Re: [ccp4bb] Advice on Over-expressing and Purifying Metalloproteins
Nothing out of the ordinary there. You can try to induce overnight (for 20 hours or so) at 15C. And you can try to reduce the concentration of IPTG if excess protein is going to inclusion bodies anyways. Collect whatever protein is soluble and ignore your inclusion bodies. The His tags allow you to capture the protein easily from large batches of cell culture and cell lysate. As long as you get some soluble protein in good condition, you could just make more of it and not worry too much with optimizing the expression conditions. Jose. Buz Barstow b...@mac.com wrote: Dear all, I am trying to purify a metalloprotein (a hydrogenase) using affinity chromatography. I have produced two tagged versions of the enzyme: one with an N-terminal 6x histidine affinity tag, and the other with a C-terminal 6x his-tag. The tagged proteins are both tied to an IPTG-inducible promoter. When trying to express and purify the N-terminal tagged protein, I have found that almost all of the expressed protein goes into inclusion bodies when the culture is grown at 37 or at 30 degrees C. When the culture is grown at 20 degrees C, a small amount of protein can be found in the cell extract. Unfortunately, as the enzyme has several oxygen-sensitive metal clusters, we do not believe that the protein can be refolded from the inclusion bodies. Could you offer some advice on how to express and purify this protein and reduce the quantity of protein found in inclusion bodies? Thanks! and all the best, --Buz -- *** Jose Antonio Cuesta-Seijo Biophysical Chemistry Group Department of Chemistry University of Copenhagen Tlf: +45-35320261 Universitetsparken 5 DK-2100 Copenhagen, Denmark ***
Re: [ccp4bb] Fo-Fo Difference Map
Ian Tickle replied: (freezing soaking can easily induce 1% and sometimes 5% change), is surely the reason that Fo-Fo maps never caught on! I've been curious about this unit cell constant mis-matching for a while. If I understood well, Perutz tried to exploit the effect for phasing prior to heavy atom methods. As the unit cell changes, the diffraction peaks move, intensities change, and the fourier transform of the molecule is sampled in between the Bragg peaks. Has anyone tried to persue the idea since then? Cheers, Jon See, eg, Bragg Perutz, Proc. R. Soc. Lond. A 22 July 1952 vol. 213 no. 1115 425-435. doi: 10.1098/rspa.1952.0136
[ccp4bb] How to predict the secondary structure from a protein sequence..??
Dear all, i have protein sequence and how can i predict the secondary structure from the sequence..??? Thank u hsn.
Re: [ccp4bb] How to predict the secondary structure from a protein sequence..??
Hi Hussain, Psipred is a good a place as any to start: http://bioinf.cs.ucl.ac.uk/psipred/ Cheers, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 5 May 2010 11:50, Hussain Bhukyagps hsn...@yahoo.com wrote: Dear all, i have protein sequence and how can i predict the secondary structure from the sequence..??? Thank u hsn.
Re: [ccp4bb] How to predict the secondary structure from a protein sequence..??
Exapsy (http://www.expasy.ch/tools/#proteome) also has a whole section about secondary structure prediction. Tim On Wed, May 05, 2010 at 04:20:13PM +0530, Hussain Bhukyagps wrote: Dear all, i have protein sequence and how can i predict the secondary structure from the sequence..??? Thank u hsn. -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
[ccp4bb] low resolution secondary structural restraints
Hi - I am refining a low (3.7Å) structure with refmac(5.5.0091), and am having trouble maintaining some secondary structure elements. I would like to restrain the H-bonding in clear secondary structural elements, which should help prevent carbonyls in helices from flipping out, etc, but have not had success doing this so far. As I understand it, it is not correct to put a LINK restraint for hydrogen bonds in refmac, and instead the HYDBND keyword should be used. How do we specify these HYDBND restraints (putting them in the header of XYZIN doesn't seem to work), and is there a (simple) way to decrease the tolerance for breaking these bonds? Thanks, Greg -- Department of Biophysics Johns Hopkins University 302 Jenkins Hall 3400 N. Charles St. Baltimore, MD 21218 Phone: (410) 516-7850 (office) Phone: (410) 516-3476 (lab) Fax: (410) 516-4118 gdbow...@jhu.edu
Re: [ccp4bb] Advice on Over-expressing and Purifying Metalloproteins
Several suggestions: Try tagging with a solubility-enhancing tag like GST or NusA. If using a pET vector system, try "leaky" _expression_. The T7 promoter is not well-repressed (except in pLysS systems) and you can get low but very significant levels of _expression_ without induction. One protein system I have worked with expresses very well under these conditions, and the low rate of _expression_ discourages inclusion body formation. You can attempt to clone tandem, triple, or quadruple repeats of your gene behind a promoter. This works well for the _expression_ of the alpha subunit of Trp synthase. Supposedly, the gene repeats tie up transcription machinery and slow down the rate of mRNA and protein production. Whatever. It works. You have already tried lower temperature, and this often helps to slow down overexpression and increase the soluble fraction of protein. You could try going even lower in temperature and overexpressing longer. Good luck! Cheers. On 5/4/2010 5:11 PM, Buz Barstow wrote: Dear all, I am trying to purify a metalloprotein (a hydrogenase) using affinity chromatography. I have produced two tagged versions of the enzyme: one with an N-terminal 6x histidine affinity tag, and the other with a C-terminal 6x his-tag. The tagged proteins are both tied to an IPTG-inducible promoter. When trying to express and purify the N-terminal tagged protein, I have found that almost all of the expressed protein goes into inclusion bodies when the culture is grown at 37 or at 30 degrees C. When the culture is grown at 20 degrees C, a small amount of protein can be found in the cell extract. Unfortunately, as the enzyme has several oxygen-sensitive metal clusters, we do not believe that the protein can be refolded from the inclusion bodies. Could you offer some advice on how to express and purify this protein and reduce the quantity of protein found in inclusion bodies? Thanks! and all the best, --Buz -- Roger S. Rowlett 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
Re: [ccp4bb] Advice on Over-expressing and Purifying Metalloproteins
try JM109(DE3) instead of BL21(DE3), combined with low temperature induction - for several proteins this has yielded us soluble protein instead of inclusion bodies. I think this is because JM109(DE3) grows a bit slower. Mark J van Raaij mvr...@ibmb.csic.es http://webspersoais.usc.es/mark.vanraaij/ researcherID: B-3678-2009 On 5 May 2010, at 14:54, Roger Rowlett wrote: Several suggestions: • Try tagging with a solubility-enhancing tag like GST or NusA. • If using a pET vector system, try leaky expression. The T7 promoter is not well-repressed (except in pLysS systems) and you can get low but very significant levels of expression without induction. One protein system I have worked with expresses very well under these conditions, and the low rate of expression discourages inclusion body formation. • You can attempt to clone tandem, triple, or quadruple repeats of your gene behind a promoter. This works well for the expression of the alpha subunit of Trp synthase. Supposedly, the gene repeats tie up transcription machinery and slow down the rate of mRNA and protein production. Whatever. It works. • You have already tried lower temperature, and this often helps to slow down overexpression and increase the soluble fraction of protein. You could try going even lower in temperature and overexpressing longer. Good luck! Cheers. On 5/4/2010 5:11 PM, Buz Barstow wrote: Dear all, I am trying to purify a metalloprotein (a hydrogenase) using affinity chromatography. I have produced two tagged versions of the enzyme: one with an N-terminal 6x histidine affinity tag, and the other with a C-terminal 6x his-tag. The tagged proteins are both tied to an IPTG-inducible promoter. When trying to express and purify the N-terminal tagged protein, I have found that almost all of the expressed protein goes into inclusion bodies when the culture is grown at 37 or at 30 degrees C. When the culture is grown at 20 degrees C, a small amount of protein can be found in the cell extract. Unfortunately, as the enzyme has several oxygen-sensitive metal clusters, we do not believe that the protein can be refolded from the inclusion bodies. Could you offer some advice on how to express and purify this protein and reduce the quantity of protein found in inclusion bodies? Thanks! and all the best, --Buz -- Roger S. Rowlett 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
Re: [ccp4bb] low resolution secondary structural restraints
Hi Greg, you can generate secondary structure restraints definitions for Refmac using this command: phenix.secondary_structure_restraints model.pdb format=refmac Pavel. On 5/5/10 4:33 AM, Gregory Bowman wrote: Hi - I am refining a low (3.7Å) structure with refmac(5.5.0091), and am having trouble maintaining some secondary structure elements. I would like to restrain the H-bonding in clear secondary structural elements, which should help prevent carbonyls in helices from flipping out, etc, but have not had success doing this so far. As I understand it, it is not correct to put a LINK restraint for hydrogen bonds in refmac, and instead the HYDBND keyword should be used. How do we specify these HYDBND restraints (putting them in the header of XYZIN doesn't seem to work), and is there a (simple) way to decrease the tolerance for breaking these bonds? Thanks, Greg -- Department of Biophysics Johns Hopkins University 302 Jenkins Hall 3400 N. Charles St. Baltimore, MD 21218 Phone: (410) 516-7850 (office) Phone: (410) 516-3476 (lab) Fax: (410) 516-4118 gdbow...@jhu.edu mailto:gdbow...@jhu.edu
[ccp4bb] software to represent raw reflections in hkl zones
Hi, I have problems solving the structure of a protein crystal which seems to be disordered. In order to investigate the disorder it would be useful to have a precision photograph that shows reflections only in the [0kl] plane. Does anyone know software that can transform raw data to give intensity distribution in distinct zones of hkl? Many Thanks for your help, Tillmann
Re: [ccp4bb] software to represent raw reflections in hkl zones
hklview will generate pseudo precession images Sent from my iPhone On 05/05/2010, at 11:03 PM, Tillmann Heinisch tillmann.heini...@unibas.ch wrote: Hi, I have problems solving the structure of a protein crystal which seems to be disordered. In order to investigate the disorder it would be useful to have a precision photograph that shows reflections only in the [0kl] plane. Does anyone know software that can transform raw data to give intensity distribution in distinct zones of hkl? Many Thanks for your help, Tillmann
Re: [ccp4bb] software to represent raw reflections in hkl zones
to my knowledge hklview just works with integrated data whereas I need to plot raw intensities along h, k and l to investigate reflection streakings. I heard such software is routinely used in small molecule crystallography. Tillmann On May 5, 2010, at 3:18 PM, David Briggs wrote: Hi Tillmann Will the CCP4 program HKLview do what you want? http://www.ccp4.ac.uk/html/hklview.html Cheers, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 5 May 2010 14:03, Tillmann Heinisch tillmann.heini...@unibas.ch wrote: Hi, I have problems solving the structure of a protein crystal which seems to be disordered. In order to investigate the disorder it would be useful to have a precision photograph that shows reflections only in the [0kl] plane. Does anyone know software that can transform raw data to give intensity distribution in distinct zones of hkl? Many Thanks for your help, Tillmann
Re: [ccp4bb] Advice on Over-expressing and Purifying Metalloproteins
I have found that if you give a cold shock ( 4 degrees for 30 mins-1 hr) before low temperature induction it helps to keep proteins soluble. Ivan
Re: [ccp4bb] software to represent raw reflections in hkl zones
Hi Tillmann, what do you mean by 'raw intensities' as opposed to integrated data? Would xprep be an option for you? It reads XDS_ASCII.HKL, but that's of course after integration. But it should be easy to convert any (non-binary) file containing raw intensities into an hkl-file that you can read with xprep!? rlatt might be another program you are looking for. Tim On Wed, May 05, 2010 at 03:33:40PM +0200, Tillmann Heinisch wrote: to my knowledge hklview just works with integrated data whereas I need to plot raw intensities along h, k and l to investigate reflection streakings. I heard such software is routinely used in small molecule crystallography. Tillmann On May 5, 2010, at 3:18 PM, David Briggs wrote: Hi Tillmann Will the CCP4 program HKLview do what you want? http://www.ccp4.ac.uk/html/hklview.html Cheers, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 5 May 2010 14:03, Tillmann Heinisch tillmann.heini...@unibas.ch wrote: Hi, I have problems solving the structure of a protein crystal which seems to be disordered. In order to investigate the disorder it would be useful to have a precision photograph that shows reflections only in the [0kl] plane. Does anyone know software that can transform raw data to give intensity distribution in distinct zones of hkl? Many Thanks for your help, Tillmann -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] software to represent raw reflections in hkl zones
I think he's looking for a program which will extract a plane from the raw 3D reciprocal space, as sampled by the raw images (ie before integration, but with the plane defined by the indexed lattice). That's a much harder job Phil On 5 May 2010, at 16:50, Tim Gruene wrote: Hi Tillmann, what do you mean by 'raw intensities' as opposed to integrated data? Would xprep be an option for you? It reads XDS_ASCII.HKL, but that's of course after integration. But it should be easy to convert any (non-binary) file containing raw intensities into an hkl-file that you can read with xprep!? rlatt might be another program you are looking for. Tim On Wed, May 05, 2010 at 03:33:40PM +0200, Tillmann Heinisch wrote: to my knowledge hklview just works with integrated data whereas I need to plot raw intensities along h, k and l to investigate reflection streakings. I heard such software is routinely used in small molecule crystallography. Tillmann On May 5, 2010, at 3:18 PM, David Briggs wrote: Hi Tillmann Will the CCP4 program HKLview do what you want? http://www.ccp4.ac.uk/html/hklview.html Cheers, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 5 May 2010 14:03, Tillmann Heinisch tillmann.heini...@unibas.ch wrote: Hi, I have problems solving the structure of a protein crystal which seems to be disordered. In order to investigate the disorder it would be useful to have a precision photograph that shows reflections only in the [0kl] plane. Does anyone know software that can transform raw data to give intensity distribution in distinct zones of hkl? Many Thanks for your help, Tillmann -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] software to represent raw reflections in hkl zones
As Phil says, constructing an undistorted slice through reciprocal space is much harder than displaying integrated intensities. The Bruker APEX2 software does this nicely and I understand that they can also convert MAR CCD and possibly some other frame formats to Bruker format, which presumably would be necessary to apply it to your data. Although intended for - and widely used by - small molecule crystallographers this should work equally well for macromolecules. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Wed, 5 May 2010, Phil Evans wrote: I think he's looking for a program which will extract a plane from the raw 3D reciprocal space, as sampled by the raw images (ie before integration, but with the plane defined by the indexed lattice). That's a much harder job Phil On 5 May 2010, at 16:50, Tim Gruene wrote: Hi Tillmann, what do you mean by 'raw intensities' as opposed to integrated data? Would xprep be an option for you? It reads XDS_ASCII.HKL, but that's of course after integration. But it should be easy to convert any (non-binary) file containing raw intensities into an hkl-file that you can read with xprep!? rlatt might be another program you are looking for. Tim On Wed, May 05, 2010 at 03:33:40PM +0200, Tillmann Heinisch wrote: to my knowledge hklview just works with integrated data whereas I need to plot raw intensities along h, k and l to investigate reflection streakings. I heard such software is routinely used in small molecule crystallography. Tillmann On May 5, 2010, at 3:18 PM, David Briggs wrote: Hi Tillmann Will the CCP4 program HKLview do what you want? http://www.ccp4.ac.uk/html/hklview.html Cheers, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 5 May 2010 14:03, Tillmann Heinisch tillmann.heini...@unibas.ch wrote: Hi, I have problems solving the structure of a protein crystal which seems to be disordered. In order to investigate the disorder it would be useful to have a precision photograph that shows reflections only in the [0kl] plane. Does anyone know software that can transform raw data to give intensity distribution in distinct zones of hkl? Many Thanks for your help, Tillmann -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] software to represent raw reflections in hkl zones
Hi this is what I thought. I would imagine that you'd want to combine the 2D information in the X-ray images in a dataset into a 3D solid figure, then look at slices through this for the RL projection you were interested in. If you indexed the dataset first you should be able to get the right orientations... I don't know anything that does this for single crystal X-ray work, but maybe something from cryo EM single particle reconstruction could be adapted? On 5 May 2010, at 17:10, Phil Evans wrote: I think he's looking for a program which will extract a plane from the raw 3D reciprocal space, as sampled by the raw images (ie before integration, but with the plane defined by the indexed lattice). That's a much harder job Phil On 5 May 2010, at 16:50, Tim Gruene wrote: Hi Tillmann, what do you mean by 'raw intensities' as opposed to integrated data? Would xprep be an option for you? It reads XDS_ASCII.HKL, but that's of course after integration. But it should be easy to convert any (non-binary) file containing raw intensities into an hkl-file that you can read with xprep!? rlatt might be another program you are looking for. Tim On Wed, May 05, 2010 at 03:33:40PM +0200, Tillmann Heinisch wrote: to my knowledge hklview just works with integrated data whereas I need to plot raw intensities along h, k and l to investigate reflection streakings. I heard such software is routinely used in small molecule crystallography. Tillmann On May 5, 2010, at 3:18 PM, David Briggs wrote: Hi Tillmann Will the CCP4 program HKLview do what you want? http://www.ccp4.ac.uk/html/hklview.html Cheers, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 5 May 2010 14:03, Tillmann Heinisch tillmann.heini...@unibas.ch wrote: Hi, I have problems solving the structure of a protein crystal which seems to be disordered. In order to investigate the disorder it would be useful to have a precision photograph that shows reflections only in the [0kl] plane. Does anyone know software that can transform raw data to give intensity distribution in distinct zones of hkl? Many Thanks for your help, Tillmann -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
[ccp4bb] software to represent raw reflections in hkl zones
This 'much harder job' is accomplished, too, in the comprehensive CrysAlisPro data-analysis suite from Oxford Diffraction. Again: not just from images in the OD format, but from a wide variety of others, also. So, if we could assist in this case, Tillmann, then we would be pleased to... Marcus Winter. (Oxford Diffraction Ltd.) -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of George M. Sheldrick Sent: 05 May 2010 17:20 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] software to represent raw reflections in hkl zones As Phil says, constructing an undistorted slice through reciprocal space is much harder than displaying integrated intensities. The Bruker APEX2 software does this nicely and I understand that they can also convert MAR CCD and possibly some other frame formats to Bruker format, which presumably would be necessary to apply it to your data. Although intended for - and widely used by - small molecule crystallographers this should work equally well for macromolecules. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Wed, 5 May 2010, Phil Evans wrote: I think he's looking for a program which will extract a plane from the raw 3D reciprocal space, as sampled by the raw images (ie before integration, but with the plane defined by the indexed lattice). That's a much harder job Phil On 5 May 2010, at 16:50, Tim Gruene wrote: Hi Tillmann, what do you mean by 'raw intensities' as opposed to integrated data? Would xprep be an option for you? It reads XDS_ASCII.HKL, but that's of course after integration. But it should be easy to convert any (non-binary) file containing raw intensities into an hkl-file that you can read with xprep!? rlatt might be another program you are looking for. Tim On Wed, May 05, 2010 at 03:33:40PM +0200, Tillmann Heinisch wrote: to my knowledge hklview just works with integrated data whereas I need to plot raw intensities along h, k and l to investigate reflection streakings. I heard such software is routinely used in small molecule crystallography. Tillmann On May 5, 2010, at 3:18 PM, David Briggs wrote: Hi Tillmann Will the CCP4 program HKLview do what you want? http://www.ccp4.ac.uk/html/hklview.html Cheers, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 5 May 2010 14:03, Tillmann Heinisch tillmann.heini...@unibas.ch wrote: Hi, I have problems solving the structure of a protein crystal which seems to be disordered. In order to investigate the disorder it would be useful to have a precision photograph that shows reflections only in the [0kl] plane. Does anyone know software that can transform raw data to give intensity distribution in distinct zones of hkl? Many Thanks for your help, Tillmann -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] software to represent raw reflections in hkl zones
Storing a complete 3D image set in memory is moderately challenging even for today's computers (probably several to several 10s of gigabytes, though you could perhaps cheat a bit), so programs would probably have to use old-fashioned double sort techniques to extract a zone. I wonder how the Bruker program does it Phil On 5 May 2010, at 17:20, George M. Sheldrick wrote: As Phil says, constructing an undistorted slice through reciprocal space is much harder than displaying integrated intensities. The Bruker APEX2 software does this nicely and I understand that they can also convert MAR CCD and possibly some other frame formats to Bruker format, which presumably would be necessary to apply it to your data. Although intended for - and widely used by - small molecule crystallographers this should work equally well for macromolecules. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Wed, 5 May 2010, Phil Evans wrote: I think he's looking for a program which will extract a plane from the raw 3D reciprocal space, as sampled by the raw images (ie before integration, but with the plane defined by the indexed lattice). That's a much harder job Phil On 5 May 2010, at 16:50, Tim Gruene wrote: Hi Tillmann, what do you mean by 'raw intensities' as opposed to integrated data? Would xprep be an option for you? It reads XDS_ASCII.HKL, but that's of course after integration. But it should be easy to convert any (non-binary) file containing raw intensities into an hkl-file that you can read with xprep!? rlatt might be another program you are looking for. Tim On Wed, May 05, 2010 at 03:33:40PM +0200, Tillmann Heinisch wrote: to my knowledge hklview just works with integrated data whereas I need to plot raw intensities along h, k and l to investigate reflection streakings. I heard such software is routinely used in small molecule crystallography. Tillmann On May 5, 2010, at 3:18 PM, David Briggs wrote: Hi Tillmann Will the CCP4 program HKLview do what you want? http://www.ccp4.ac.uk/html/hklview.html Cheers, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 5 May 2010 14:03, Tillmann Heinisch tillmann.heini...@unibas.ch wrote: Hi, I have problems solving the structure of a protein crystal which seems to be disordered. In order to investigate the disorder it would be useful to have a precision photograph that shows reflections only in the [0kl] plane. Does anyone know software that can transform raw data to give intensity distribution in distinct zones of hkl? Many Thanks for your help, Tillmann -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] software to represent raw reflections in hkl zones
I wrote a little jiffy for doing this some years ago: http://bl831.als.lbl.gov/~jamesh/pickup/adsc2pdb.com However, I should note that this program relies on the DPS program: dps_peaksearch to pick spots on each image in your data set. These spots are then transformed into reciprocal space coordinates in the form of a PDB file, which you can view and rotate around with your favorite graphics program. At the time I wrote it, the most popular program was O, and so the jiffy writes input files for that program. If you don't have DPS, then a similar jiffy for converting the *.spt files that MOSFLM generates during the autoindexing process is here: http://bl831.als.lbl.gov/~jamesh/pickup/spt2xyz.com This is not exactly the 2D-3D-2D re-binning program that is now being discussed as the much harder job, but I have found that transforming peaks will do in most cases. After all, you are generally looking for how the spots transform anyway. Even this will make a very large PDB file in typical cases. The main caveat here is that my jiffy is only set up by default to work with a particular image type (ADSC) from a detector and spindle set up in the usual way (the way it is at my beamline). This is because I very rarely have access to other kinds of images. I think this underlies the reason why a general program for converting any diffraction image into reciprocal space does not exist: you have to know the camera geometry, and camera geometries are poorly documented. Specifically, given the fast and slow dimensions of the image file, you need to know not just the pixel size, but the beam center. The latter has at least 32 different conventions to represent it. Nearly all are used by one facility or another, but no image file format tells you which one it is using. For example, the beam center is usually given as two numbers, but 0 0 can be any of the four image corners (depending on the beamline), and X and Y could be fast or slow. You also need to know if you are in cameraman view or sample view. Both are popular, but invert the hand of the world if you get it wrong. Oh yes! and you also need to know the orientation and rotation direction of the spindle. This is often given as vertical vs horizontal and clockwise vs anticlockwise (which is not enough information), but most spindles are not precisely 90 degrees to the x-ray beam, and yes that does impact precisely where the spots fall. The detector is also not usually perfectly square with the beam either, and is even sometimes purposefully offset at a 2theta angle. Add to all this the heated debates over which direction is X, Y or Z (some think that X is the spindle axis, others (like me) think that X is the X-ray beam), and I think you can see the scope of this problem. Phil makes a good point about memory management problems, but I think that pales in comparison to the headaches of getting a poor, defenseless biologist with a stack of images to come up with the 14 numbers needed to uniquely describe the camera geometry. This is probably why most people just write a program for their favorite image format (the one down the hall). Part of the problem, I think, is that there are more combinations of conventions than there are degrees of freedom in the camera. You really only need 14 numbers (4 vectors and two scales) to completely describe an image file from a flat 2D detector and spindle (independent of XYZ conventions). But that is a topic for another time -James Holton MAD Scientist George M. Sheldrick wrote: As Phil says, constructing an undistorted slice through reciprocal space is much harder than displaying integrated intensities. The Bruker APEX2 software does this nicely and I understand that they can also convert MAR CCD and possibly some other frame formats to Bruker format, which presumably would be necessary to apply it to your data. Although intended for - and widely used by - small molecule crystallographers this should work equally well for macromolecules. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Wed, 5 May 2010, Phil Evans wrote: I think he's looking for a program which will extract a plane from the raw 3D reciprocal space, as sampled by the raw images (ie before integration, but with the plane defined by the indexed lattice). That's a much harder job Phil On 5 May 2010, at 16:50, Tim Gruene wrote: Hi Tillmann, what do you mean by 'raw intensities' as opposed to integrated data? Would xprep be an option for you? It reads XDS_ASCII.HKL, but that's of course after integration. But it should be easy to convert any (non-binary) file containing raw intensities into an hkl-file that you can read with xprep!? rlatt might be another program you are looking for. Tim On Wed, May 05, 2010 at 03:33:40PM
Re: [ccp4bb] Fo-Fo Difference Map
On Wednesday 05 May 2010 12:49:34 am Jon Wright wrote: I've been curious about this unit cell constant mis-matching for a while. If I understood well, Perutz tried to exploit the effect for phasing prior to heavy atom methods. As the unit cell changes, the diffraction peaks move, intensities change, and the fourier transform of the molecule is sampled in between the Bragg peaks. Has anyone tried to persue the idea since then? Acta Cryst. (2000). A56, 596-605[ doi:10.1107/S010876730001031X ] On possible extensions of X-ray crystallography through diffraction-pattern oversampling J. Miao and D. Sayre Acta Cryst. (2000). D56, 1312-1315[ doi:10.1107/S090744498970 ] The oversampling phasing method J. Miao, J. Kirz and D. Sayre Acta Cryst. (2003). A59, 577-583[ doi:10.1107/S0108767303021123 ] Phase extension in crystallography using the iterative Fienup-Gerchberg-Saxton algorithm and Hilbert transforms J. S. Wu and J. C. H. Spence Cheers, Jon See, eg, Bragg Perutz, Proc. R. Soc. Lond. A 22 July 1952 vol. 213 no. 1115 425-435. doi: 10.1098/rspa.1952.0136 -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] Advice on Over-expressing and Purifying Metalloproteins
English et al. Recombinant and in vitro expression systems for hydrogenases: new frontiers in basic and applied studies for biological and synthetic H2 production. Dalton Trans. (2009) (45) pp. 9970-8 I've never tried to express a hydrogenase, but I've read that it's very hard. Do you have all necessary assembly proteins and/or are you expressing in an organism that has a closely related hydrogenase? -Joel On Wed, May 5, 2010 at 8:14 AM, xaravich ivan xaravich.i...@gmail.comwrote: I have found that if you give a cold shock ( 4 degrees for 30 mins-1 hr) before low temperature induction it helps to keep proteins soluble. Ivan
Re: [ccp4bb] software to represent raw reflections in hkl zones
Tillmann, I wrote a little jiffy some months ago, to go from raw images to a pseudo-precession photo, in the context of labelit.index. I'll dig it out and post a link to the program... Nick Tillmann Heinisch wrote: Hi, I have problems solving the structure of a protein crystal which seems to be disordered. In order to investigate the disorder it would be useful to have a precision photograph that shows reflections only in the [0kl] plane. Does anyone know software that can transform raw data to give intensity distribution in distinct zones of hkl? Many Thanks for your help, Tillmann
