfaces might come together to exclude water, which might lead to shrinkage. #2 could exert osmotic force, as water diffuses out of the crystal to the bulk solvent resulting in lower hydrostatic pressure in the solvent channels. Pressure on the surface of the crystal could then cause shrinkage. in #3, dehydration can reduce the volume of solution to the point where it is insufficient to fill the solvent channels. If surface tension prevents air from entering the crystals, atmospheric pressure on the surface of the crystal will promote shrinkage. I think in some cases annealing actually works by dehydration #3. We reported such a case in JMB 351, 573-597 (buried in Methods at the end of the paper, and disc p579). We have diffraction images before and after annealing, and not only the resolution improved dramatically but the cell volume decreased by 18%. And the structure showed a new crystal contact was formed. We're pretty sure this is dehydration because of the volume change and because a similar effect on diffraction and cell param could be obtained (with the same batch of crystals or a few other batches, in other cases something else was limiting and the improvement was not so significant) by simply holding the crystal in the air for 60-90 sec before plunging in LN2. It seems odd that annealing would cause dehydration- you would expect massive condensation on the crystal as it thaws - but I think the heat capacity of the crystal and the amount of ice (if any) to be melted is so small that it reaches room temp before much condensation occurs. Then the down-draft caused by the cold copper pin (sitting vertical with crystal upward) drew enough warm dry air over it to dehydrate. Juan Sanchez-Weatherby wrote: Dear all, From Leonid's reply earlier you can see a problem some of us have been having for a while now, when looking for literature regarding dehydration. Most of you that perform dehydration either don't consider it happening or don't report it in great detail in your publications. This is only understandable because it isn't the focus of your work and it only helps you get to where you want to get to. I'm trying to get an up to date picture of what is out there but I haven't got the time or eyes to go through everyone's methods to pick the couple of lines that describe your particular method. I really want to find out what is being done to be able to give people better advice. So: Could people out there that think that in their particular projects dehydration/hydration had an effect send me a ref. or a short description? (can be done outside the BB to not spam everyone) I will duly acknowledge everyone!! By dehydration I mean: 1 Soaking with increasing concentration of precipitants or salts 2 By equilibrating against a new precipitant or salt (by vapour diffusion or dialysis) 3 By letting the drops dry (controlled or uncontrolled) 4 by using an FMS/HC1/MicroRT or any other gadget 5 By some other magical trick you may have Thank you all for your help, Regards Juan Juan Sanchez-Weatherby, PhD Beamline Scientist - I02 Macromolecular Crystallography Group Diamond Light Source Ltd Diamond House DR1.64 Harwell Science and Innovation Campus RAL, Chilton, Didcot Oxfordshire OX11 0DE United Kingdom Tel: +44 (0)1235 778661 Mob:+44 (0)7795 641259 Fax:+44 (0)1235 778052 juan.sanchez-weathe...@diamond.ac.uk http://www.diamond.ac.uk -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Leonid Sazanov Sent: 15 January 2013 19:32 To: ccp4bb Subject: Re: [ccp4bb] crystal dehydration In case if dehydration needs to be done slowly and under tight control of all parameters, one possibility is to use micro-dialysis buttons. We used it for a large membrane protein complex and diffraction improved from ~7 to 2.7 A. The crystal is fished
Re: [ccp4bb] changing resolution bins statistics in CORRECT.LP
Excellent. Thanks a lot for the clarifications. ciao, s On Jan 16, 2013, at 4:14 PM, Kay Diederichs kay.diederi...@uni-konstanz.de wrote: Sebastiano, CORRECT does scale the data. XSCALE is only needed for two or more datasets, or if other requirements exist - you named one. The two varieties of FRIEDEL'S_LAW differ in their rejections! HTH, Kay Sebastiano Pasqualato sebastiano.pasqual...@gmail.com schrieb: Thanks to Kay and Tim or the feedback. The reason I wanted to get statistics from the CORRECT step of XDS is that I have refined a structure using the mtz output by the GO.COM automatic reduction routine of SLS beamline PXIII, which does not involve a scaling step (I discovered recently). I was willing to have the integration statistics of the reflection file I used in the refinement's high resolution bin. I will definitely give xprep a try. Another question that raised by looking deeper into their automatic procedure (thanks Meitian for the help) is that when integrating with XDS CORRECT keeping the FRIEDEL'S_LAW=TRUE or =FALSE I get a different number of reflections in the final mtz. In my case, if I run the same XDS.INP script and change only the FRIEDEL'S_LAW flag, I obtain: =TRUE: 11551 reflections * Resolution Range : 0.000430.11138 ( 48.225 - 2.996 A ) * Sort Order : 1 2 3 0 0 * Space group = 'P 3 2 1' (number 150) OVERALL FILE STATISTICS for resolution range 0.000 - 0.111 === Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 29 0 100.00 13.7 13.7 48.22 3.00 H H 2 NONE 0 16 0 100.00 4.7 4.7 48.22 3.00 H K 3 NONE -32 32 0 100.00 0.5 12.2 48.22 3.00 H L 4 NONE1.4 292.0 0 100.0019.4019.40 48.22 3.00 F FP 5 NONE0.1 4.3 0 100.00 0.70 0.70 48.22 3.00 Q SIGFP 6 NONE0.0 1.0 0 100.00 0.95 0.95 48.22 3.00 I FreeRflag No. of reflections used in FILE STATISTICS11551 =FALSE: 11643 reflections * Cell Dimensions : (obsolete - refer to dataset cell dimensions above) 100.5450 100.5450 96.4500 90. 90. 120. * Resolution Range : 0.000430.11138 ( 48.225 - 2.996 A ) * Sort Order : 1 2 3 0 0 * Space group = 'P 3 2 1' (number 150) OVERALL FILE STATISTICS for resolution range 0.000 - 0.111 === Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. LowHigh label 1 ASC 0 29 0 100.00 13.7 13.7 48.22 3.00 H H 2 NONE 0 16 0 100.00 4.7 4.7 48.22 3.00 H K 3 NONE -32 32 0 100.00 0.5 12.2 48.22 3.00 H L 4 NONE1.4 291.9 0 100.0019.4019.40 48.22 3.00 F FP 5 NONE0.1 4.3 0 100.00 0.67 0.67 48.22 3.00 Q SIGFP 6 NONE -13.613.269 99.41-0.01 0.69 48.22 3.00 D DANO 7 NONE0.0 5.769 99.41 1.13 1.13 48.22 3.00 Q SIGDANO 8 NONE 0 2 0 100.00 0.0 0.0 48.22 3.00 Y ISYM 9 NONE0.0 1.0 0 100.00 0.95 0.95 48.22 3.00 I FreeRflag No. of reflections used in FILE STATISTICS11643 Aren't they supposed to be the exact same number? Thanks, ciao, s On Jan 16, 2013, at 1:12 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Sebastiano, you could use xprep to get the statistics in user defined resolution shells. Out of curiosity: Would you mind sharing why you want to do this and why you don't want to use the XSCALE statistics instead? The statistics are probably more meaningful after scaling, I guess. Best, Tim On 01/15/2013 04:22 PM, Sebastiano Pasqualato wrote: Hi all, I was wondering if XDS allows to change the number of resolution bins appearing in the table: SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION of CORRECT.LP. Please, note that I am not referring to the table output by XSCALE, in which you can change the resolution bins with the keyword RESOLUTION_SHELLS=, but rather the table output by the CORRECT job of XDS. Thanks in advance, ciao, Sebastiano - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla -
[ccp4bb] 18 Days to the deadline: Please apply for RapiData 2013, a course on Data Collection and Structure Solving at the NSLS.
We are offering RapiData 2013, the fifteenth offering of our popular course: Rapid Data Collection and Structure Solving at the NSLS: A Practical Course in Macromolecular X-Ray Diffraction Measurement The course will be held 21-26 April 2013: http://www.bnl.gov/RapiData/. Students could be at any level from advanced undergraduate to full professor. The course should accommodate 48 students total. We encourage all students to bring their own specimens for data collection, and to bring old data for the data-reduction and structure-solving tutorials. Please read the Course Announcement at http://www.bnl.gov/RapiData/. You'll see that many experts in the field will be available for lectures and tutorials. You'll find the application materials on the Course Application tab at this site. For the eleventh time we will hold a short lecture course on the fundamentals of crystallography for roughly five hours on Sunday 21 April. The body of the RapiData course really requires that students have a healthy knowledge of crystallography. For potential students who have some experience but are shaky about fundamentals, this course will help. There will be a small additional fee for the fundamentals course, to pay for Saturday night accomodations and food on Sunday morning and noon. Latin American Scientists: Several scholarships are available, from the International Union of Crystallography, to pay partial travel and subsistence costs for Latin-American students and junior faculty (under 40 yrs). Please apply for the course, and then contact R. Sweet (sw...@bnl.gov) if you are interested in applying for a scholarship. In accordance with the standards of the International Union of Crystallography, we observe the basic policy of non-discrimination, affirming the right and freedom of scientists to associate in international scientific activity without regard to such factors as citizenship, religion, creed, political stance, ethnic origin, race, colour, language, age, or gender, in accordance with the Statutes of the International Council for Science. At this course no barriers will exist beyond the application procedure that would prevent the participation of bona fide scientists. Please apply or send your students to our course, Bob Sweet, Sonya Kiss, and Alex Soares Course Announcement at http://www.bnl.gov/RapiData/ = Robert M. Sweet E-Dress: sw...@bnl.gov Group Leader, PXRR: Macromolecular ^ (that's L Crystallography Research Resource at NSLSnot 1) http://px.nsls.bnl.gov/ Photon Sciences and Biology Dept Mail Stop, Bldg 463 Brookhaven Nat'l Lab. Phones: Upton, NY 11973631 344 3401 (Office) U.S.A. 631 344 2741 (Facsimile) =
[ccp4bb] CCP4 package and update managers: relocating installations
Hi, I had exactly the same problem. I wanted to install ccp4 on a Mac Server then export it to other devices. What I found was that the setup script needed to be edited to reflect the new location. (/Applications/Software/ccp4-6.3.0/ccp4.app/Contents/Resources/ccp4.sh) However ….. There doesn't seem to be a $CCP4HOME (or similar) set, such that multiple lines need to be edited. Also, some updates seem to rewrite this script for /Applications directory the whole process needs to be repeated. (similar needs to be done for arpwarp … and most mac installations !) I'm fairly new to MacOS and maybe there is a better way of doing this ….. Sid Dr K S Sidhu Department of Biochemistry 1/61 Henry Wellcome Building Lancaster Road Leicester LE1 9HN Tel: 0116 229 7237 On 16 Jan 2013, at 15:27, Chris Richardson chris.richard...@icr.ac.ukmailto:chris.richard...@icr.ac.uk wrote: Dear CCP4, While we're talking about the package and update managers, I have a question. Is there any way to install the CCP4 suite in a non-standard location under OS X and still use the package manager and update facilities? With a large number of Macs it's much easier for me to have CCP4 living in a single, network-mounted directory than on each machine. Under Linux, the package manager and updater allow me to put things where I like. Under OS X the package manager wants to put things in /Applications and I can't edit that, despite it being in a GUI element that looks like an input field. Regards, Chris -- Dr Chris Richardson :: Sysadmin, structural biology, icr.ac.ukhttp://icr.ac.uk The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network.
Re: [ccp4bb] side question re crystal dehydration
Dear Enrico, I agree that in broad terms even bad weather (happens a lot here) could be considered a change in humidity. I'm not trying to claim that every time someone changes something in their drop dehydration is to be blamed. But it is a fact that by simply opening your drop you are causing a change and that change may be the difference between good and bad diffraction. I'm trying to capture people's experiments where they know for a fact that something they did (that could be attributed to a change in humidity) made the difference between having the dataset they wanted or not. The way I've meant to understand hydration is double. Firstly the very clear case where deliberately the sample has been dried and then allowed to hydrate again or where the sample has been hydrated above it starting state. It has been documented a couple of times with the FMS and I think at least once with the HC1. Secondly the case where (and it happens) people soak/cryo-protect) in a solution that has less osmotic power (and lower measurable RH) than that of the original solution. It is done very frequently when for example people add well solution to their nice crystal (and protein and ML where quite different) or add a less dehydrating cryo-protectant solution than the sample had in the first place. And yes cryo-protection by its very nature is a dehydration process it mainly works by hydrogen bonding water so it isn't available for nucleation. Another matter is weather a measurable effect can be linked to the process done to the samples. For example, you can dehydrate lysozyme (to about 70%) and not see any difference in lattice parameter, mosaicity, etc (but it is very dehydrated). VM and any other measure is just the physical consequence of a lattice shrinking where your solvent/protein ratio is changing. The key is weather the change is useful or not. For example photosystem I or II (can't remember exactly) had a very clear pattern correlating approximate lattice parameter with resolution limit in deposited structures. As resolution improved lattice parameters where smaller. Coincidently they also show an improvement with controlled dehydration. I'm not looking for anecdotes I am looking for cases where people are confident that process A gave them something and process B gave them something better and that they might have also observed a lattice change, space group shift, etc. I know there are lots of good cases out there but I can't get hold of them. I hope this is clearer, Juan Juan Sanchez-Weatherby, PhD Beamline Scientist - I02 Tel: +44 (0)1235 778661 Mob:+44 (0)7795 641259 -Original Message- From: Enrico Stura [mailto:est...@cea.fr] Sent: 16 January 2013 13:51 To: ccp4bb; Sanchez-Weatherby, Juan (DLSLtd,RAL,DIA) Subject: Re: [ccp4bb] side question re crystal dehydration Juan, Humidity variation is what vapour diffusion crystallization achieves. In your list of all possible dehydration methods you would end up classifying all vapour diffusion experiments as a case of dehydration. After nucleation, crystals continue to grow and the drop continues to become more concentrated in precipitant. I agree you are completely right! The concept of crystal hydration is very complicated: Crystals are grown in liquid water and often analysed in vitrified water with a solvent-hydrogen bond network that is different from that in the liquid state. What does this mean in terms of hydration? The use of cryo-protectant alter the solvent hydrogen bonding pattern. What does this mean in terms of hydration? VM, the Matthews coefficient, defined as the crystal volume per unit of protein molecular weight is a a measure of hydration? So if the VM is the same the hydration is the same? I agree that all the methods that you mention will affect hydration of the crystals, but the way that X-ray crystallography is carried out today cannot avoid it. Crystal dehydration must be defined as an explicit effort to use methodology designed to alter the hydration of crystals, preferably using a defined measured and controlled relative humidity value. As you start to consider badly defined systems, you will also have badly defined hydration. Enrico. On Wed, 16 Jan 2013 14:18:05 +0100, Juan Sanchez-Weatherby juan.sanchez-weathe...@diamond.ac.uk wrote: Dear all, From Leonid's reply earlier you can see a problem some of us have been having for a while now, when looking for literature regarding dehydration. Most of you that perform dehydration either don't consider it happening or don't report it in great detail in your publications. This is only understandable because it isn't the focus of your work and it only helps you get to where you want to get to. I'm trying to get an up to date picture of what is out there but I haven't got the time or eyes to go through everyone's methods to pick the
Re: [ccp4bb] protein degradation in crystal
Could you identify the cleavage sites by protein sequencing and design new constructs (truncated versions) accordingly? It might improve your crystal quality to get better resolution. Joe On Wed, Jan 16, 2013 at 9:22 AM, Tom Murray-Rust tom.murray.r...@gmail.comwrote: Just to add to Herman's suggestions, if you are trying to crystallise a protease then you could also try using the S195A variant rather than an inhibitor. This would certainly be the case if you ever want to co-crystallise in a substrate, as PPACK (or the like) would occupy the active site cleft and prevent formation of the protease-substrate complex. Tom ** Hi John, This is really an amazing wild west story: the man who crystallizes faster than his protease! I really must compliment you with how you successfully performed these experiments! Unfortunately, proteins usually do not crystallize that fast (at least not in my hands), so in these cases other methods have to be used. As has mentioned before, protease inhibitors are the way to go. Especially with autolysis, as one protease cuts another one, the speed of the reaction goes with the square of the protease concentration. Whereas in dilute solutions not much happens, as soon as you start to concentrate towards crystallization conditions, say 10 mg/ml, degradation suddenly goes very fast. There are 2 cases to consider: 1) the protein you want to crystallize is a protease and is destroying itself. In this case you need to cocrystallize with a potent and specific inhibitor. With serine proteases, Wolfram Bode was very successful by using chloromethylketone-containing peptides (e.g. PPACK). These compounds would make covalent links with both the active site serine and histidine, effectively killing any protease activity. 2) the protein you want to crystallize is not a protease and it is a contaminant which is causing the problems. In this case I would add a protease inhibitor coctail in an earlier step of the purification to block the protease before the final purification steps. I would also add some small broad protease inhibitor e.g. PMSF to the protein solution used for crystallization. Herman -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *John Domsic *Sent:* Wednesday, January 16, 2013 2:22 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] protein degradation in crystal Hi Lisa, Speed is definitely a big factor here. With a protein I work with I can get large crystals in myriad conditions that only diffract to about 4-5 Ang. What I ended up doing was taking these crystals and seeding entire screens. I found that not only would crystals appear sooner but it revealed novel crystallization conditions. These seeded crystals would appear within minutes as Preben described and diffracted to better than 2 Ang. Another thought would be to try limited proteolysis to see if you can identify a more stable construct. -John -- John Domsic Postdoctoral Fellow Gene Expression and Regulation Program The Wistar Institute Philadelphia, PA 19104 On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth j.p.mo...@ncmm.uio.no wrote: Dear Lisa It is not uncommon to see breakdown products when you run crystals on gel. Espesially if they are older crystals, sometimes you even see higher molecular bands, these are probably due to intra molecular cross links formed over time. If you are worried about stability, try to increase the crystallization speed, we have one example where we see a clear difference in both crystal quality and even space group depending on when we fish the crystals. The crystals appear within 5 min, the best quality data sets come from crystals we fish after only 30-60 min. You may also have a little protease contamination of course, to prevent this add protease inhibitor, or DTT, or EDTA to you protein before you set it up. cheers Preben On 1/16/13 12:14 PM, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several samll bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 %2B47%202284%200794 http://www.jpmorth.dk -- Skype: tom.murray.rust Twitter: tmurrayrust
Re: [ccp4bb] how many metal sites
Zn is a very electron rich atom so a 2.3 A resolution data set should be a fine experiment to determine the number of fully occupied metal sites. It is always hard to be sure about screen shots of density, but it looks to me that you only have evidence for one zinc here. In my opinion, it is not useful to build models that don't make sense. Your zinc cluster does not make chemical sense to me and the atoms are not in the density. I suspect that you built this cluster, and not the obvious model with fewer zinc atoms, simply because you wanted to match the magic number of four. Use the things you know with confidence as your guide. Dale Tronrud On 01/16/13 11:15, ruisher hu wrote: Hi, Dear All, I recently got a dataset about 2.3 A resolution, however, I got some trouble assigning the metal sites. It suppose to have multiple binding site(possibly four) around those four glu residues in the center (see the attached figure), however, it shows up a huge single positive density ,clustered in the binding center. The signal is pretty strong and I think zn is definitely there. When I tried to put four zns around, the geometry doesn't look very good and there is still some positive density in the center (although get weaker) and the bfactor of metals are high like 100. Does anyone know what's going on?Does it mean only one single site in the middle?Or maybe just metals are too mobile? What's the best way to tell how many metal sites are actually there?Which experiment can I use to test? Thanks very much. Best, R On Wed, Nov 7, 2012 at 9:29 AM, SD Y ccp4...@hotmail.com mailto:ccp4...@hotmail.com wrote: Dear all, I have a related question to the one I have posted low resolution and SG, on which I am still working based on the suggestions I have got. The model I have used, has Zn co-ordinated well in tetrahydral fashion by 3 cys and 1 His residues. They have add Zn in to their experiment. In my 3.4 A structure (I am still working on right SG), initial maps show very strong positive density (sigma=6.5) at the place of Zn ( https://www.dropbox.com/s/4jd6gdor87ab9lj/Zn-coordination.png). I have not used Zn in my experiment. I could only suspect Tryptone and yeast extract which I used to make media. I would like to know how likely this positive density belongs to Zn? How to reason the presence of Zn when its not been used? Is there is any way to confirm if its Zn. If this is not Zn, what else could it be? Any thing I could try to rule out or in Zn or other ions. I appreciate your help and suggestions. Sincerely, SDY
Re: [ccp4bb] how many metal sites
The geometry is difficult to see from a static image, but it looks like you possibly have a His(2)Glu(4) coordination environment around what looks like one likely metal ion, based on the difference density. Zn(II) is typcially (but not always) tetrahedral with Zn-O/N bond distances of approximately 2.0 A. You should look carefully at the coordination environment of the putative metal ion to determine if it is tetrahedral, pentacoordinate (like a trigonal bipyramid), or octahedral. Zn(II) can adopt all of those coordination environments, but octahedral environment could be a lot of different metals, e.g., Fe(II), etc. Octahedral bond lengths for metal-bound ligands will be different from tetrahedral or pentacoordinate bond lengths. BTW, both Zn and Fe are very common adventitious metal ions in protein preps, and are very difficult to exclude. You have options for directly identifying the metal in the home lab: If the metal is bound tight enough (and it may be stoichiometric) then (1) ICP-OES or (2) ICP-MS or (3) TXRF of a protein sample should be able to detect its presence. TXRF is really nice in that there is no prep and it will work on a few uL of sample, but the others are also quite sensitive, and will only require 10-50 uL of sample depending on your protein concentration. When you are a building a metalloenzyme model you should really have some solid evidence that a metal ion is present by (1) inclusion in the crystallization medium, (2) direct determination by an analytical technique, (3) UV-visible spectroscopy (when appropriate--obviously Zn(II) is d10 and silent in the visible d-d transition wavelength range) and/or (4) appropriate coordination geometry and bond lengths. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 1/16/2013 2:15 PM, ruisher hu wrote: Hi, Dear All, I recently got a dataset about 2.3 A resolution, however, I got some trouble assigning the metal sites. It suppose to have multiple binding site(possibly four) around those four glu residues in the center (see the attached figure), however, it shows up a huge single positive density ,clustered in the binding center. The signal is pretty strong and I think zn is definitely there. When I tried to put four zns around, the geometry doesn't look very good and there is still some positive density in the center (although get weaker) and the bfactor of metals are high like 100. Does anyone know what's going on?Does it mean only one single site in the middle?Or maybe just metals are too mobile? What's the best way to tell how many metal sites are actually there?Which experiment can I use to test? Thanks very much. Best, R
Re: [ccp4bb] how many metal sites
On Wed, Jan 16, 2013 at 2:53 PM, Roger Rowlett rrowl...@colgate.edu wrote: When you are a building a metalloenzyme model you should really have some solid evidence that a metal ion is present by (1) inclusion in the crystallization medium, (2) direct determination by an analytical technique, (3) UV-visible spectroscopy (when appropriate--obviously Zn(II) is d10 and silent in the visible d-d transition wavelength range) and/or (4) appropriate coordination geometry and bond lengths. What about: (5) anomalous scattering (i.e. anomalous difference map)? Even on a home source I suspect Zn should still be visible, and at shorter wavelengths this should certainly be the case if the anomalous data are reasonably good and complete. The coordination geometry and bond lengths aren't necessarily going to be definitive at this resolution, although I agree that it should be approximately tetrahedral. -Nat
Re: [ccp4bb] how many metal sites
*** This message has been scanned by the InterScan for CSC SSM by IICT security policy and found to be free of known security risks. *** I agree with Dale that your four metals are not in the density and also don't seem to make sense in terms of chemistry. Based on your residue placement and electron density, it looks more like a non-heme manganese catalase structure where two metal ions bind with very similar environment. This reference may help you. Archives of Biochemistry and Biophysics Volume 525, Issue 2, 15 September 2012, Pages 111–120 Anthony - Dr. Anthony Addlagatta Center for Chemical Biology Indian Institute of Chemical Technology [IICT] Tarnaka, Hyderabad AP-500 607, INDIA Tel:91-40-27191812 Web: https://sites.google.com/site/chembioliict/home/dr-anthony-addlagatta-1 -- Original Message --- From: Dale Tronrud det...@uoxray.uoregon.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 16 Jan 2013 14:14:50 -0800 Subject: Re: [ccp4bb] how many metal sites *** This message has been scanned by the InterScan for CSC SSM at IICT and found to be free of known security risks. *** Zn is a very electron rich atom so a 2.3 A resolution data set should be a fine experiment to determine the number of fully occupied metal sites. It is always hard to be sure about screen shots of density, but it looks to me that you only have evidence for one zinc here. In my opinion, it is not useful to build models that don't make sense. Your zinc cluster does not make chemical sense to me and the atoms are not in the density. I suspect that you built this cluster, and not the obvious model with fewer zinc atoms, simply because you wanted to match the magic number of four. Use the things you know with confidence as your guide. Dale Tronrud On 01/16/13 11:15, ruisher hu wrote: Hi, Dear All, I recently got a dataset about 2.3 A resolution, however, I got some trouble assigning the metal sites. It suppose to have multiple binding site(possibly four) around those four glu residues in the center (see the attached figure), however, it shows up a huge single positive density ,clustered in the binding center. The signal is pretty strong and I think zn is definitely there. When I tried to put four zns around, the geometry doesn't look very good and there is still some positive density in the center (although get weaker) and the bfactor of metals are high like 100. Does anyone know what's going on?Does it mean only one single site in the middle?Or maybe just metals are too mobile? What's the best way to tell how many metal sites are actually there?Which experiment can I use to test? Thanks very much. Best, R On Wed, Nov 7, 2012 at 9:29 AM, SD Y ccp4...@hotmail.com mailto:ccp4...@hotmail.com wrote: Dear all, I have a related question to the one I have posted low resolution and SG, on which I am still working based on the suggestions I have got. The model I have used, has Zn co-ordinated well in tetrahydral fashion by 3 cys and 1 His residues. They have add Zn in to their experiment. In my 3.4 A structure (I am still working on right SG), initial maps show very strong positive density (sigma=6.5) at the place of Zn ( https://www.dropbox.com/s/4jd6gdor87ab9lj/Zn-coordination.png). I have not used Zn in my experiment. I could only suspect Tryptone and yeast extract which I used to make media. I would like to know how likely this positive density belongs to Zn? How to reason the presence of Zn when its not been used? Is there is any way to confirm if its Zn. If this is not Zn, what else could it be? Any thing I could try to rule out or in Zn or other ions. I appreciate your help and suggestions. Sincerely, SDY --- End of Original Message --- This Mail Scanned by ClamAV and Spammassassin
[ccp4bb] Unit Cell of Ensemble must be orthogonal
Dear all, When I used PHASER to do a molecular replacement and define ensemble via map (mtz file), the program stoped very soon. The log file showed that Unit Cell of Ensemble must be orthogonal. I tried many ways to generate the map file, but the results are the same. Can anyone help me to solve this problem? Thanks a lot! Wei
[ccp4bb] Crystallization buffer pH optimization
Hello, Shameful and sorry for asking this simple question, it looks like this when first starting a new setup in so-called structural biology. I remmeber a book of, probably, Hampton, in which there are tables of pH optimization for many buffers. For example for buffer TRIS, the table will list how many drops NaOH/HCl needed to change pH from 7.0 to 7.2, etc. This is useful for crystallization pH optimization, where can I buy or download this info? Alternatives? Thank you very much! Mike
Re: [ccp4bb] Crystallization buffer pH optimization
Hi Mike, check out the EZ screen builder from Emerald Bio, that is a really nice tool for this purpose: http://www.emeraldbiosystems.com/escreentoolkit/escreen.html Select your screen, your hit condition, then hit 'optimize' and you will have a screen that will vary the precipitant concentration in the horizontal and the pH in the vertical direction. You can modify the optimization screen as you like, change concentrations, the pH range, add components etc. You can then either order the screen from EmeraldBio, or download a pipetting protocol to make it from your own stock solutions. Good luck! Jan -- Jan Abendroth Emerald BioStructures Seattle / Bainbridge Island WA, USA home: Jan.Abendroth_at_gmail.com work: JAbendroth_at_embios.com http://www.emeraldbios.com On Jan 16, 2013, at 9:38 PM, Mike John perturb-w...@hotmail.com wrote: Hello, Shameful and sorry for asking this simple question, it looks like this when first starting a new setup in so-called structural biology. I remmeber a book of, probably, Hampton, in which there are tables of pH optimization for many buffers. For example for buffer TRIS, the table will list how many drops NaOH/HCl needed to change pH from 7.0 to 7.2, etc. This is useful for crystallization pH optimization, where can I buy or download this info? Alternatives? Thank you very much! Mike
