Re: [ccp4bb] precipitates
Dear Amit -- On 30 Sep 2008, at 14:16, amit sharma wrote: I am trying to crystallize a protein with a peptide attached to it via a 9aa linker. I am mostly getting precipitates in conditions carrying salts. I have tried optimization screens by varying the pH and salt concentrations, but in vain. Can anyone please suggest ways by which I could optimize the conditions further? Among all the possibilities, you can: - try to crystallize without salt ; - change the temperature ; - dilute your protein ; - add some isopropanol (or other) to make the solution smoother ; - change your linker size ; - ... HTH Kind regards. -- Leo -- Chavas Leonard, Ph.D. @ home Research Associate Marie Curie Actions Fellow Faculty of Life Sciences The University of Manchester The Michael Smith Building Oxford Road Manchester Lancashire M13 9PT Tel: +44(0)161-275-1586 e-mail: [EMAIL PROTECTED] http://personalpages.manchester.ac.uk/staff/leonard.chavas/
Re: [ccp4bb] precipitates
Dear All, Many thanks for the suggestions. Cheers, Amit 2008/10/1 Chavas Leo [EMAIL PROTECTED] Dear Amit -- On 30 Sep 2008, at 14:16, amit sharma wrote: I am trying to crystallize a protein with a peptide attached to it via a 9aa linker. I am mostly getting precipitates in conditions carrying salts. I have tried optimization screens by varying the pH and salt concentrations, but in vain. Can anyone please suggest ways by which I could optimize the conditions further? Among all the possibilities, you can: - try to crystallize without salt ; - change the temperature ; - dilute your protein ; - add some isopropanol (or other) to make the solution smoother ; - change your linker size ; - ... HTH Kind regards. -- Leo -- Chavas Leonard, Ph.D. @ home Research Associate Marie Curie Actions Fellow Faculty of Life Sciences The University of Manchester The Michael Smith Building Oxford Road Manchester Lancashire M13 9PT Tel: +44(0)161-275-1586 e-mail: [EMAIL PROTECTED] http://personalpages.manchester.ac.uk/staff/leonard.chavas/ -- Amit Sharma, Ph.D. Research Associate, Department of Biology, University of York, YO10 5DD UK
[ccp4bb] Anomalous correlation plot in scala
Dear all, I'm analyzing some SAD data sets and I would like to compare them in terms of anomalous correlation. Is there a way in scala (or in some other program) to calculate a correlation plot like the file_correlplot.xmgr using two different data sets (or better two different runs) instead of having scala using the randomly generated two-half data sets ? Cheers Daniele -- Daniele de Sanctis, PhD Macromolecular Crystallography Group ESRF, Grenoble, France Tel 33 (0)4 76 88 2869
[ccp4bb] to extract protein sequence from a rebuilt pdb file
Hi, all I am currently rebuilting my structure in coot, and the protein sequence is around 98% rebuilt yet. I am looking for a way to extract protein sequence from the rebuilt.pdb so that I could see clearly which part need to be rebuilt by sequence alignment. Any ideas about this? thanks Best Jane
Re: [ccp4bb] to extract protein sequence from a rebuilt pdb file
echo sequence single | pdbset xyzin rebuilt.pdb sequence rebuilt.seq On 1 Oct 2008, at 09:40, Jane Bailey wrote: Hi, all I am currently rebuilting my structure in coot, and the protein sequence is around 98% rebuilt yet. I am looking for a way to extract protein sequence from the rebuilt.pdb so that I could see clearly which part need to be rebuilt by sequence alignment. Any ideas about this? thanks Best Jane
Re: [ccp4bb] Anomalous correlation plot in scala
If the two datasets are assigned to different datasets in the mtz file (from mosflm, pointless or rebatch), or assigned to different datasets in the scala then the correlation coefficients are calculated between datasets, but not the correlplot. Maybe I should add it ... Phil Evans On 1 Oct 2008, at 08:30, Daniele de Sanctis wrote: Dear all, I'm analyzing some SAD data sets and I would like to compare them in terms of anomalous correlation. Is there a way in scala (or in some other program) to calculate a correlation plot like the file_correlplot.xmgr using two different data sets (or better two different runs) instead of having scala using the randomly generated two-half data sets ? Cheers Daniele -- Daniele de Sanctis, PhD Macromolecular Crystallography Group ESRF, Grenoble, France Tel 33 (0)4 76 88 2869
Re: [ccp4bb] to extract protein sequence from a rebuilt pdb file
You can do this quickly in Coot: CalculateScriptingScheme And a scripting window opens. See the Coot user manual (1) for detailed scheme instructions, but the command now is in the form: (print-sequence imol) imol comes from the number shown in the display manager to the left of the model name. e.g.: (print-sequence 0) ...for imol 0. The sequences of each chain are output to the terminal. If you have only python support in Coot: CalculateScriptingPython print_sequence(0) is the pythonized version of the command for imol 0. (1) http://www.ysbl.york.ac.uk/~emsley/coot/doc/user-manual.html#SEC79 2008/10/1 Jane Bailey [EMAIL PROTECTED] Hi, all I am currently rebuilting my structure in coot, and the protein sequence is around 98% rebuilt yet. I am looking for a way to extract protein sequence from the rebuilt.pdb so that I could see clearly which part need to be rebuilt by sequence alignment. Any ideas about this? thanks Best Jane -- Mark BROOKS Telephone: 0169157968 Fax: 0169853715 Institut de Biochmie et de Biophysique Moleculaire et Cellulaire UMR8619 - Bât 430 - Université de Paris-Sud 91405 Orsay CEDEX Skype: markabrooks
[ccp4bb] resuspending precipitated protein
Hello, following on from a previous topic about precipitating protein, but I believe a distinct caveat of this warranting a separate thread, I would like to know people's experiences in trying to get precipitated protein back into solution? Is there a way or are there many ways? Any experiences would be welcome, yours, Andy
[ccp4bb] Bin Number (0 -19) Put in Mtz file in Place of Rfree Flag -and- 5MC in DNA
I've tried several ways to get an Rfree flag into my mtz file...converting from a CNS to mtz (with existing flags) and scalepack to mtz and etc etc...with the same result, i.e. that the bin number that the reflection is in (0-19) is put in mtz file as the Rfree flag and not 0 or 1. I was wondering if this has to do with array size or something. This is a 1.42A dataset with 125982 reflections. In addition, I am trying to get refmac to recognize a 5MC as a DNA base with proper links in the backbone etc...what is the best way to do this? -- John R. Horton, Ph.D. Assistant Professor (Research) E- mail:[EMAIL PROTECTED] Emory University School of MedicineVoice- mail: 404-727-8492 Department of Biochemistry FAX:404-727-3746 Rollins Research Center, Suite G239 1510 Clifton Road Atlanta, GA 30322 --
Re: [ccp4bb] Bin Number (0 -19) Put in Mtz file in Place of Rfree Flag -and- 5MC in DNA
John R. Horton wrote: I've tried several ways to get an Rfree flag into my mtz file...converting from a CNS to mtz (with existing flags) and scalepack to mtz and etc etc...with the same result, i.e. that the bin number that the reflection is in (0-19) is put in mtz file as the Rfree flag and not 0 or 1. I was wondering if this has to do with array size or something. This is a 1.42A dataset with 125982 reflections. In MTZ files, free-R flags are usually from 0-19 (or 0-N, if you are not using a 5% free set). There is a good reason for this - if you are not sure whether your FreeR is meaningful or not, you can re-run your structure solution using a different, non-overlapping FreeR set just by telling each individual program to use a set other than 0 as the test set. By this means you can do a full cross-validation. Has anyone ever used this in practice? I don't know. But it is a good design feature.
Re: [ccp4bb] Anomalous correlation plot in scala
Dear Daniele, you could 'misuse' RSTATS: cad hklin1 data1.mtz hklin2 data2.mtz hklout cad.mtz e LABI FILE 1 E1=DANO E2=SIGDANO LABO FILE 1 E1=DANO1 E2=SIGDANO2 LABI FILE 2 E1=DANO E2=SIGDANO LABO FILE 2 E1=DANO2 E2=SIGDANO2 e rstats hklin cad.mtz e LABI FP=DANO1 SIGFP=SIGDANO1 FC=DANO2 SIGFC=SIGDANO2 NOAB CYCL 0 OUTP NOHKL END e _should_ work and give you those tables ... Or use SFTOOLS on that cad.mtz with the CORREL command. Cheers Clemens On Wed, Oct 01, 2008 at 09:30:32AM +0200, Daniele de Sanctis wrote: Dear all, I'm analyzing some SAD data sets and I would like to compare them in terms of anomalous correlation. Is there a way in scala (or in some other program) to calculate a correlation plot like the file_correlplot.xmgr using two different data sets (or better two different runs) instead of having scala using the randomly generated two-half data sets ? Cheers Daniele -- Daniele de Sanctis, PhD Macromolecular Crystallography Group ESRF, Grenoble, France Tel 33 (0)4 76 88 2869 -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] mtz library in windows
Dear Alex The exact form taken by the external symbols depends on the compiler settings. The Compaq Fortran 6.6 compiler defaulted to adding an @ to the function name, followed by a number which was the sum of the sizes in bytes of the function arguments. This practice was not followed in later versions of the compiler, after the rights to it had been bought by Intel. We currently use the Intel 10 compiler to build the suite. You need to adjust your Project properties to get the external symbols right. Go to Project, Properties, Fortran, External Procedures and try adjusting the Calling Convention entry. 'C, Reference' is probably the one to use. (At least this is what you do with later versions of the compiler. It's a few years since I last used CVF6.6 so you might need to make minor modifications to these instructions. I assume you are running through the gui. If you are running CVF 6.6 from the command line, please get back to me and I'll try to dig out the command line way of doing this). Norman Stein CCP4 From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Alex XIE Sent: 01 October 2008 03:07 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] mtz library in windows Dear all, I am using CCP4 mtz library and other libraries to develop a program. Because I am using a PC at home, I am actually using the CCP4 in windows. However, when I link the mtz library(hence libccp4c.lib), it happens that the compiler complains error LNK2001: unresolved external symbol [EMAIL PROTECTED] and some other functions that should be in libccp4c not found. I checked the project setting(I am using compaq visual fortran 6.6), I have included libccp4c, libccp4f in the library, and library path in /link/input/Additional Library Path, so I don't understand why the compiler still cannot resolve the functions. Can anybody help, please? Your suggestion is greatly appreciated. Alex
Re: [ccp4bb] to extract protein sequence from a rebuilt pdb file
Hi Jane, Qingping Xu wrote a python script that will extract the sequence from the PDB file and align it with a fasta file. The script depends on biopython and clustalw. Regards, Mitch -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Miller, Mitchell D. Sent: Tuesday, October 03, 2006 1:57 PM To: Bottomley, Matthew; [EMAIL PROTECTED] Subject: RE: [ccp4bb]: PDB to 1 letter code *** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk *** Hi Matt, Qingping Xu wrote a python script that will compare a PDB file with a fasta file and corrects ala/gly to the correct amino acid on output. The script depends on biopython and clustalw. If you are interested I made a copy available, http://smb.slac.stanford.edu/~mmiller/seqcheck.py http://biopython.org/ http://www.ebi.ac.uk/clustalw/ Regards, Mitch From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jane Bailey Sent: Wednesday, October 01, 2008 1:40 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] to extract protein sequence from a rebuilt pdb file Hi, all I am currently rebuilting my structure in coot, and the protein sequence is around 98% rebuilt yet. I am looking for a way to extract protein sequence from the rebuilt.pdb so that I could see clearly which part need to be rebuilt by sequence alignment. Any ideas about this? thanks Best Jane
Re: [ccp4bb] to extract protein sequence from a rebuilt pdb file
Thanks, I have found out that swiss pdb viewer could easily export sequence, thanks all. On Wed, Oct 1, 2008 at 5:49 PM, S. Thiyagarajan [EMAIL PROTECTED] wrote: Hi I would take it pdb deposition site (rcsb.org) and feed the pdb into the validation server. That gives out the sequence of the input pdb regards == S. Thiyagarajan == == #5344 Scott Hall == == Department of Biochemistry and Molecular Biology == == School of Medicine, Wayne State University == == 540 E. Canfield St. == == Detroit, MI 48201 U.S.A == == Phone: +1-313-577-0618 (lab) == +1-248-412-3758 (Cell) == --- On *Wed, 10/1/08, Jane Bailey [EMAIL PROTECTED]* wrote: From: Jane Bailey [EMAIL PROTECTED] Subject: [ccp4bb] to extract protein sequence from a rebuilt pdb file To: CCP4BB@JISCMAIL.AC.UK Date: Wednesday, October 1, 2008, 4:40 AM Hi, all I am currently rebuilting my structure in coot, and the protein sequence is around 98% rebuilt yet. I am looking for a way to extract protein sequence from the rebuilt.pdb so that I could see clearly which part need to be rebuilt by sequence alignment. Any ideas about this? thanks Best Jane
Re: [ccp4bb] Putting ligand in the protein structure
On Sep 30, 2008, at 9:56, Anshul Awasthi wrote: Hi all the crystallographers, I am trying to solve a structure of a protein with some inhibitor. I want to know how I can put in my inhibitor in the density map of the data i got. I can see some density in the active site where the inhibitor should be. I generated the topoly file of the inhibitor (in both pdba nd refmac5 top formats) from the Dundee PRODRG server. Now do i need to incorporate the structure of the inhibitor in ccp4 or can i do in coot?? I am not sure of how to do it. As one of many alternatives you can use ARP/wARP Ligand Fit from the CCP4I (provided you downloaded and installed the current version of ARP/wARP) and then input your protein structure, the observed data, and the PDB of your ligand. The script will calculate the mFo-DFc map, find the most likely site for the ligand, and then fir the ligand there, and refine it in real space. Tassos ANy sugegstion will be very valuable for me.
Re: [ccp4bb] Putting ligand in the protein structure
The best why that I have put inhibitors in is by using the sketcher program in CCP4. Its a little unwieldy at first but if you get the hang of it, it provides pdb's and library files that can be directly inputed into refmac and Coot. The Coot ligand find function does a pretty good job of initially placing inhibitors in density. The only thing to be careful of is that sketcher tends to label hydrogens in a problematic way for large ligands. It labels the hydrogen based on the carbon number that it is attached too. Problems tend to occur if the hydrogens get three digit labels. Refmac won't necessarily recognize these causing problems with the cif file. If this problem occurs re-label the hydrogens to two digit numbers. Unless your using a ligand that requires more than 100 hydrogens. Then you will have to build it a different way. Scott On Wed, Oct 1, 2008 at 3:38 PM, Anastassis Perrakis [EMAIL PROTECTED]wrote: On Sep 30, 2008, at 9:56, Anshul Awasthi wrote: Hi all the crystallographers, I am trying to solve a structure of a protein with some inhibitor. I want to know how I can put in my inhibitor in the density map of the data i got. I can see some density in the active site where the inhibitor should be. I generated the topoly file of the inhibitor (in both pdba nd refmac5 top formats) from the Dundee PRODRG server. Now do i need to incorporate the structure of the inhibitor in ccp4 or can i do in coot?? I am not sure of how to do it. As one of many alternatives you can use ARP/wARP Ligand Fit from the CCP4I (provided you downloaded and installed the current version of ARP/wARP) and then input your protein structure, the observed data, and the PDB of your ligand. The script will calculate the mFo-DFc map, find the most likely site for the ligand, and then fir the ligand there, and refine it in real space. Tassos ANy sugegstion will be very valuable for me. -- Scott D. Pegan, Ph.D. Senior Research Specialist Center for Pharmaceutical Biotechnology University of Illinois at Chicago
Re: [ccp4bb] mtz library in windows
Hi, Stein, I tried your method but still found compiling errors. The compiler complains a library not found. I searched the web and found that the library only exists in the Intel Fortran Compiler. My decision is that I will switch to IFC. Thanks for your message. It is very useful. Alex On Wed, Oct 1, 2008 at 10:15 PM, Stein, ND (Norman) [EMAIL PROTECTED]wrote: Dear Alex The exact form taken by the external symbols depends on the compiler settings. The Compaq Fortran 6.6 compiler defaulted to adding an @ to the function name, followed by a number which was the sum of the sizes in bytes of the function arguments. This practice was not followed in later versions of the compiler, after the rights to it had been bought by Intel. We currently use the Intel 10 compiler to build the suite. You need to adjust your Project properties to get the external symbols right. Go to Project, Properties, Fortran, External Procedures and try adjusting the Calling Convention entry. 'C, Reference' is probably the one to use. (At least this is what you do with later versions of the compiler. It's a few years since I last used CVF6.6 so you might need to make minor modifications to these instructions. I assume you are running through the gui. If you are running CVF 6.6 from the command line, please get back to me and I'll try to dig out the command line way of doing this). Norman Stein CCP4 -- *From:* CCP4 bulletin board [mailto:[EMAIL PROTECTED] *On Behalf Of *Alex XIE *Sent:* 01 October 2008 03:07 *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] mtz library in windows Dear all, I am using CCP4 mtz library and other libraries to develop a program. Because I am using a PC at home, I am actually using the CCP4 in windows. However, when I link the mtz library(hence libccp4c.lib), it happens that the compiler complains error LNK2001: unresolved external symbol [EMAIL PROTECTED] and some other functions that should be in libccp4c not found. I checked the project setting(I am using compaq visual fortran 6.6), I have included libccp4c, libccp4f in the library, and library path in /link/input/Additional Library Path, so I don't understand why the compiler still cannot resolve the functions. Can anybody help, please? Your suggestion is greatly appreciated. Alex
[ccp4bb] R32 data
Dear all, I have set of data (~2.7A). After indexing, HKL2000 suggests a rhombohedral space group or C centered monoclinic space group. I tried molecular replacement with R32 ( 109.55 109.55 155.28 90.00 90.00 120.00) and C2 (121.092 109.31581.53790.00097.874 90.000) (both have good merging statistics), but did not get any solution. I also tried P1 ( 81.55381.52781.52384.202 84.13284.156) but did get any solution ( wt structure is known and the only difference with the wt structure is that the new structure is complexed with a smaller protein). I expect that there'll be some domain movement so searched for individual domains in phaser. The self rotation function in P1 showed both two fold and three fold peaks but they were both off the centre. The data does not appear to be twinned. I am curious to know if it is normal for cell dimensions to drastically vary from one space group to another. I am running out of ideas, could I be dealing with a wrong space group or pseudosymetry? Any suggestions would be appreciated. Thanks. Josiah.
[ccp4bb] Protein-DNA complex prepartion for crystallization
Dear All Sorry for non-crystallography query. I am working on DNA binding protein, while mixing DNA with protein for preparing Protein-DNA complex for crystallization, protein is precipitating. pI of the protein is 9.3 and in 15 mM HEPES 7.0, 150mM NaCl and 5% glycerol. Concentration of the protein used for mixing with DNA is 8 mg/mL. DNA to protein molar ratio is 1.2. Please advise me how to prevent precipitation. Is changing of buffer pH and adding divalent cation like MgCl2 can help in preventing precipitation? Thanking You Rajakumara E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) Get your preferred Email name! Now you can @ymail.com and @rocketmail.com. http://mail.promotions.yahoo.com/newdomains/aa/
[ccp4bb] Pittsburgh Diffraction Conference - deadline reminder
Greetings: This is a reminder that the deadline for submitting an abstract for the PDC poster session has been extended to October 10th. Also, the deadline for reserving a hotel room at the Holiday Inn University Center at the special discounted rate of $119/night is October 8th. For more information on accommodations please visit this webpage: http://www.pittdifsoc.org/PDC_2008/accommodations.htm. The conference schedule is available here: http://www.pittdifsoc.org/PDC_2008/schedule.htm. Aina Cohen From: Cohen, Aina E. Sent: Wednesday, August 27, 2008 2:53 PM To: Subject: FW: Pittsburgh Diffraction Conference - 2008 Dear Colleague: On behalf of the program committee, you and your colleagues are cordially invited to join us for the 66th Annual Pittsburgh Diffraction Conference to be held from October 30th - November 1st, 2008 in Pittsburgh, Pennsylvania at the Holiday Inn University Center. The goal of the conference is to bring together researchers in all areas of fundamental and applied diffraction and crystallographic research to present current topics. The program of the 2008 conference includes diffraction phasing, structure refinement and validation, synchrotron data collection, environmental science, materials science, chemistry and combined methods for structural biology research including NMR, Cryo-EM, EXAFS, and Raman spectroscopy. Conference speakers include: Sandy Asher, Department of Chemistry, University of Pittsburgh Pavel Afonine, The Berkeley Center for Structural Biology, LBNL Guillermo Calero, Department of Structural Biology, University of Pittsburgh Tom Caradoc-Davies, Protein Crystallography, Australian Synchrotron Paul Carey, Department of Biochemistry, Case Western Reserve University Angela R. Criswell, Rigaku, USA Yu-Sheng Chen, Microcrystallography, ChemMatCARS, APS James Conway, Department of Structural Biology, University of Pittsburgh Bryan Craven, Department of Chemistry, Indiana University of Pennsylvania Angela Gronenborn, Department of Structural Biology, University of Pittsburgh Bill Furey, Biocrystallography, VA Medical Center, University of Pittsburgh Roger Hendrix, Department of Biological Sciences, University of Pittsburgh Xinguo Hong, MacCHESS, Cornell High Energy Synchrotron Source Li-Wei Hung, The Berkeley Center for Structural Biology, LBNL Christian Jelsch, LCM3B, Nancy University, France Patrick Loria, Biophysical Physical Chemistry, Yale University Jianpeng Ma, Biochemistry, Baylor College of Medicine Michael D. Moore, Department of Pharmaceutics, Duquesne University Jianjun Pan, Department of Physics, Carnegie Mellon University Kanagalaghatta Rajashankar, NE-CAT, APS / Cornell University Kaustubh Sinha, Department of Biological Sciences, Carnegie Mellon University John Rose, Biochemistry Molecular Biology, University of Georgia Nathaniel Rosi, Department of Chemistry, University of Pittsburgh Glenn Waychunas, Molecular Geochemistry Nanoscience Group, LBNL Hui Wu, NIST Center for Neutron Research Junko Yano, Physical Biosciences, Lawrence Berkeley National Laboratory For more information about the conference program please visit the conference website: http://www.pittdifsoc.org/PDC_2008/schedule.htm http://www.pittdifsoc.org/PDC_2008/schedule.htm . Poster sessions and social events, included in the program, provide many opportunities for formal and informal discussions with the speakers. The Chung Soo Yoo Award will be given to graduate students for best poster presentation. To insure maximum participation, the cost of attending the conference has been kept to a minimum. The conference will be preceded by a workshop for researchers intrigued by or new to crystallography titled, ‘Crystallography Made Easy through Automation’ on October 29th at the University of Pittsburgh, Structural Biology Department. This workshop will introduce what structural information crystallography can provide, the basics of crystallographic techniques (protein crystallization, x-ray diffraction data collection, and data analysis) and then demonstrate how these steps may be simplified by taking advantage of the automated facilities available at Stanford Synchrotron Radiation Laboratory and the Hauptman Woodward Medical Research Institute. The workshop is open to 24 participants and advanced registration is required. For more information and to register, please visit the workshop website: http://www-conf.slac.stanford.edu/crystallography http://www- conf.slac.stanford.edu/crystallography . Discounted accommodations for the Pittsburgh Diffraction Conference and workshop attendees are available at the Holiday Inn University Center. The Holiday Inn is holding rooms at this discounted rate until October 8th. For additional information and instructions, please view our website at: http://www.pittdifsoc.org/PDC_2008/ http://www.pittdifsoc.org/PDC_2008/ or
Re: [ccp4bb] resuspending precipitated protein
6M Guanidiniumhydrochloride works pretty good, the question is only will you be able to refold it correctly ? I believe a better approach is to avoid precipitation in the first place and optimize your purification procedure in such a way that it works. When do you observe precipitate ? Dialysis, then us e a fast desalting column instead. Jürgen On 1 Oct 2008, at 06:12, ANDY DODDS wrote: Hello, following on from a previous topic about precipitating protein, but I believe a distinct caveat of this warranting a separate thread, I would like to know people's experiences in trying to get precipitated protein back into solution? Is there a way or are there many ways? Any experiences would be welcome, yours, Andy - Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
[ccp4bb] SOme wquestions about refining.......
Hello everyone, Please forgive my ignorance about the field of crystallography. I have recently started to process the diffraction data of a protein with a ligand attached to it. we have the structure of this protein already known and also known with different ligands. the way I did it is as flllows: I process the images in mosflm and then integrate them using scala inccp4i. After that I do molecular replacement by phaser. and finally refine it using refmac. Am I doing it correctly? Now since I am completely new to all this, I have not much clue about the outputs I get and if they are correct or not. What are the key things you should be looking at in the log files generated by phaser and refmac? Till now I get overall R factor of 29.32 and Rfree of 34.94. Is this fine or can I improve it more? If yes then what do I do to further improve the statistics? What should be the RmsBond and Rms Angle values? Since the structure is already solved a number of times with different ligands, what final values should i aim for to get a decent density maps in which I can fit my ligand structure. Sorry for being so naive. Any sort of information or papers or links would be very helpful. Thanks a lot. AA
Re: [ccp4bb] Protein-DNA complex prepartion for crystallization
Hi Raj, I would definitely 10 mM MgCl2. I would also increases NaCl conc. as 200-250 mM. Also you can try 5 mM DTT. Good Luck Dev Devendra B. Srivastava Postdoctoral Associate Laboratory of Molecular Biophysics, The Rockefeller University 1230 York Avenue New York, NY-10021, USA Phone: 212-327-7478 (Lab) 339-222-7670 (Mobile) On Wed, Oct 1, 2008 at 6:52 PM, E rajakumar [EMAIL PROTECTED] wrote: Dear All Sorry for non-crystallography query. I am working on DNA binding protein, while mixing DNA with protein for preparing Protein-DNA complex for crystallization, protein is precipitating. pI of the protein is 9.3 and in 15 mM HEPES 7.0, 150mM NaCl and 5% glycerol. Concentration of the protein used for mixing with DNA is 8 mg/mL. DNA to protein molar ratio is 1.2. Please advise me how to prevent precipitation. Is changing of buffer pH and adding divalent cation like MgCl2 can help in preventing precipitation? Thanking You Rajakumara E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) Get your preferred Email name! Now you can @ymail.com and @rocketmail.com. http://mail.promotions.yahoo.com/newdomains/aa/
Re: [ccp4bb] SOme wquestions about refining.......
Hi AA, On 1 Oct 2008, at 20:34, AA wrote: Hello everyone, Please forgive my ignorance about the field of crystallography. I have recently started to process the diffraction data of a protein with a ligand attached to it. we have the structure of this protein already known and also known with different ligands. the way I did it is as flllows: I process the images in mosflm and then integrate them using scala inccp4i. After that I do molecular replacement by phaser. and finally refine it using refmac. Am I doing it correctly? Yes that is correct but don't stop here. Now since I am completely new to all this, I have not much clue about the outputs I get and if they are correct or not. What are the key things you should be looking at in the log files generated by phaser and refmac? Till now I get overall R factor of 29.32 and Rfree of 34.94. this is a solid indication that the MR actually worked - as you would expect as you already know the structure. Is this fine or can I improve it more? You should next use the program Refmac and refine your molecular replacement solution to your highest resolution dataset you have. If yes then what do I do to further improve the statistics? As a rule of thumb multiply the highest resolution limit to which your data was processed/collected by the factor of ten this or better is what you are aiming for with the Rfactor, the gap between Rwork and Rfree should not exceed 5% e.g. Rfactor =25 and Rfree=30 What should be the RmsBond and Rms Angle values? Since the structure is already solved a number of times with different ligands, what final values should i aim for to get a decent density maps in which I can fit my ligand structure. Sorry for being so naive. Any sort of information or papers or links would be very helpful. Do you have someone at your university who you could bug directly ? It's much easier to learn from someone with experience than to read something. Good luck ! Jürgen Thanks a lot. AA - Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
