Re: [ccp4bb] Superpose
Hi Eugene, thanks for your response. Yes, the program is LSQKAB. No aligment is pursued, the aim is just to superpose models and get all the information as tables. Looking at old jobs (CCP4 6.2: LSQKAB version 6.2 : 13/03/09), it seems it always did the same, the results on xyz and B rms just a subset of the atoms (most) being superposed are written to the log file. This is independent on the specific PDB files in use (I have tested a few only)can this be confirmed by you/circumvented by the user (i.e. by changing the com file) or eventualy solved? Thanks again! H Quoting Eugene Krissinel eugene.krissi...@stfc.ac.uk: I would say the guess is a wrong one. SS superposition is used only as a seed for further refinement. SSM (superpose) outputs all residues (not all atoms) and marks those which get aligned. Then, superposition is done using aligned residues. Alignment != superposition. As to LSQKAB, it's pure superposition, not alignment AFAIK. Alignment should be given to LSQKAB in form of atom pairs that should be used for superposition. No idea what is wrong in this example though. Eugene On 9 May 2014, at 23:33, Tim Gruene wrote: Dear H, you are referring to superpose, but your logfile lists the output from LSQKAB, which are two different programs. According to the man-page of superpose, it uses secondary structure elements for superposition, so maybe the missing atoms are those not part of a helix and not part of a strand. Just a guess. Best, Tim On 05/09/2014 08:05 PM, Horacio Botti wrote: Dear all, Why does (if it is suppossed to do so) Superpose output results for a subset of atoms only? See a summary of log file below (just the top lines, data on atoms, and final data and message). In the example, results for residues 69-76 are absent, other atoms are absent as well, but the number of atoms that were skipped in the analysis is 0 (NUMBER OF ATOMS EXCLUDED BY RADCHK IS 0). Of course, the atoms for these residues are present in the pdb files and no alternative conformations are being modeled. If you display a table from the log graph window, you find xyz RMS for all atoms including the missed atoms. I also need info on B RMS, which are not displayed in log graph. What can I do to get a complete log file? Thanks in advance! H #CCP4I VERSION CCP4Interface 2.2.0 #CCP4I SCRIPT LOG superpose #CCP4I DATE 09 May 2014 09:21:04 #CCP4I USER hbotti #CCP4I PROJECT .. #CCP4I JOB_ID 30 #CCP4I SCRATCH /tmp/hbotti #CCP4I HOSTNAME pxf8.ipmont.lan #CCP4I PID 8268 ### ### ### ### CCP4 6.3: LSQKAB version 6.3 : 13/03/09## ### User: hbotti Run date: 9/ 5/2014 Run time: 09:21:04 Please reference: Collaborative Computational Project, Number 4. 1994. The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50, 760-763. as well as any specific reference in the program write-up. Data line--- title .. Data line--- fit res MAIN 66 to 544 chain B Data line--- match 66 to 544 chain C Data line--- output xyz rms deltas Data line--- end LSQKAB RUN .. OPEN FILES AS REQUESTED Opening coordinate file of model to be moved Logical name: XYZIN2 File name: ... PDB file is being opened on unit 1 for INPUT. MATRICES DERIVED FROM CRYST1 CARD IN COORDINATE FILE RF RO 0.010 -0.000 0.002 0.000 96.790 0.000 -19.210 -0.000 -0.000 0.008 -0.000 -0.0000.000 133.030 0.000 0.000 0.000 -0.000 0.009 -0.0000.000 0.000 106.024 -0.000 -0.000 0.000 -0.000 1.000 -0.000 0.000 -0.000 1.000 Logical name: XYZOUT File name: .. Opening coordinate file of fixed model. Logical name: XYZIN1 File name: ... PDB file is being opened on unit 3 for INPUT. MATRICES DERIVED FROM CRYST1 CARD IN COORDINATE FILE RF RO 0.010 -0.000 0.002 0.000 96.610 0.000 -18.928 -0.000 -0.000 0.008 -0.000 -0.0000.000 132.960 0.000 0.000 0.000 -0.000 0.009 -0.0000.000 0.000 106.156 -0.000 -0.000 0.000 -0.000 1.000 -0.000 0.000 -0.000 1.000 FORMATTED UNKNOWN file opened on unit 7 Logical name: RMSTAB, Filename: . FORMATTED UNKNOWN file opened on unit 8 Logical name: DELTAS, Filename: .. - NO MATCH FOR WORKCD ATOM - 142CA B IN REFRCD FILE ** ZERO OCCUPANCIES IN WORKING SET ** 0.0 ** ZERO OCCUPANCIES IN REFERENCE SET ** 0.0 LSFIT ATOMS IN WORKING MOLECULE(1915 TO BE REFINED
[ccp4bb] Superpose
Dear all, Why does (if it is suppossed to do so) Superpose output results for a subset of atoms only? See a summary of log file below (just the top lines, data on atoms, and final data and message). In the example, results for residues 69-76 are absent, other atoms are absent as well, but the number of atoms that were skipped in the analysis is 0 (NUMBER OF ATOMS EXCLUDED BY RADCHK IS 0). Of course, the atoms for these residues are present in the pdb files and no alternative conformations are being modeled. If you display a table from the log graph window, you find xyz RMS for all atoms including the missed atoms. I also need info on B RMS, which are not displayed in log graph. What can I do to get a complete log file? Thanks in advance! H #CCP4I VERSION CCP4Interface 2.2.0 #CCP4I SCRIPT LOG superpose #CCP4I DATE 09 May 2014 09:21:04 #CCP4I USER hbotti #CCP4I PROJECT .. #CCP4I JOB_ID 30 #CCP4I SCRATCH /tmp/hbotti #CCP4I HOSTNAME pxf8.ipmont.lan #CCP4I PID 8268 ### ### ### ### CCP4 6.3: LSQKAB version 6.3 : 13/03/09## ### User: hbotti Run date: 9/ 5/2014 Run time: 09:21:04 Please reference: Collaborative Computational Project, Number 4. 1994. The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50, 760-763. as well as any specific reference in the program write-up. Data line--- title .. Data line--- fit res MAIN 66 to 544 chain B Data line--- match 66 to 544 chain C Data line--- output xyz rms deltas Data line--- end LSQKAB RUN .. OPEN FILES AS REQUESTED Opening coordinate file of model to be moved Logical name: XYZIN2 File name: ... PDB file is being opened on unit 1 for INPUT. MATRICES DERIVED FROM CRYST1 CARD IN COORDINATE FILE RF RO 0.010 -0.000 0.002 0.000 96.790 0.000 -19.210 -0.000 -0.000 0.008 -0.000 -0.0000.000 133.030 0.000 0.000 0.000 -0.000 0.009 -0.0000.000 0.000 106.024 -0.000 -0.000 0.000 -0.000 1.000 -0.000 0.000 -0.000 1.000 Logical name: XYZOUT File name: .. Opening coordinate file of fixed model. Logical name: XYZIN1 File name: ... PDB file is being opened on unit 3 for INPUT. MATRICES DERIVED FROM CRYST1 CARD IN COORDINATE FILE RF RO 0.010 -0.000 0.002 0.000 96.610 0.000 -18.928 -0.000 -0.000 0.008 -0.000 -0.0000.000 132.960 0.000 0.000 0.000 -0.000 0.009 -0.0000.000 0.000 106.156 -0.000 -0.000 0.000 -0.000 1.000 -0.000 0.000 -0.000 1.000 FORMATTED UNKNOWN file opened on unit 7 Logical name: RMSTAB, Filename: . FORMATTED UNKNOWN file opened on unit 8 Logical name: DELTAS, Filename: .. - NO MATCH FOR WORKCD ATOM - 142CA B IN REFRCD FILE ** ZERO OCCUPANCIES IN WORKING SET ** 0.0 ** ZERO OCCUPANCIES IN REFERENCE SET ** 0.0 LSFIT ATOMS IN WORKING MOLECULE(1915 TO BE REFINED) ATOMS IN REFERENCE MOLECULE CENTROID OF WORKING MOLECULE : 10.597 -61.757 22.304 CENTROID OF WORKING MOLECULE :(fractional)0.151 -0.464 0.210 CENTROID OF REFERENCE MOLECULE: 13.208 -36.008 70.253 CENTROID OF REFERENCE MOLECULE:(fractional)0.266 -0.271 0.662 Distance between CENTROIDS : 54.488 Direction cosines of vector between CENTROIDS: -0.048 -0.473 -0.880 NUMBER OF ATOMS EXCLUDED BY RADCHK IS 0 RMS B DISPLACEMENT = 13.133 AVERAGE B DISPLACEMENT = -8.353 RMS XYZ DISPLACEMENT = 0.360 AVERAGE XYZ DISPLACEMENT = 0.245 MAXIMUM XYZ DISPLACEMENT = 4.237 ROTATION MATRIX: -0.80795 -0.52390 0.26974 -0.52128 0.42202 -0.74173 0.27475 -0.73988 -0.61407 TRANSLATION VECTOR IN AS-16.5998512.1225335.34469 TRANSLATION VECTOR IN fractions of cell edge-0.106590 0.0911740.332951 Natom2 ROTATED CDSATOMId2 Natom1AtomId1 Bdiff XYZDiff 3906 42.25 -30.59 71.01 N SER B 66 7814N C 66 12.0861.362 3907 43.27 -30.91 72.03 CA SER B 66 7815CA C 669.6492.287 3908 43.70 -29.70 72.95 C SER B 66 7816C C 66 10.0091.369 3909 44.77 -29.07 72.82 O SER B 66 7817O C 667.790
Re: [ccp4bb] Superpose
I should have said that this is the comparison of two crystaline states of the same molecule, the sequences are identical. Best H hbo...@pasteur.edu.uy ha escrito: Dear all, Why does (if it is suppossed to do so) Superpose output results for a subset of atoms only? See a summary of log file below (just the top lines, data on atoms, and final data and message). In the example, results for residues 69-76 are absent, other atoms are absent as well, but the number of atoms that were skipped in the analysis is 0 (NUMBER OF ATOMS EXCLUDED BY RADCHK IS 0). Of course, the atoms for these residues are present in the pdb files and no alternative conformations are being modeled. If you display a table from the log graph window, you find xyz RMS for all atoms including the missed atoms. I also need info on B RMS, which are not displayed in log graph. What can I do to get a complete log file? Thanks in advance! H #CCP4I VERSION CCP4Interface 2.2.0 #CCP4I SCRIPT LOG superpose #CCP4I DATE 09 May 2014 09:21:04 #CCP4I USER hbotti #CCP4I PROJECT .. #CCP4I JOB_ID 30 #CCP4I SCRATCH /tmp/hbotti #CCP4I HOSTNAME pxf8.ipmont.lan #CCP4I PID 8268 ### ### ### ### CCP4 6.3: LSQKAB version 6.3 : 13/03/09## ### User: hbotti Run date: 9/ 5/2014 Run time: 09:21:04 Please reference: Collaborative Computational Project, Number 4. 1994. The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50, 760-763. as well as any specific reference in the program write-up. Data line--- title .. Data line--- fit res MAIN 66 to 544 chain B Data line--- match 66 to 544 chain C Data line--- output xyz rms deltas Data line--- end LSQKAB RUN .. OPEN FILES AS REQUESTED Opening coordinate file of model to be moved Logical name: XYZIN2 File name: ... PDB file is being opened on unit 1 for INPUT. MATRICES DERIVED FROM CRYST1 CARD IN COORDINATE FILE RF RO 0.010 -0.000 0.002 0.000 96.790 0.000 -19.210 -0.000 -0.000 0.008 -0.000 -0.0000.000 133.030 0.000 0.000 0.000 -0.000 0.009 -0.0000.000 0.000 106.024 -0.000 -0.000 0.000 -0.000 1.000 -0.000 0.000 -0.000 1.000 Logical name: XYZOUT File name: .. Opening coordinate file of fixed model. Logical name: XYZIN1 File name: ... PDB file is being opened on unit 3 for INPUT. MATRICES DERIVED FROM CRYST1 CARD IN COORDINATE FILE RF RO 0.010 -0.000 0.002 0.000 96.610 0.000 -18.928 -0.000 -0.000 0.008 -0.000 -0.0000.000 132.960 0.000 0.000 0.000 -0.000 0.009 -0.0000.000 0.000 106.156 -0.000 -0.000 0.000 -0.000 1.000 -0.000 0.000 -0.000 1.000 FORMATTED UNKNOWN file opened on unit 7 Logical name: RMSTAB, Filename: . FORMATTED UNKNOWN file opened on unit 8 Logical name: DELTAS, Filename: .. - NO MATCH FOR WORKCD ATOM - 142CA B IN REFRCD FILE ** ZERO OCCUPANCIES IN WORKING SET ** 0.0 ** ZERO OCCUPANCIES IN REFERENCE SET ** 0.0 LSFIT ATOMS IN WORKING MOLECULE(1915 TO BE REFINED) ATOMS IN REFERENCE MOLECULE CENTROID OF WORKING MOLECULE : 10.597 -61.757 22.304 CENTROID OF WORKING MOLECULE :(fractional)0.151 -0.464 0.210 CENTROID OF REFERENCE MOLECULE: 13.208 -36.008 70.253 CENTROID OF REFERENCE MOLECULE:(fractional)0.266 -0.271 0.662 Distance between CENTROIDS : 54.488 Direction cosines of vector between CENTROIDS: -0.048 -0.473 -0.880 NUMBER OF ATOMS EXCLUDED BY RADCHK IS 0 RMS B DISPLACEMENT = 13.133 AVERAGE B DISPLACEMENT = -8.353 RMS XYZ DISPLACEMENT = 0.360 AVERAGE XYZ DISPLACEMENT = 0.245 MAXIMUM XYZ DISPLACEMENT = 4.237 ROTATION MATRIX: -0.80795 -0.52390 0.26974 -0.52128 0.42202 -0.74173 0.27475 -0.73988 -0.61407 TRANSLATION VECTOR IN AS-16.5998512.1225335.34469 TRANSLATION VECTOR IN fractions of cell edge-0.1065900.091174 0.332951 Natom2 ROTATED CDSATOMId2 Natom1AtomId1 Bdiff XYZDiff 3906 42.25 -30.59 71.01 N SER B 66 7814N C 66 12.0861.362 3907 43.27 -30.91 72.03 CA SER B 66 7815CA C 669.6492.287 3908 43.70 -29.70 72.95 C SER B 66
[ccp4bb] Not to refine B factors, not to refine coordinates
Dear all, hi!! I need help in order to use Refmac in two particular ways. On one hand I would like to ask the program not to refine B factor values (whichever the selected refinement mode was) and in the other I would like to ask it not to refine coordinates (whichever the selected refinement mode was). How can I do this? There must be more than one way...? Pros and cons... Clarifications: It is not about setting initial B values neither to set Bavg to a certain value so that scaling goes well. Thks in advance! Horacio attachment: hbotti.vcf
Re: [ccp4bb] Extra positive density seen after TLS refinement?
Dear Naveed A Nadvi I think that your results highlight the fact that modelling the disorder/complex ordering of your crystal is relevant and in general, that we should take care in optimizing B factor refinement as a strong factor for model improvement. In this sense I would not relay in one TLS group definition, even though you have obtain better results comparing no TLS vs TLS. Try common sense definitions also: does the protein have a hinge between two domains, use the domains as TLS groups, etc. Then, look for optimal NCS between TLS groups also. Best wishes Horacio Quoting Naveed Ahmed Nadvi nnad2...@uni.sydney.edu.au: Dear Crystallographers, Thank you for your responses. The density map levels were 0.11e/A^3 (3.00 A) for both images with and without TLS refinement. When I superposed the deposited structure I could see the extra density was due to water moleucles that were seen in the higher resolution deposited structure. It is so interesting how I could not see them in my data without doing TLS. Performing the TLS refinement improved overall parameters: no TLS/TLS R0.2425/0.2334 R-free 0.2809/0.2748 RMS BondLen 0.0092/0.0074 RMS BondAngle1.1812/1.1407 ChirVol 0.0806/0.0779 My question is, do the positive density seen after TLS refinement justify adding these solvent molecules especially when they were not observed without TLS refinement? Thank you once again for your insights! Naveed From: Ethan Merritt [merr...@u.washington.edu] Sent: Sunday, 19 February 2012 3:10 PM To: Naveed Ahmed Nadvi Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] Extra positive density seen after TLS refinement? On Saturday, 18 February 2012, Naveed A Nadvi wrote: Dear crystallographers, I am fairly new in crystallographic work and structure determination, but I thought this would be the best place to post my questions. We had collected structural data for a protein that diffracted to 3 A. We had used a previously deposited structure (1.5 A) for molecular replacement. Our final structure used NCS restraints refinement over 4 chains within the assymetric unit. We were able to assign some water moleules using COOT and subsequently removed 'bad waters' manually. We used automated settings when dealing with these water molecules. In all cases these water molecules were found in the same positions as the initial structure (1.5 A) that we had used as a search model. This gave us confidence in the placement of our water molecules. Finally we had run validation tools (MolProbity) and our structure was found to be with Molprobity score within the 100th percentile. We then performed a TLS refinement (from TLSMD) to further improve R values. We used the final MolProbity-validated structure using 8 TLS groups and using PureTLS with constant B factor (50). We are observing large positive densities from the subsequent REFMAC5 refinement that are otherwise not observed in the absence of TLS refinement. Is it possible that the peaks are not higher in terms of absolute electron level, but only in terms of RMSD? That is, if the TLS treatment cleans up the map everywhere, then whatever peaks are left will deviate more from the (now lower) mean value even though their absolute size is the same. In other words, the 3 sigma contours in your first map may be more like 6 sigma contours in your second (cleaner) map. My questions are: 1) Is TLS suitable for our dataset (3 A)? There is no universal answer to that. You just have to test for yourself each time. Certainly TLS can help a lot at 3A for some structures. In general the more anisotropy is present, the more it helps to include it in your model somehow - and TLS is a cheap way to include it in your model. But if your structure does not have much anisotropy, then adding TLS to describe it won't have much effect. 2) Is TLS refinement independent of NCS refinement or should I define my NCS based on the 8 TLS groups? They are not the same thing at all. 3) Is it normal to see extra positive density after TLS refinement and what does it mean? See possible explanation above. Ethan 4) We had PEG4000 and Tris in our crystallization buffer. Could these 'blobs' represent these molecules or short water chains? I have attached images of the largest blob. Any comments and suggestions would be highly appreciated. Kind regards, Naveed A Nadvi Faculty of Pharmacy, University of Sydney, Australia
Re: [ccp4bb] On pKa of Aspartic acid
Dear all, for further discussion I believe that using the 0-14 pH scale assumes water activity of pure water, something that is surely not matched in the surface or pocket of a protein, so keeping this in mind I always prefer to speak about apparent pKa of a group if talking about a non solvent-exposed group. Horacio 2012/2/7 Roger Rowlett rrowl...@colgate.edu No. Kw = [H3O+][OH-] = 1 x 10^-14 at 25 deg C. So at pH 7.0, you have 10^-7 M each at equilibrium no matter how you slice it or whatever else is in solution. If equilibrium [H3O+] goes up [OH-] goes down commensurately. The pKa of water as an acid is based on Kw and water's effective concentration of 55 M in pure water. This pKa is used to compare the instrinsic acidity of water to other weak acids. Water is an exceptionally weak acid or base. ___ 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 2/7/2012 3:42 PM, Kevin Jin wrote: Oops, It should be: [H3O+]/[OH-]= 50/50 Kw = [H3O+][OH-], pH = pKa +log ([OH-]/[H2O]) H3O+ concentration of pure water is 10^-7 mol/L total H+ = 55.5M * 10^-7 = 5.55* 10^-6 mole. Is this right? Regards, Kevin On Tue, Feb 7, 2012 at 12:13 PM, Zachary Wood z...@bmb.uga.edu z...@bmb.uga.edu wrote: Hi Kevin, Hate to point this out, but under pH 7.0, the protonation state of water is not 50:50, and it is not a good acid. The H30+ concentration of pure water is 10^-7 Molar. In pure water (assuming 55.5 M) only 1:555,000,000 water molecules is in the protonated, charged state (H3O+). This is why when an enzyme uses water in its mechanism as a nucleophile, base, or acid, there is usually an acid/base catalyst or metal that protonates or deprotonates the water to 'activate it'. Best regards, Z *** Zachary A. Wood, Ph.D. Assistant Professor Department of Biochemistry Molecular Biology University of Georgia Life Sciences Building, Rm A426B 120 Green Street Athens, GA 30602-7229 Office: 706-583-0304 Lab:706-583-0303 FAX: 706-542-1738 *** On Feb 7, 2012, at 11:22 AM, Kevin Jin wrote: As we know, the pKa of water is 15.7. Under pH 7.0, its protonation should be 50/50. In this case, we may need to consider water in two formats: H2O vs. H3O+ When we say water as acid, it usually stands for H3O+ in chemistry. In chemical equation, H+ represents H3O+. In enzyme catalysis, water as a general acid sounds reasonable under pH 7.0. In some famous paper, water has been concluded as the general base (pKa 15.7) to deprotonate an alpha hydrogen (pKa ~ 22) or a hydrogen from a sp3 hybridized carbon (pKa ~36). This logic may need to be reconsidered. . Recently, I have read papers for pKa perturbation. I am also interested in the general base of Asp and Glu in enzyme catalysis. I will be very happy to read your paper in the future. Regards, Kevin Jin On Tue, Feb 7, 2012 at 3:48 AM, Deepak Oswal deepos...@gmail.com deepos...@gmail.com wrote: Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids’ ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal -- Horacio Botti PhD MD Protein Crystallography Unit Institut Pasteur of Montevideo
Re: [ccp4bb] Reasoning for Rmeas or Rpim as Cutoff
Dear all Perhaps a bit off of theme, just an example about resolution cut-off mean I/sigma(I) = 2 for dmin = 3.35 A (please have a look at the attached pdf) I would trust in I/s(I) = 2 (in this case it worked), but why not to determine what is information after the model has been refined to some extent using lower I/s(I) and then cutting the resolution by 0.05-0.1 A? Delta R and (from procheck) Avg-G factors did well. Note that Rfree improved by using data from higher resolution. Perhaps if Rmeas or Rpim were bad at that resolution (3.35 A) the story would be different. Best regards, Horacio Quoting John R Helliwell jrhelliw...@gmail.com: Dear Jacob, As an editor I am always mindful that an article is finally under the authors' names. That said the reader always deserves to know at what diffraction resolution average intensities (cease to) exist. The usual statistical practice to do that is to use a given quantity's (ie in this case a reflection intensity) sigma. Good effort is made in data processing programs to protect the quality of the estimate of each reflection intensity's sigma notably the chi square test. Thus I request that the diffraction resolution where I/sig(I) crosses 2.0 is quoted in the article, if it is not there already. I agree that 2.0 is arbitrary but it is more 'lenient' than the usual '3sigma' statistical test. Sometimes the title or abstract has to be changed to follow this criterion; eg 'The structure of xxx is determined to 2.4 Angstrom resolution' type of title has to be consistent with the above criterion. I do not follow an 'Rmerge must be less than x% rule'. I think the above follows reasonable general statistical practice, whilst permitting authors reasonable freedom, and also protects the (more innocent) readers of articles. I am aware that the 'correlation coefficient' between randomly portioned parts of data sets is being increasingly discussed, this parameter also having general statistical validity. I am monitoring discussion on this carefully. It has long been a good way of assessing the statistical quality of anomalous differences for example; to my knowledge introduced by Michael Rossmann many years ago. Best wishes, John On Fri, Jan 27, 2012 at 5:55 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Clarification: I did not mean I/sigma of 2 per se, I just meant I/sigma is more directly a measure of signal than R values. JPK On Fri, Jan 27, 2012 at 11:47 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Dear Crystallographers, I cannot think why any of the various flavors of Rmerge/meas/pim should be used as a data cutoff and not simply I/sigma--can somebody make a good argument or point me to a good reference? My thinking is that signal:noise of 2 is definitely still signal, no matter what the R values are. Am I wrong? I was thinking also possibly the R value cutoff was a historical accident/expedient from when one tried to limit the amount of data in the face of limited computational power--true? So perhaps now, when the computers are so much more powerful, we have the luxury of including more weak data? JPK -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** -- Professor John R Helliwell DSc resolution-meanI.pdf Description: Adobe PDF document
[ccp4bb] Crystallization robot and trypsin
Dear all We may use a Honeybee 963 robot to screen crystallization conditions for trypsin-containing protein samples and we are worried about robot contamination by residual protease. How do you normally clean robots when using this kind of sample? Your suggestions/recommendations will be appreciated. Thks!! Horacio Botti Unit of Protein Crystallography, Institut Pasteur of Montevideo, Uruguay. PS: below you have an old short CCP4 discussion: [ccp4bb]: Crystallization robots and protease. * /To/: ccp...@dl.ac.uk mailto:ccp4bb%40dl.ac.uk * /Subject/: [ccp4bb]: Crystallization robots and protease. * /From/: Marc Graille marc.grai...@ibbmc.u-psud.fr mailto:marc.graille%40ibbmc.u-psud.fr * /Date/: Mon, 19 Dec 2005 13:21:36 +0100 * /Sender/: owner-ccp...@dlmail1.dl.ac.uk mailto:owner-ccp4bb%40dlmail1.dl.ac.uk * /User-agent/: Debian Thunderbird 1.0.2 (X11/20050602) *** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk *** Dear all, I have a question regarding the use of robotics to screen for crystallization conditions for proteases. Does anyone have already used robots on proteases? If yes, have you experienced any protease contaminant in the robot pipes, which could have affected the results on other projects performed during the next few days ?? I mean that we cannot exclude that a contaminant could digest the protein we are working on and yield crystals of a fragment of the studied protein. We are hesitating in using our crystallization robots on proteases as we are afraid to have some contaminant in the pipes that will disturb all our future experiments!!! Any advice about how to clean the robot syringes after use of proteases are welcome!!! Regards, Marc -- Marc Graille, PhD Equipe de Genomique Structurale Institut de Biochimie et de Biophysique Moleculaire et Cellulaire (IBBMC) CNRS UMR8619 Bat 430 Universite Paris Sud 91405 Orsay Cedex Tel: 0169155047 *** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk *** Dear Marc We recommend cleaning dispensing tips with Hellmanex II from the German company Hellma. It's for cleaning cuvettes. You can buy it from VWR and others. Hellmanex is a mildly alkaline solution with surfactants etc. It has no enzymes in it, but you do need to flush with buffer to get rid of the alkali. Previously, users reported that cleaning with methanol mixed with concentrated HCl worked very well in extreme cases. Our robot, the Oryx, uses only one tip for protein. This is semi-disposable. Using one tip and touching off the drops has the great advantage that virtually no protein is wasted, and drops as low as 20 nl can be dispensed, even containing 50% glycerol! We recommend that users keep tips that have been used for proteases separately, and clean them after use. (The system comes with 8 tips and replacements cost 45 USD.) Also we recommend that tips are thoroughly cleaned once a fortnight for average use in any case. I hope this is helpful. Sincerely Patrick -- patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart, James Smith http://douglas.co.uk or http://www.douglasinstruments.com Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 attachment: hbotti.vcf
Re: [ccp4bb] Dehydration treatments
Hi Israel! The phenomena you describe seems to be related to cryoprotection, but you don't say anything about collection temperature or cryoprotection used. Controlled-slow dehydration is one way of achieving optimized cryocooling of specially fragile crystals. I would recommend you to read Garman's works, particularly the one that follows: Cryocooling of macromolecular crystals: optimization methods EF Garman? - Methods in enzymology, 2003 Enjoy Horacio Quoting Israel Sanchez israelsan...@gmail.com: Hi folks, I am currently impressed by the efficiency of dehydration treatments over the diffraction capacity of our crystals in one particular condition. Without any treatment the crystals seldom diffract to 20-30A but in our last synchrotron trip the very same crystals, after been incubated with increasing concentration of low molecular weight PEGs diffracted to 6A. I was wondering if anyone has studied these effects in a systematic way. Does anyone on the ccp4bb knows references or has any experience/pseudo-religious believes that do not care to share with the community about this particular topic? Thank you very much in advance -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK
Re: [ccp4bb] OT: Covalent modification of Cys by reducing agents?
Dear Mike BME readily autooxidizes (need for metal traces and dissolved O2). Is yours a metalloprotein? Is your buffer contaminated with metals? Those situations would make the case a bit different. If not, unless your BME stock is already oxidized, blocking of the accesible thiols with BME should take some time. If you treat your protein for 40 min with fresh BME you should not observe thiol blocking. If you let the preparation to stay for several days, even at 4-6 °C you may observe the blocking that you may be observing. If you want to prevent Cys blocking you can also change to DTT (it is a dithiol, does not readily form mixed disulfides) and use it with caution (for thiol reduction it is advisable to use stoichiometric DTT (with respect to the number of Cys you need to reduce) and 10 fold excess of BME, look for their redox potentials). Take care of not over-reducing your protein if internal disulfide bonds are expected. Once reduced I suggest you to remove any reducing agent and store the protein at -80 °C. External Cys can be easily oxidized, they are highly expossed to metals and oxidants (H2O2, BME disulfides, etc). Diffusion is for sure much faster than SS bond formation, although some cys react at almost diffusion-controlled rates with oxidants (is yours a thiol'dependen t peroxidase?) You can take a look at the following reference (advertising): 2011. Factors Affecting Protein Thiol Reactivity and Specificity in Peroxide Reduction. Chem Res Toxicol. Metals can contaminate bad quality materials (water, salts, buffers, etc), take care of that too. If you need to control the redox state of your protein you should use DTNB (Ellman´s reagent), or DTDPy, to measure accesible reduced thiol groups. Good luck! Horacio Quoting Kendall Nettles knett...@scripps.edu: We see BME adducts in all of our estrogen receptor structures, though we don't always put them in the models. Sometimes we only see one or two atoms of the adduct, and in others it is completely ordered. We only see it on the solvent accessible cysteines. We do it on purpose. We used to treat the protein with iodoacetic acid to generate uniform modification of the cysteines, but then we realized we could get then same homogeneity with 20-50mM BME. Kendall Nettles On Apr 15, 2011, at 4:09 PM, Michael Thompson mi...@chem.ucla.edu wrote: Hi All, I was wondering if anyone knew whether or not it is possible for reducing agents with thiol groups, such as DTT or beta-mercaptoethanol (BME), to form covalent S-S bonds with Cys residues, particularly solvent-exposed Cys? I have some puzzling biochemical results, and in the absence of a structure (thus far), I was wondering if this might be something to try to control for. I have never heard of this happening (or seen a structure where there was density for this type of adduct), but I can't really think of a good reason for why this wouldn't happen. Especially for something like BME, where the molecule is very much like the Cys sidechain and seems to me like it should have similar reactivity. The only thing I can think of is if there is a kinetic effect taking place. Perhaps the rate of diffusion of these small molecules is much faster that the formation of the S-S bond? Does anyone know whether or not this is possible, and why it does or does not happen? Thanks, Mike -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
