[ccp4bb] suitable buffer for CD studies
Sorry for a simple and non-CCP4 question. I have determined the structures of three different mutants of a thermostable protein by X-ray crystallography method. I feel that Mg2+ has a role in protein stability. So I want to perform a thermal denaturation study by CD spectroscopy both in presence and absence of Mg2+ ion. In this regards, what should be the suitable buffer for CD studies? May I use PBS buffer ? Since phosphate sequester divalent cations like Mg2+. Is it advisable to use PBS buffer. If so, what is maximum concentration of Mg2+ ion that can be used say e.g. 5 mM? My protein was in Tris buffer and lyophilized and have theoretical pI =4.56 and maximum activity at pH 8.4. Thanking you in advances, harsh
Re: [ccp4bb] Resolution limit of index in XDS
Hello, The way I do it is by manually editing the SPOT.XDS file (generated by the COLSPOT step). Spots are arranged by order of decreasing intensity in that file. So if you do down the file, select an appropriate intensity cutoff and then remove all spots below that value, it will have the effect of selecting a resolution cutoff (think of the plot of I vs. resolution) but you won't know what cutoff this corresponds to unless you do a careful analysis of the resulting SPOT.XDS file. HTH, Fred. On 19/03/13 20:53, Niu Tou wrote: Dear All, Is there any command can set the resolution limit for index step in XDS? I only found a keyword INCLUDE_RESOLUTION_RANGE, but it looks to be a definition of resolution range after index step as it says: INCLUDE_RESOLUTION_RANGE=20.0 0.0 !Angstroem; used by DEFPIX,INTEGRATE,CORRECT Thanks! Niu -- Fred. Vellieux (B.Sc., Ph.D., hdr) ouvrier de la recherche IBS / ELMA 41 rue Jules Horowitz F-38027 Grenoble Cedex 01 Tel: +33 438789605 Fax: +33 438785494
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Dear Edward, I think you gave a very good summary of what might have happened in the crystals. If the two domains diffract like a single crystal and interfere, F's are added, if they diffract like two different crystals, I's are added. In fact, one should model the special protein molecule in two alternative orientations, just like one does with individual amino acids that have multiple conformations. Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Edward A. Berry Sent: Tuesday, March 19, 2013 7:19 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree Maybe this thread still needs some more pedagogy/explanation for those newbies and for biologist/ wanna-be crystallographers like me. My original reaction was- if the true space group is P21 you wouldn't want to expand from data reduced in higher symmetry, because you would be enforcing that higher symmetry. But if it were simply a case of P21 symmetry, with three molecules in the AU, that happened to have a beta angle of 90, merging statistics would have prevented reducing the data in p222 in the first place. Does order/disorder mean that the third molecule is actually present in two different orientations with equal occupancy, so that on the average it does obey the higher symmetry? Like our heme on a special position in the bacterioferritin paper? And structure factors add because the two orientations are present in the same domain, whereas with twinning the two orientations are present in different domains that diffract like separate crystals, and the resulting intensities add on the film? herman.schreu...@sanofi.com wrote: If it is crystal packing disorder (F's added instead of I's), the switches between the alternative conformations need to be very frequent, to be within coherent range, so I would asume that the alternative conformations will be present in equal proportions. Still the alternatives need to be modeled somehow and if this can be conveniently done in a lower symmetry spacegroup this would not artificially lower the free R-factors. As Phoebe mentioned, ignoring the higher symmetry relations and repicking the free Rflags at lower symmetry would lead free reflections to be linked to the working set, leading to too low Rfree values. However, with perfect packing disorder, no extra information would be gained by reprocessing in lower symmetry (in contrast to cases with pseudo symmetry). My 2 cents, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Phoebe A. Rice Sent: Tuesday, March 19, 2013 4:49 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree oops, I should have expanded my comments to include the sort of funky lattice order-disorder Zbyszek so cleverly diagnosed. Scratch that perfect twinning comment in my last message. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phoebe A. Rice [pr...@uchicago.edu] Sent: Tuesday, March 19, 2013 10:34 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree Hi Zbyszek, If the issue is perfect twinning, I agree - good point! But you don't want to confuse people who simply have nearly-but-not-quite crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of newbies read the BB). We had a case of P31 that was so close to P61 we actually solved the molecular replacement problem in P61, then expanded it back and re-rigid-bodied it. We've played similar games with translational pseudo-symmetry (ignoring the weak spots at first). In cases like that it is important to properly reprocess the data in the lower symmetry space group (or smaller unit cell) because there is real information in those small differences. However, the point about Rfree holds for twinning or rotational pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry operators, not re-picked in the lower symmetry space group. Phoebe ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek Otwinowski [zbys...@work.swmed.edu] Sent: Tuesday, March 19, 2013 9:37 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree It is a clear-cut case of crystal packing disorder. The tell-tale sign is that data can be merged in the higher-symmetry lattice, while the number of molecules in the asymmetric unit (3 in P21) is not
Re: [ccp4bb] How to convert file format from CNS to CCP4
Dear steffi, Can you give me a link? I did not find it in google. Thank you! Wei 在 2013-03-19 20:55:42，Stefanie Becker stefanie.bec...@uni-konstanz.de 写道： Am Dienstag, 19. März 2013 04:37 CET, Wei Feng ccp4...@hotmail.com schrieb: Hi! don't know if you already got the answer by now, but there is a small program called map2mtz that should do it (i think you can just google it) good luck! steffi Dear all, I have used CNS to calculate the experimental phase of my structure. After Heavy-atom search, Heavy-atom refinement/SAD phasing and SAD Phasing - Density Modification - Selection of Map. Some files outputted: sad_phase2.hkl sad_phase2.sdb density_modify.hkl density_modify.map density_modify.mask ... I want to use these files to do the model building, but I do not know how to do it in CNS. So I want to convert these files to CCP4 format and do the model building by ARP/Warp, but I do not know which files should be converted and which software can be used to convert the file format from CNS to CCP4. Thank you for your time! Wei
Re: [ccp4bb] Resolution limit of index in XDS
Dear Tim, but probably I should adres this to Kai Diederichs, not including the resolution cutoff in COLSPOT and IDXREF is a feature of XDS I do not understand at all. For most cases, it may not matter since only the strong spots are used, but what are the advantages? In fact there are disadvantages, especially when dealing with poorly diffracting difficult data sets: -when a crystallographer imposes a resolution limit, there are usually good reasons for it. -outside the resolution limit, there may be ice rings or contaminating salt spots, which make the autoindexing fail. -when processing 900 frame Pilatus data sets, running COLSPOT on the complete detector surface takes significantly longer then running it only on the center region. Of course, one could fudge a resolution cutoff by translating resolution into pixels and then calculating a TRUSTED_REGION, or manually editing the SPOT.XDS file, but this is a lot of extra and in my view unneccessary work. I would really consider using the resolution cutoff for COLSPOT as well. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Gruene Sent: Tuesday, March 19, 2013 11:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Resolution limit of index in XDS -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Niu, indexing relies on strong reflections only, that is (in very brieft) why INCLUDE_RESOLUTION_RANGE indeed does not affect the relections collected in COLSPOT which in turn are used by IDXREF. You can work around this, however, by making use of TRUSTED_REGION and set it to e.g. 0.7 or 0.6 (you can use adxv to translate resolution into pixel and then calculate the fraction you need to set the second number in TRUSTED_REGION to (or the first if you want to exclude the inner resolution reflections - I remember one data set where this was essential for indexing - DNA was involved there) Best, Tim On 03/19/2013 08:53 PM, Niu Tou wrote: Dear All, Is there any command can set the resolution limit for index step in XDS? I only found a keyword INCLUDE_RESOLUTION_RANGE, but it looks to be a definition of resolution range after index step as it says: INCLUDE_RESOLUTION_RANGE=20.0 0.0 !Angstroem; used by DEFPIX,INTEGRATE,CORRECT Thanks! Niu - -- 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/ iD8DBQFRSOFJUxlJ7aRr7hoRAo6TAKC+BePgeODbDyngO7N8vCE4CnjxmQCfS5cP srShHNz1sDK0EMHSbE3fDwA= =kAwf -END PGP SIGNATURE-
Re: [ccp4bb] How to convert file format from CNS to CCP4
Dear Wei, There is a CCP4 program called Convert to/modify/extend MTZ which you can use to convert various file types (notably SHELX, CNS/XPLOR, mmCIF, MULTAN and TNT) to the MTZ format. Look for it in the Reflection Data Utilities module. http://www.ccp4.ac.uk/dist/share/ccp4i/help/modules/mtz_utils.html cheers Ganesh Le 20/03/13 10:15, Wei Feng a écrit : Dear steffi, Can you give me a link? I did not find it in google. Thank you! Wei 在 2013-03-19 20:55:42，Stefanie Becker stefanie.bec...@uni-konstanz.de mailto:stefanie.bec...@uni-konstanz.de 写道： Am Dienstag, 19. März 2013 04:37 CET, Wei Feng ccp4...@hotmail.com mailto:ccp4...@hotmail.com schrieb: Hi! don't know if you already got the answer by now, but there is a small program called map2mtz that should do it (i think you can just google it) good luck! steffi Dear all, I have used CNS to calculate the experimental phase of my structure. After Heavy-atom search, Heavy-atom refinement/SAD phasing and SAD Phasing - Density Modification - Selection of Map. Some files outputted: sad_phase2.hkl sad_phase2.sdb density_modify.hkl density_modify.map density_modify.mask ... I want to use these files to do the model building, but I do not know how to do it in CNS. So I want to convert these files to CCP4 format and do the model building by ARP/Warp, but I do not know which files should be converted and which software can be used to convert the file format from CNS to CCP4. Thank you for your time! Wei
Re: [ccp4bb] suitable buffer for CD studies
I would use a low affinity metal ion binding buffer like MOPS, HEPES, or MES. The Good buffers all have fairly low metal-ion affinity. Phosphates will be problematic because of magnesium phosphate formation. 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 3/20/2013 2:59 AM, Harsh Bansia wrote: Sorry for a simple and non-CCP4 question. I have determined the structures of three different mutants of a thermostable protein by X-ray crystallography method. I feel that Mg2+ has a role in protein stability. So I want to perform a thermal denaturation study by CD spectroscopy both in presence and absence of Mg2+ ion. In this regards, what should be the suitable buffer for CD studies? May I use PBS buffer ? Since phosphate sequester divalent cations like Mg2+. Is it advisable to use PBS buffer. If so, what is maximum concentration of Mg2+ ion that can be used say e.g. 5 mM? My protein was in Tris buffer and lyophilized and have theoretical pI =4.56 and maximum activity at pH 8.4. Thanking you in advances, harsh
Re: [ccp4bb] suitable buffer for CD studies
However, these buffers generally absorb in the far UV (180-200nm) which is the most interesting part for discriminating the secondary structure distribution. Even if this is not the primary interest, the 220nm points one might want to use for a denaturation study can have quite some background contributions from the organic buffers. Some alternative such as a thermofluor stability assay might not have the problems with organic buffers nor with the presence/absence of Mg++? BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger Rowlett Sent: Wednesday, March 20, 2013 1:07 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] suitable buffer for CD studies I would use a low affinity metal ion binding buffer like MOPS, HEPES, or MES. The Good buffers all have fairly low metal-ion affinity. Phosphates will be problematic because of magnesium phosphate formation. 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 3/20/2013 2:59 AM, Harsh Bansia wrote: Sorry for a simple and non-CCP4 question. I have determined the structures of three different mutants of a thermostable protein by X-ray crystallography method. I feel that Mg2+ has a role in protein stability. So I want to perform a thermal denaturation study by CD spectroscopy both in presence and absence of Mg2+ ion. In this regards, what should be the suitable buffer for CD studies? May I use PBS buffer ? Since phosphate sequester divalent cations like Mg2+. Is it advisable to use PBS buffer. If so, what is maximum concentration of Mg2+ ion that can be used say e.g. 5 mM? My protein was in Tris buffer and lyophilized and have theoretical pI =4.56 and maximum activity at pH 8.4. Thanking you in advances, harsh
[ccp4bb] PDB crawler
Hi all, just wondering if anybody is aware of a service similar to PubCrawler (http://pubcrawler.gen.tcd.ie/), but for PDB structures, released and unreleased. Basically a weekly email that would alert you of freshly deposited structures that match some keywords. Thanks in advance, ciao Sebastiano -- 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
Re: [ccp4bb] PDB crawler
Hi Sebastiano, Phyre Alarm would do something similar to what you suggest. http://www.sbg.bio.ic.ac.uk/phyre2/html/help.cgi?id=help/phyrealarm HTH, Dave David C. Briggs PhD http://about.me/david_briggs On 20 March 2013 12:40, Sebastiano Pasqualato sebastiano.pasqual...@gmail.com wrote: Hi all, just wondering if anybody is aware of a service similar to PubCrawler (http://pubcrawler.gen.tcd.ie/), but for PDB structures, released and unreleased. Basically a weekly email that would alert you of freshly deposited structures that match some keywords. Thanks in advance, ciao Sebastiano -- 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
Re: [ccp4bb] suitable buffer for CD studies
Harsh, This article describes common buffers for CD on page 2: http://www.ncbi.nlm.nih.gov/pubmed/17406547 Or this article on page 8: http://www.ncbi.nlm.nih.gov/pubmed/16027053 It seems like phosphate is the best, because it has low absorption at 180-200nm region. From organic buffers Tris/H2SO4 seems to the the best. As I remember, Cl absorbs a lot in that UV region. Vitali On Wed, Mar 20, 2013 at 1:59 AM, Harsh Bansia spideysp...@gmail.com wrote: Sorry for a simple and non-CCP4 question. I have determined the structures of three different mutants of a thermostable protein by X-ray crystallography method. I feel that Mg2+ has a role in protein stability. So I want to perform a thermal denaturation study by CD spectroscopy both in presence and absence of Mg2+ ion. In this regards, what should be the suitable buffer for CD studies? May I use PBS buffer ? Since phosphate sequester divalent cations like Mg2+. Is it advisable to use PBS buffer. If so, what is maximum concentration of Mg2+ ion that can be used say e.g. 5 mM? My protein was in Tris buffer and lyophilized and have theoretical pI =4.56 and maximum activity at pH 8.4. Thanking you in advances, harsh
Re: [ccp4bb] suitable buffer for CD studies
Tris-sulfate might be acceptable for studies in the presence of magnesium ion. Na-MES is tolerable at low concentrations and shorter path lengths if a cutoff of 190-200 nm or so is acceptable. Chloride is indeed problematic in the far UV. ___ 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 3/20/2013 9:32 AM, Vitali Stanevich wrote: Harsh, This article describes common buffers for CD on page 2: http://www.ncbi.nlm.nih.gov/pubmed/17406547 Or this article on page 8: http://www.ncbi.nlm.nih.gov/pubmed/16027053 It seems like phosphate is the best, because it has low absorption at 180-200nm region. From organic buffers Tris/H2SO4 seems to the the best. As I remember, Cl absorbs a lot in that UV region. Vitali On Wed, Mar 20, 2013 at 1:59 AM, Harsh Bansia spideysp...@gmail.com mailto:spideysp...@gmail.com wrote: Sorry for a simple and non-CCP4 question. I have determined the structures of three different mutants of a thermostable protein by X-ray crystallography method. I feel that Mg2+ has a role in protein stability. So I want to perform a thermal denaturation study by CD spectroscopy both in presence and absence of Mg2+ ion. In this regards, what should be the suitable buffer for CD studies? May I use PBS buffer ? Since phosphate sequester divalent cations like Mg2+. Is it advisable to use PBS buffer. If so, what is maximum concentration of Mg2+ ion that can be used say e.g. 5 mM? My protein was in Tris buffer and lyophilized and have theoretical pI =4.56 and maximum activity at pH 8.4. Thanking you in advances, harsh
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Re: [ccp4bb] PDB crawler
Hi, I think that what you're looking for (or close to it) is available on the pdb site. Through your PDB login you can do a search according to keyword(s) and ask to repeat the search periodically. You'll get an alert in the e-mail. I actually use it for some time. Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sebastiano Pasqualato [sebastiano.pasqual...@gmail.com] Sent: Wednesday, March 20, 2013 2:40 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] PDB crawler Hi all, just wondering if anybody is aware of a service similar to PubCrawler (http://pubcrawler.gen.tcd.ie/), but for PDB structures, released and unreleased. Basically a weekly email that would alert you of freshly deposited structures that match some keywords. Thanks in advance, ciao Sebastiano -- 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
[ccp4bb] COOT running slow after upgrading to CENTOS 6.4 from 6.3
Hello, We have a curious situation here: after upgrading CentOS 6.3 to 6.4, COOT runs slowly every time after the go to atom command is executed - every rotation of the molecules takes nearly 1 second to finish. But if the go to atom command is never sent then the speed is normal. A COOT running in a virtual machine windows XP on the same computer behaves OK though. It doesn't seem to be a conflict with the CentOS 6.4 either, because COOT on another computer with newly installed CentOS 6.4 runs perfectly fine. So I guess it might has to do with some libraries that COOT might use. The computer with trouble have python upgraded to 2.7 from the python source code when it was running CentOS 6.3, could this be a problem? Zhijie
Re: [ccp4bb] COOT running slow after upgrading to CENTOS 6.4 from 6.3
Hi Zhijie, our system administrator has warned me sternly against touching RHEL 6.4. The same might hold true for CentOS. I would be interested to hear from anyone running 6.4 successfully. Andreas On 20/03/2013 2:42, Zhijie Li wrote: Hello, We have a curious situation here: after upgrading CentOS 6.3 to 6.4, COOT runs slowly every time after the go to atom command is executed - every rotation of the molecules takes nearly 1 second to finish. But if the go to atom command is never sent then the speed is normal. A COOT running in a virtual machine windows XP on the same computer behaves OK though. It doesn't seem to be a conflict with the CentOS 6.4 either, because COOT on another computer with newly installed CentOS 6.4 runs perfectly fine. So I guess it might has to do with some libraries that COOT might use. The computer with trouble have python upgraded to 2.7 from the python source code when it was running CentOS 6.3, could this be a problem? Zhijie -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
[ccp4bb] SUMMARY: PDB crawler
Hi Boaz, that is definitely what I was looking for. Thank you very much. Other suggestions, on weekly queries based on your favourite protein sequence were given by Gerard Kleyvegt (http://bip.weizmann.ac.il/noop/NOOP_seqalert.html) and David Briggs (http://www.sbg.bio.ic.ac.uk/phyre2/html/help.cgi?id=help/phyrealarm) Ciao, Sebastiano On Mar 20, 2013, at 3:41 PM, Boaz Shaanan bshaa...@exchange.bgu.ac.il wrote: Hi, I think that what you're looking for (or close to it) is available on the pdb site. Through your PDB login you can do a search according to keyword(s) and ask to repeat the search periodically. You'll get an alert in the e-mail. I actually use it for some time. Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sebastiano Pasqualato [sebastiano.pasqual...@gmail.com] Sent: Wednesday, March 20, 2013 2:40 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] PDB crawler Hi all, just wondering if anybody is aware of a service similar to PubCrawler (http://pubcrawler.gen.tcd.ie/), but for PDB structures, released and unreleased. Basically a weekly email that would alert you of freshly deposited structures that match some keywords. Thanks in advance, ciao Sebastiano -- 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 -- 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
Re: [ccp4bb] suitable buffer for CD studies
I have found it is best to test the absorption of your buffer in the wavelength range you are interested in. If you are going to do a temperature study with CD perhaps at 222 nm, then test your buffer there with your UV spec. You want to have little or no absorption. Or do the range 200-270 nm and choose a buffer that does not absorb in that range. For example we thought Bicine pH 8.5 with BME would be ok for CD studies. Bicine pH 7 did not absorb at 222 nm, but 8.5 gave a high background. bME really absorbs We ended up using PBS with a smidgeon of BME. Also you should use a protein concentration that gives you an absorption of 1.0 at the wavelength of interest This is crucial to keep noise out of your data. Also the CD dynod voltage should be monitored and keep it below 400 for protein studies Here is an excellent reference: Kelly, Ness and Price (2005) How to study proteins using circular dichroism Biochemica et Biophysica Acta *1751*, 119-139 Good Luck, Gloria On Wed, Mar 20, 2013 at 1:59 AM, Harsh Bansia spideysp...@gmail.com wrote: Sorry for a simple and non-CCP4 question. I have determined the structures of three different mutants of a thermostable protein by X-ray crystallography method. I feel that Mg2+ has a role in protein stability. So I want to perform a thermal denaturation study by CD spectroscopy both in presence and absence of Mg2+ ion. In this regards, what should be the suitable buffer for CD studies? May I use PBS buffer ? Since phosphate sequester divalent cations like Mg2+. Is it advisable to use PBS buffer. If so, what is maximum concentration of Mg2+ ion that can be used say e.g. 5 mM? My protein was in Tris buffer and lyophilized and have theoretical pI =4.56 and maximum activity at pH 8.4. Thanking you in advances, harsh
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Dear Zbyszek, I am concerned that the unmerged data would be bypassed and not preserved in your recommendation. I also find it counter intuitive that the merged data would then be unmerged into a lower symmetry and be better than the unmerged data; there is I imagine some useful reference or two you can direct me to that may well correct my lack of understanding. Thirdly I think this a very likely useful case to preserve the raw diffraction images. All best wishes, John Prof John R Helliwell DSc On 19 Mar 2013, at 14:37, Zbyszek Otwinowski zbys...@work.swmed.edu wrote: It is a clear-cut case of crystal packing disorder. The tell-tale sign is that data can be merged in the higher-symmetry lattice, while the number of molecules in the asymmetric unit (3 in P21) is not divisible by the higher symmetry factor (2, by going from P21 to P21212). From my experience, this is more likely a case of order-disorder than merohedral twinning. The difference between these two is that structure factors are added for the alternative conformations in the case of order-disorder, while intensities (structure factors squared) are added in the case of merohedral twinning. Now an important comment on how to proceed in the cases where data can be merged in a higher symmetry, but the structure needs to be solved in a lower symmetry due to a disorder. !Such data needs to be merged in the higher symmetry,assigned R-free flag, and THEN expanded to the lower symmetry. Reprocessing the data in a lower symmetry is an absolutely wrong procedure and it will artificially reduce R-free, as the new R-free flags will not follow data symmetry! Moreover, while this one is likely to be a case of order-disorder, and these are infrequent, reprocessing the data in a lower symmetry seems to be frequently abused, essentially in order to reduce R-free. Generally, when data CAN be merged in a higher symmetry, the only proper procedure in going to a lower-symmetry structure is by expanding these higher-symmetry data to a lower symmetry, and not by rescaling and merging the data in a lower symmetry. Zbyszek Otwinowski Dear all, We have solved the problem. Data processing in P1 looks better (six molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules in ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round of refinement (without put waters, ligands, etc.). Indeed, there were one more molecule in ASU, but the over-merged data in an orthorhombic lattice hid the correct solution. Thank you very much for all your suggestions, they were very important to solve this problem. Cheers, Andrey 2013/3/15 Andrey Nascimento andreynascime...@gmail.com *Dear all,* *I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure.* *A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel???* *I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf* * * *Do someone have an explanation or solution for this?* * * *Cheers,* *Andrey* Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353
[ccp4bb] Scaling with SCALA high and low resolution data sets
Dear All, we have two data sets at about 0.9 and 1.9 Ang. resolution collected from a single crystal. Integration with iMosflm seems to be fine like the scaling within each of the data sets. When we try to merge and scale both of them with 'Scala' we get extremely high scale factors for the lower resolution images varying between approximately 30 and 200! Do we need to pay attention to some particular options for running the program(s)? Thank you, Kyriacos e-mail: petra...@imbb.forth.gr
Re: [ccp4bb] Scaling with SCALA high and low resolution data sets
On 03/20/13 13:25, Kyriacos Petratos wrote: Dear All, we have two data sets at about 0.9 and 1.9 Ang. resolution collected from a single crystal. Integration with iMosflm seems to be fine like the scaling within each of the data sets. When we try to merge and scale both of them with 'Scala' we get extremely high scale factors for the lower resolution images varying between approximately 30 and 200! Do we need to pay attention to some particular options for running the program(s)? Thank you, Kyriacos e-mail: petra...@imbb.forth.gr Is this a space group with alternate settings? Perhaps the two data sets were indexed with different settings. Did you limit the low resolution for the 0.9 A data set to exclude overloads? -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
[ccp4bb] column tube
Dear Colleagues, Apologies for the non-crystallographic question but it very likely that someone out there may have the answer. I have a chromatography column. Unfortunately the company does not provide a replacement for the plastic tube, which should not cost more than 100 €. The cost for a new column is more than 1500 €! Does anybody know if there is a company that could provide custom-made plastic tubing for chromatography columns? Thanks in advance for your answers, George Kontopidis Assοciate Professor of Biochemistry Veterinary School, University of Thessaly Karditsa 43100, Greece Tel: +30 24410 66017 Mob: +30 69 342 643 75 e-mail: mailto:gkontopi...@vet.uth.gr gkontopi...@vet.uth.gr web site: http://www.vet.uth.gr/english/
Re: [ccp4bb] PDB crawler
Hi Sebastino Both PDB and PBDe have facebook pages. PDBe updates the page weekly with some snippets as well. On Wed, Mar 20, 2013 at 3:41 PM, Boaz Shaanan bshaa...@exchange.bgu.ac.ilwrote: Hi, I think that what you're looking for (or close to it) is available on the pdb site. Through your PDB login you can do a search according to keyword(s) and ask to repeat the search periodically. You'll get an alert in the e-mail. I actually use it for some time. Cheers, Boaz *Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710* ** ** * * -- *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sebastiano Pasqualato [sebastiano.pasqual...@gmail.com] *Sent:* Wednesday, March 20, 2013 2:40 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] PDB crawler Hi all, just wondering if anybody is aware of a service similar to PubCrawler ( http://pubcrawler.gen.tcd.ie/), but for PDB structures, released and unreleased. Basically a weekly email that would alert you of freshly deposited structures that match some keywords. Thanks in advance, ciao Sebastiano -- 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
Re: [ccp4bb] column tube
Hi George, What is the plastic tube? Is it part of the end adaptors or it is part of the tubing? What type of plastic it is? If you can provide a photograph of the broken part maybe someone can give you more useful suggestions. Zhijie From: George Kontopidis Sent: Wednesday, March 20, 2013 2:28 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] column tube Dear Colleagues, Apologies for the non-crystallographic question but it very likely that someone out there may have the answer. I have a chromatography column. Unfortunately the company does not provide a replacement for the plastic tube, which should not cost more than 100 €. The cost for a new column is more than 1500 €! Does anybody know if there is a company that could provide custom-made plastic tubing for chromatography columns? Thanks in advance for your answers, George Kontopidis Assοciate Professor of Biochemistry Veterinary School, University of Thessaly Karditsa 43100, Greece Tel: +30 24410 66017 Mob: +30 69 342 643 75 e-mail: gkontopi...@vet.uth.gr web site: http://www.vet.uth.gr/english/
Re: [ccp4bb] column tube
Hi George, I think that part can be made from an aluminum or a steel tube with proper OD. The ID seems only need to be large enough to allow the glass tube to fit in - depending what is allowed by the two black end adaptors. You can check your engineering department or some machine shops to find some one to cut the tubes, ground the ends and machine the threads for you. Of course you won't be able to see through the wall now, but it will be a very strong column. The expense I think would mainly be labor. Zhijie From: George Kontopidis Sent: Wednesday, March 20, 2013 3:27 PM To: 'Zhijie Li' Subject: RE: [ccp4bb] column tube Dear Zhijie, I am looking for the external clear plastic tube of Bio-rad UNO Q1 Column 720-0001 Column information: http://www.bio-rad.com/prd/en/GR/LSR/PDP/d825a6ab-49cf-4140-91c3-ea131c886b74/UNO-Monolith-Anion-Exchange-Columns see also attached the broken plastic tube. Unfortunately the company does not provide me with additional information regarding plastic type. Although we know that the plastic should stand 700 psi pressure. thanks George From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Zhijie Li Sent: Wednesday, March 20, 2013 9:13 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] column tube Hi George, What is the plastic tube? Is it part of the end adaptors or it is part of the tubing? What type of plastic it is? If you can provide a photograph of the broken part maybe someone can give you more useful suggestions. Zhijie From: George Kontopidis Sent: Wednesday, March 20, 2013 2:28 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] column tube Dear Colleagues, Apologies for the non-crystallographic question but it very likely that someone out there may have the answer. I have a chromatography column. Unfortunately the company does not provide a replacement for the plastic tube, which should not cost more than 100 €. The cost for a new column is more than 1500 €! Does anybody know if there is a company that could provide custom-made plastic tubing for chromatography columns? Thanks in advance for your answers, George Kontopidis Assοciate Professor of Biochemistry Veterinary School, University of Thessaly Karditsa 43100, Greece Tel: +30 24410 66017 Mob: +30 69 342 643 75 e-mail: gkontopi...@vet.uth.gr web site: http://www.vet.uth.gr/english/
Re: [ccp4bb] Resolution limit of index in XDS
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Herman, the short answer might be that at the stage of COLSPOT the term 'resolution' has a limited meaning because COLSPOT does not rely on the experimental setup like distance and beam direction, so the term 'resolution limit' is conceptually not applicable at this stage. Indexing does often not require the full data set, you can reduce the SPOT_RANGE if you are worried about processing time, or by a multi-CPU machine. One of the great advantages of XDS is that it asks you to think at a level higher than the average MS-Windows user while processing your data, so the effort to figure out the three numbers to set the TRUSTED_REGION is in line with the philosphy of XDS as I understand it. But you are right, I do not have access to the source of XDS and I am not the person to address a request to. Kind regards, Tim On 03/20/2013 10:29 AM, herman.schreu...@sanofi.com wrote: Dear Tim, but probably I should adres this to Kai Diederichs, not including the resolution cutoff in COLSPOT and IDXREF is a feature of XDS I do not understand at all. For most cases, it may not matter since only the strong spots are used, but what are the advantages? In fact there are disadvantages, especially when dealing with poorly diffracting difficult data sets: -when a crystallographer imposes a resolution limit, there are usually good reasons for it. -outside the resolution limit, there may be ice rings or contaminating salt spots, which make the autoindexing fail. -when processing 900 frame Pilatus data sets, running COLSPOT on the complete detector surface takes significantly longer then running it only on the center region. Of course, one could fudge a resolution cutoff by translating resolution into pixels and then calculating a TRUSTED_REGION, or manually editing the SPOT.XDS file, but this is a lot of extra and in my view unneccessary work. I would really consider using the resolution cutoff for COLSPOT as well. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Gruene Sent: Tuesday, March 19, 2013 11:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Resolution limit of index in XDS Dear Niu, indexing relies on strong reflections only, that is (in very brieft) why INCLUDE_RESOLUTION_RANGE indeed does not affect the relections collected in COLSPOT which in turn are used by IDXREF. You can work around this, however, by making use of TRUSTED_REGION and set it to e.g. 0.7 or 0.6 (you can use adxv to translate resolution into pixel and then calculate the fraction you need to set the second number in TRUSTED_REGION to (or the first if you want to exclude the inner resolution reflections - I remember one data set where this was essential for indexing - DNA was involved there) Best, Tim On 03/19/2013 08:53 PM, Niu Tou wrote: Dear All, Is there any command can set the resolution limit for index step in XDS? I only found a keyword INCLUDE_RESOLUTION_RANGE, but it looks to be a definition of resolution range after index step as it says: INCLUDE_RESOLUTION_RANGE=20.0 0.0 !Angstroem; used by DEFPIX,INTEGRATE,CORRECT Thanks! Niu - -- 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/ iD8DBQFRSjVtUxlJ7aRr7hoRApZhAJ9RFBs8D9NGjgLY3KOoNHhNtdOWggCgj7U0 zY7jEFDYZfl0Umb9E1Bzs1U= =+HjR -END PGP SIGNATURE-
Re: [ccp4bb] COOT running slow after upgrading to CENTOS 6.4 from 6.3
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Zhijie, this sounds like a graphics driver issue. You can use the command 'lspci | grep -i vga' to find out what graphics chip you have on the computer, and you should check /var/log/Xorg.0.log what driver is actually used. If it is an NVIDIA card and the system uses the nouveau kernel module, try and install the driver from the NVIDIA homepage. Best, Tim On 03/20/2013 03:42 PM, Zhijie Li wrote: Hello, We have a curious situation here: after upgrading CentOS 6.3 to 6.4, COOT runs slowly every time after the go to atom command is executed - every rotation of the molecules takes nearly 1 second to finish. But if the go to atom command is never sent then the speed is normal. A COOT running in a virtual machine windows XP on the same computer behaves OK though. It doesn't seem to be a conflict with the CentOS 6.4 either, because COOT on another computer with newly installed CentOS 6.4 runs perfectly fine. So I guess it might has to do with some libraries that COOT might use. The computer with trouble have python upgraded to 2.7 from the python source code when it was running CentOS 6.3, could this be a problem? Zhijie - -- 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/ iD8DBQFRSjZrUxlJ7aRr7hoRAmtcAKDMMuaV4jJIPxiLHpxrjqzMqH0GKwCgw179 e4KL5/FfJAGARe8d4igTByc= =4NYm -END PGP SIGNATURE-
Re: [ccp4bb] Scaling with SCALA high and low resolution data sets
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Kyriacos, as David has pointed out different indexing possibilites might be a problem. Could you run the mtz-files from mosflm through pointless (and then aimless instead of scala)? pointless compares the different indexing possibilities and picks the one where the data fit best. Regards, Tim On 03/20/2013 06:25 PM, Kyriacos Petratos wrote: Dear All, we have two data sets at about 0.9 and 1.9 Ang. resolution collected from a single crystal. Integration with iMosflm seems to be fine like the scaling within each of the data sets. When we try to merge and scale both of them with 'Scala' we get extremely high scale factors for the lower resolution images varying between approximately 30 and 200! Do we need to pay attention to some particular options for running the program(s)? Thank you, Kyriacos e-mail: petra...@imbb.forth.gr - -- 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/ iD8DBQFRSjcvUxlJ7aRr7hoRAhZbAKDl++SESOqHcqqDdfA+bO0LvAnr0gCfZXyg RVHYwqqwZx6E+3b9sXlZzvY= =RJC3 -END PGP SIGNATURE-
Re: [ccp4bb] suitable buffer for CD studies
Sent from my iPad On 20/03/2013, at 7:59 PM, Harsh Bansia spideysp...@gmail.com wrote: Sorry for a simple and non-CCP4 question. I have determined the structures of three different mutants of a thermostable protein by X-ray crystallography method. I feel that Mg2+ has a role in protein stability. So I want to perform a thermal denaturation study by CD spectroscopy both in presence and absence of Mg2+ ion. In this regards, what should be the suitable buffer for CD studies? May I use PBS buffer ? Since phosphate sequester divalent cations like Mg2+. Is it advisable to use PBS buffer. If so, what is maximum concentration of Mg2+ ion that can be used say e.g. 5 mM? My protein was in Tris buffer and lyophilized and have theoretical pI =4.56 and maximum activity at pH 8.4. Thanking you in advances, harsh
Re: [ccp4bb] suitable buffer for CD studies
Hi Harsh Something like sodium borate at pH 9.0 could be an alternative to phosphate buffers. If you are looking at thermal unfolding above 220nm, then the choice of buffer is less critical as many buffers and additives are problematic only below 200nm. If your samples require high salt concentrations, I routinely use NaF as an alternative. It is transparent in the wavelength range relevant for far UV CD spectral collection. Kavestri Y. Kingston Laboratory - Structural Biology Group University of Auckland On 20/03/2013, at 7:59 PM, Harsh Bansia spideysp...@gmail.com wrote: Sorry for a simple and non-CCP4 question. I have determined the structures of three different mutants of a thermostable protein by X-ray crystallography method. I feel that Mg2+ has a role in protein stability. So I want to perform a thermal denaturation study by CD spectroscopy both in presence and absence of Mg2+ ion. In this regards, what should be the suitable buffer for CD studies? May I use PBS buffer ? Since phosphate sequester divalent cations like Mg2+. Is it advisable to use PBS buffer. If so, what is maximum concentration of Mg2+ ion that can be used say e.g. 5 mM? My protein was in Tris buffer and lyophilized and have theoretical pI =4.56 and maximum activity at pH 8.4. Thanking you in advances, harsh
Re: [ccp4bb] suitable buffer for CD studies
Why not thermal denaturation in the presence of Sypro Orange and a realtime PCR machine ? Crowther et al. Use of thermal melt curves to assess the quality of enzyme preparations. Anal Biochem (2010) vol. 399 (2) pp. 268-275 Or (shameless advertisement): Hain et al. Structural characterization and inhibition of the Plasmodium Atg8-Atg3 interaction. Journal of Structural Biology (2012) vol. 180 (3) pp. 551-62 2.9.2. Thermal shift assay Mutant and wild type proteins were tested at 1 mg/mL in a reaction containing 30 lL of protein and 1 lL of a 1:30 dilution of SYPROÒ orange dye. Fluorescent measurements were done in triplicates in a CFX96 thermal cycler (BioRad) from 20 to 80 °C over a period of 60 min and the melting temperature was determined from the maximum of the first derivative of the melting curve. You will need less protein compared to the CD melt. Jürgen On Mar 20, 2013, at 8:56 PM, Kavestri wrote: Hi Harsh Something like sodium borate at pH 9.0 could be an alternative to phosphate buffers. If you are looking at thermal unfolding above 220nm, then the choice of buffer is less critical as many buffers and additives are problematic only below 200nm. If your samples require high salt concentrations, I routinely use NaF as an alternative. It is transparent in the wavelength range relevant for far UV CD spectral collection. Kavestri Y. Kingston Laboratory - Structural Biology Group University of Auckland On 20/03/2013, at 7:59 PM, Harsh Bansia spideysp...@gmail.commailto:spideysp...@gmail.com wrote: Sorry for a simple and non-CCP4 question. I have determined the structures of three different mutants of a thermostable protein by X-ray crystallography method. I feel that Mg2+ has a role in protein stability. So I want to perform a thermal denaturation study by CD spectroscopy both in presence and absence of Mg2+ ion. In this regards, what should be the suitable buffer for CD studies? May I use PBS buffer ? Since phosphate sequester divalent cations like Mg2+. Is it advisable to use PBS buffer. If so, what is maximum concentration of Mg2+ ion that can be used say e.g. 5 mM? My protein was in Tris buffer and lyophilized and have theoretical pI =4.56 and maximum activity at pH 8.4. Thanking you in advances, harsh .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] suitable buffer for CD studies
One of the other things you need to be concerned about with thermal melts is the change in buffer pKa as temperature varies (I seem to remember this being called the beta factor). Phosphate is used for CD melts regularly because its pKa is fairly invariant with temperature. (A good reference is Data for Biochemical Research by Dawson, Ch. 18). Acetate also shares this invariance but the Good buffers often do not. This is of course a concern with the Spyro Orange experiment as well. Matthew Merski Shoichet Group UCSF
Re: [ccp4bb] suitable buffer for CD studies
Yep, mostly you should stay away from Tris as this is the worst buffer system when playing with temperature changes. Tris for example has a ∆pKa/10˚C -0.31 Good, N.E. (1986) Biochemistry 5, 467 Jürgen P.S. @Matthew, was this what you meant by the Good buffers often not ? or just a coincidence ? On Mar 20, 2013, at 9:57 PM, Matthew Merski wrote: One of the other things you need to be concerned about with thermal melts is the change in buffer pKa as temperature varies (I seem to remember this being called the beta factor). Phosphate is used for CD melts regularly because its pKa is fairly invariant with temperature. (A good reference is Data for Biochemical Research by Dawson, Ch. 18). Acetate also shares this invariance but the Good buffers often do not. This is of course a concern with the Spyro Orange experiment as well. Matthew Merski Shoichet Group UCSF .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
