Re: [ccp4bb] Anisotropic data and an extremely long c axis
A first concern with that extreme anisotropy is at the integration and scaling stages. Large swaths of your detector are empty of reflections, but they will still bias the way reference profiles are calculated at integration; while the lots of reflections with intensities around 0 (but with significant sigmas) will bias the statistics for scaling the real reflections (real as in having intensity). You are better off excluding all the parts of the detector that do not contain significant intensities (and taking a hit in completeness) than trying to correct for all the 0 intensities. While the optimum would be to limit integration to an elliptical area of the detector containing the significant intensities, I am not aware of any program that can do this elegantly. In my experience, the strong data almost always end in the horizontal direction of the detector. And in many cases, the significant area while really elliptical, can be approximated to a rectangle, with the long edges horizontal. In that case, a simple trick is to use the excluded areas in the integration programs to limit integration to your rectangle. In Denzo I usually did this by playing with the detector distance, so that the diffraction ends at the edges of the detector, effectively setting the 2 short sides of our rectangle, and set the long edges of the rectangle with the excluded rectangle and excluded circle options (a circle of very large radius centered way out of the detector can be an excellent approximation to a straight line for cutting purposes). I am sure most programs will allow for equivalent ways of limiting integration to a rectangle. Once you have an mtz file with intensities, the UCLA anisotropy correction server can be a great help too. Marie Lacroix lacroix.ma...@rocketmail.com wrote: Hi, I also have a question concerning anisotropic data. Collected a data set and the best crystal gave highly anisotropic diffraction patterns ( 3.7 A - 5.8 A). So my first question is how to handle these data. I got only experience with normal data using the ccp4 suite. Are there any program specially for these kind of data? There are? The second question is how anisotropic data occur? The protein I work with has a tetragonal sg with a=b= 86.0 and an extremely long c axis of 651 A. Secondary Structure prediction suggest a lot of beta strands. How can I explain the anisotropy (for my own interest and my thesis)? Thank you very much. Marie -- *** Jose Antonio Cuesta-Seijo Biophysical Chemistry Group Department of Chemistry University of Copenhagen Tlf: +45-35320261 Universitetsparken 5 DK-2100 Copenhagen, Denmark ***
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] how to make cholesterol solution
An alternative could be to disolve your cholesterol in methanol or maybe in 50% methanol, then add it directly to the drop. Does your protein tolerate 10-20% methanol? Then you can add the methanol-cholesterol solution you your hanging drops directly and mix fast. The methanol is volatile and will end up mostly in the reservoir leaving onlt traces in the drop, while the cholesterol will stay in the water. It might precipitate at first, but should redissolve or not precipitate at all if it binds your protein. Jose Antonio Cuesta-Seijo. Jerry McCully for-crystallizai...@hotmail.com wrote: Dear ALL: Sorry for this kind of off-topic question. I am going to co-crystallize one protein with cholesterol. I read some papers saying that their protein can be pre-incubated with 1mM cholesterol in the presence of 5% (v/v) ethanol. TO do so, I first dissolved cholesterol in 100% ethanol at a concentration of 10mM. However, it is very difficult to make the dilution into 5% ethanol either just in water or some buffers. Does anyone have such experience to make cholesterol solution in normal buffers plus some ethanol? Thanks a lot, Jerry McCully _ The New Busy think 9 to 5 is a cute idea. Combine multiple calendars with Hotmail. http://www.windowslive.com/campaign/thenewbusy?tile=multicalendarocid=PID28326::T:WLMTAGL:ON:WL:en-US:WM_HMP:042010_5 -- *** Jose Antonio Cuesta-Seijo Biophysical Chemistry Group Department of Chemistry University of Copenhagen Tlf: +45-35320261 Universitetsparken 5 DK-2100 Copenhagen, Denmark ***
Re: [ccp4bb] iodine density
I am going to try to guess in this one. As you say, the positions of the iodines cannot be challenged. But maybe their occupancies can. Is there anomalous signal from iodine at your wavelenght? Are you expecting radiation damage? In any case: I see a spherical shell of low density around each one of the iodines. I think this is a fourier ripple, at 1.x times the resolution for each atom they seem to contribute negative electron density. Only that because the iodines have so many electrons, this ripple can compete with the normal possitive density of carbon atoms cancelling it out. From the online protein crystallography course from Cambridge at http://www-structmed.cimr.cam.ac.uk/Course/Convolution/convolution.html In addition, the Fourier transform of a sphere has ripples where it goes negative and then positive again, so a map computed with truncated data will also have Fourier ripples. These will be particularly strong around regions of high density, such as heavy atoms. Jose Antonio Cuesta-Seijo. Franck borel franck.bo...@ibs.fr wrote: Dear all, We have a structure with triiodothyroninne (T3 hormone) in it. The density around the ligand is very surprising. According to the omit map the iodine atoms are not anymore directly linked to the phenyl cycles (there is no more density between the iodine and the carbon). For one ligand an iodine seems now to be linked to the oxygen of the hydroxyl group (image 2a), a second one seems to be attach to the cycle through an extra atom (image 2b) and a third one seems to be too far (2.2A) to be covalently bound to the cycle. There is no reason for us to assume that such reactions may append. I would like to know if this could be something real or if it is an artefact due to the electronic properties of iodine, considering that - the data are good to a resolution of 1.75 A - the position of iodine atoms can not be challenged (I/sigma 20) Thanks for your help Franck BOREL INSTITUT DE BIOLOGIE STRUCTURALE Jean-Pierre Ebel UMR5075 CEA-CNRS-Univ. J. Fourier LCCP / Groupe Synchrotron 41, rue Jules Horowitz F-38027 Grenoble Cedex 1 - France Phone:33 (0)4 38 78 59 05 -- *** Jose Antonio Cuesta-Seijo Biophysical Chemistry Group Department of Chemistry University of Copenhagen Tlf: +45-35320261 Universitetsparken 5 DK-2100 Copenhagen, Denmark ***
Re: [ccp4bb] Vapor diffusion calculator
I would like to add that contrary to popular belief, MPD would have to be treated as a volatile in the example you give below. It vaporizes, although very slowly, as can be proven by leaving a 1uL drop of it in open air for a couple of days. Cheers, Jose Antonio Cuesta Seijo. Jacob Keller j-kell...@md.northwestern.edu wrote: Dear Crystallographers, Is anybody aware of a calculator for vapor diffusion experiments to plot concentrations of various solvent components as a function of time? For a simple example, what happens when I mix a protein solution containing 50mM NaCl 1:1 with a reservoir containing 50% MPD but no salt? Where is the vapor diffusion equilibrium, and how does the drop composition change as a function of time? More complicated would be experiments involving volatile components other than water, as I think, for example, ethanol would almost instantly equilibrate, then the water diffusion would kick in over a longer time scale. Even more complicated would be pH-dependent volatilities such as acetate. I don't think this would be impossible to figure out, but it would be nice if there were a pre-existing tool/server to do such. Regards, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** -- *** Jose Antonio Cuesta-Seijo Biophysical Chemistry Group Department of Chemistry University of Copenhagen Tlf: +45-35320261 Universitetsparken 5 DK-2100 Copenhagen, Denmark ***
[ccp4bb] What is an aceptable spread in ADP values?
Dear all, I am refining a 3.1Å structure with Phenix.refine, using two ADP groups per residue. When doing refinement in Refmac, the way to go would be to tighten the weights quite a bit to make up for the low resolution, resulting in small deviations in bond lenghts and angles from the ideal values and also in quite small ADP variations from atom to atom, typically in the range of, say, 5%. Now, doing the same refinement with Phenix.refine and 2 ADP groups per residue, the manual claims that the weights do not normally need to be touched. Judging by the values of R and R free (19.5% and 23.9%) compared to other protocols and with Refmac, it certainly does a good job. But the spread in ADP values in the refined model is strikingly high. Below is a extract from the pdb file. Note for example the jump from B=81.7 to B=163.8 for the main chains of ILE180 and LYS181, or the 163.8 to 113.5 between the main chain and the side chain of LYS181. Similar examples are all over the 1000+ residues in this structure. Is this normal? All global quality indicators look OK to me... Cheers, Jose Antonio Cuesta Seijo. ATOM 8338 N ARG F 178 65.398 30.884 -0.261 1.00 84.90 N ATOM 8339 CA ARG F 178 66.532 31.758 -0.521 1.00 84.90 C ATOM 8340 CB ARG F 178 67.576 31.628 0.583 1.00131.99 C ATOM 8341 CG ARG F 178 67.044 31.967 1.952 1.00131.99 C ATOM 8342 CD ARG F 178 68.084 32.695 2.778 1.00131.99 C ATOM 8343 NE ARG F 178 67.464 33.433 3.873 1.00131.99 N ATOM 8344 CZ ARG F 178 66.894 34.627 3.737 1.00131.99 C ATOM 8345 NH1 ARG F 178 66.865 35.222 2.551 1.00131.99 N ATOM 8346 NH2 ARG F 178 66.350 35.224 4.787 1.00131.99 N ATOM 8347 C ARG F 178 67.152 31.347 -1.839 1.00 84.90 C ATOM 8348 O ARG F 178 66.738 30.357 -2.443 1.00 84.90 O ATOM 8349 N ILE F 179 68.143 32.107 -2.287 1.00 80.99 N ATOM 8350 CA ILE F 179 68.954 31.666 -3.406 1.00 80.99 C ATOM 8351 CB ILE F 179 69.698 32.817 -4.083 1.00 53.62 C ATOM 8352 CG1 ILE F 179 68.754 33.995 -4.333 1.00 53.62 C ATOM 8353 CD1 ILE F 179 68.390 34.216 -5.806 1.00 53.62 C ATOM 8354 CG2 ILE F 179 70.308 32.325 -5.389 1.00 53.62 C ATOM 8355 C ILE F 179 69.970 30.657 -2.884 1.00 80.99 C ATOM 8356 O ILE F 179 70.677 30.914 -1.913 1.00 80.99 O ATOM 8357 N ILE F 180 70.026 29.500 -3.528 1.00 81.67 N ATOM 8358 CA ILE F 180 70.864 28.417 -3.061 1.00 81.67 C ATOM 8359 CB ILE F 180 70.161 27.066 -3.218 1.00 71.36 C ATOM 8360 CG1 ILE F 180 68.850 27.090 -2.446 1.00 71.36 C ATOM 8361 CD1 ILE F 180 68.964 27.820 -1.114 1.00 71.36 C ATOM 8362 CG2 ILE F 180 71.038 25.955 -2.707 1.00 71.36 C ATOM 8363 C ILE F 180 72.180 28.418 -3.811 1.00 81.67 C ATOM 8364 O ILE F 180 73.218 28.106 -3.240 1.00 81.67 O ATOM 8365 N LYS F 181 72.137 28.768 -5.092 1.00163.76 N ATOM 8366 CA LYS F 181 73.356 28.961 -5.872 1.00163.76 C ATOM 8367 CB LYS F 181 74.053 27.627 -6.155 1.00113.58 C ATOM 8368 CG LYS F 181 73.432 26.812 -7.280 1.00113.58 C ATOM 8369 CD LYS F 181 74.307 25.615 -7.656 1.00113.58 C ATOM 8370 CE LYS F 181 74.291 24.546 -6.571 1.00113.58 C ATOM 8371 NZ LYS F 181 75.015 23.306 -6.984 1.00113.58 N ATOM 8372 C LYS F 181 73.033 29.687 -7.172 1.00163.76 C ATOM 8373 O LYS F 181 71.889 29.673 -7.629 1.00163.76 O ATOM 8374 N LYS F 182 74.039 30.325 -7.760 1.00126.44 N ATOM 8375 CA LYS F 182 73.849 31.088 -8.988 1.00126.44 C ATOM 8376 CB LYS F 182 74.588 32.421 -8.897 1.00131.41 C ATOM 8377 CG LYS F 182 74.235 33.203 -7.652 1.00131.41 C ATOM 8378 CD LYS F 182 74.885 34.575 -7.634 1.00131.41 C ATOM 8379 CE LYS F 182 74.466 35.367 -6.393 1.00131.41 C ATOM 8380 NZ LYS F 182 75.076 36.730 -6.349 1.00131.41 N ATOM 8381 C LYS F 182 74.338 30.303 -10.192 1.00126.44 C ATOM 8382 O LYS F 182 75.506 29.930 -10.252 1.00126.44 O ATOM 8383 N LEU F 183 73.447 30.052 -11.146 1.00161.61 N ATOM 8384 CA LEU F 183 73.819 29.331 -12.360 1.00161.61 C ATOM 8385 CB LEU F 183 72.591 29.005 -13.214 1.00100.55 C ATOM 8386 CG LEU F 183 71.470 28.218 -12.534 1.00100.55 C ATOM
Re: [ccp4bb] Sigma Cutoff in HKL2000 Data Processing
I guess you mean higher redundancy/completeness in the HIGHER resolution shells. Rather than decreasing that sigma cutoff, a better solution is to increase the profile fitting radius. The default value is fine for most lab-based detectors, but too small for most (larger) synchrotron detectors. Making that value larger allows reflections further away to be included in the profile fitting, rather than including weaker reflections. In my hands this has often changed the completeness of the high resolution shell from 30% to 90% as weak reflections will no longer be ignored. There is a button in one of the graphic windows to show the profile fitting radius, it will show a circle that follows the mouse pointer around. The profile fitting radius should be made large enough that everywhere (within your resolution limit) you go, 5-10 strong reflections will be inside that circle. Jose. Ed Pozharski epozh...@umaryland.edu wrote: On Thu, 2009-10-22 at 10:18 -0400, protein.chemist protein.chemist wrote: What is the Sigma Cutoff that one should use for Data Processing using HKL2000. Since you say HKL2000, I assume that you mean the Refinement Sigma Cutoff in index tab. The parameter, imu, determines which reflections will be considered strong and used in parameter refinement and (?) profile fitting. The default value is 5.0, which is just fine for good data. I do, however, routinely set it to lower value of 3.0, since it was my observation that then you get higher redundancy/completeness in lower resolution shells. There is some evidence that the mechanism here is related to rejections due to incomplete profiles. Obviously, as you reach the outer rim, strong reflections become sparse and if you are also using the relatively small default value of the profile fitting radius, large number of reflections may be rejected because denzo can't calculate average profiles in their vicinity (I expected that in the absence of the profile the integrated intensity should be used instead, but perhaps it's not the case). Is there a minimum or maximum value. I'd say it makes no sense to go below 1.0, but you can sure try and see what happens. Upper limit is obviously defined by the point where you don't have enough strong reflections for robust refinement of parameters. The absolute values will, of course, vary from dataset to dataset. -- -- *** Jose Antonio Cuesta-Seijo Biophysical Chemistry Group Department of Chemistry University of Copenhagen Tlf: +45-35320261 Universitetsparken 5 DK-2100 Copenhagen, Denmark ***
Re: [ccp4bb] Halide soaking
Hi! Normally the cell parameters, etc change very very little. You'll only know if the bromides got in at the synchrotron by looking at the fluorescence spectrum and at the anomalous signal. Normally some will make it in and some will be in ordered sites, then it becomes mostly a question of data quality to detect it. You could also try the equivalent iodide soak. Iodine has a decent anomalous signal at the copper wavenght and thus the anomalous signal can be detected at your home source and many times the structure can be solved by SAD or SIRAS. I would also thing that conditions that give ordered iodide sites are likely to result in ordered bromide sites, although the ions are not identical. Jose. ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRS TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, ON, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: jcue...@uhnres.utoronto.ca ** On Mar 31, 2009, at 12:19 PM, tat cheung cheng wrote: Hi all I am now trying to do bromide soaking, but i am not really sure does the bromide atom enter my crystal. So is there any signs that indicate the entry of bromide atom? e.g. does the space group, cell dimension change? or just nothing change, and the bromide atom just get in? Thanks very much. T.C. Cheng Yahoo!香港提供網上安全攻略，教你如何防範黑客! 請前往 http:// hk.promo.yahoo.com/security/ 了解更多!
[ccp4bb] sparse matrix screen design
Hi all, a non-CCP4 question: Are there any good free programs for the design of one's own sparse matrix screens? I am looking for something in the lines of a set of mixtures of what gives you the best coverage of, say, seven variables with five states each in 96 experiments. Thanks, Jose. ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRS TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, ON, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: jcue...@uhnres.utoronto.ca **
Re: [ccp4bb] AW: [ccp4bb] suggestions for UV spectrometer
I also have to come in defense of the nanodrop here. I have measured up to A280 = 98 and the curve is always reasonably smooth, spikes normally mean that bubbles have formed. And proper cleaning seems to be rubbing with a kimwipe three or four times after each drop. If the last user does not clean after the last measurement, then things will dry up in the pedestals and that can of course be a problem. Also bubbles do not form if you are careful. I always use 2uL, 1uL can be too little, and even with 20% detergent I get a column and no bubbles nearly every time. You have to pipet carefully and lower the lever slowly (think of a vinyl record here). For tricky samples, use 2.2uL instead. A bubble might still form occasionally, but looking at the spectrum will quickly tell you that something went wrong. Cheers, Jose. ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRS TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, ON, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: [EMAIL PROTECTED] ** On Dec 5, 2008, at 7:00 PM, wangsa tirta ismaya wrote: Dear all, Thanks a lot for raising the issue with the not reproducibility of protein measurement with Nanodrop. We use the instrument as a workhorse in the lab. Indeed, recently I observed that the protein concentration suggested by Nanodrop is sometimes differ to the usual colorimetric measurement (Bradford method, measured with our Pharmacia's Ultrospec 2000 spectrometer). Since the cell in Nanodrop is very small, could it be due to the homogeneity of the sample in the cell? Also what I have observed, we have to be sure that the cell is cleaned properly before use. Another thing is, at high protein concentration I obtained noisy absorption curve at the peak (like a seismograf ) thus the protein concentration measured is doubtful, I have to dilute the sample to have good curve (thank God it requires only 2 mikroliter for a measurement). Well, I think nanodrop is a good, fast, and powerful instrument, however it would be better if we established a reference of our daily practice to a normal spectrometer measurement. cheers, Wangsa 2008/12/5 Martin Hallberg [EMAIL PROTECTED] Which brings us back to the Hellma TrayCell solution where you can, from the same spectrometer, have both the cuvette option and the quickness of the NanoDrop/NanoVue system. Anyone that can comment on the performance of the TrayCell from Hellma? Cheers, Martin On Dec 5, 2008, at 9:06 AM, Gregor Witte wrote: Agree! I think for crystallographic use the nanodrop is perfectly okay to see if the protein is 5mg/ml or 30mg/ml. But in fact I also do not trust our instrument if it comes to more important issues like preparing solutions for titrations or assays. And due to the small pathlength I do not trust absorptions of small concentrated samples at all. I always prefer a real 2-beam spectrophotometer (monochromators) with a quarz-cuvette and a nice pathlength. Of course, you cannot reliably measure solutions exceeding Abs 1 or maybe 1.5 OD in a spectrophotometer with 1cm pathlength. There's also one quite strange thing about the nanodrop – they sell the calibration check solution (which is some kind of yellow chromate-solution with known absorption), then you check your nanodrop with it and maybe find out that it's off to some certain extend: But then you're stuck(!), because you cannot calibrate it on your own. I guess it would be quite easy to integrate a calibration-option into the software, but at the moment the instrument tells you calibration failure and you have to call the service guys who then carry it home and calibrate it by turning of the two small screws at the top of it and then glue them with locktite. Anyway, at least for our mid to high concentrated samples the nanodrop is not showing large fluctuations so we are happy with it. But everyone using a nanodrop should check it from time to time – as I found out that ours was off more than 20% at one day - which raised some trouble of course… Cheers, Gregor Von: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Im Auftrag von Filip Van Petegem Gesendet: Donnerstag, 4. Dezember 2008 22:20 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] suggestions for UV spectrometer I want to add I absotely hate the nanodrop. We've had a demo for it, and found the readouts to be very unreliable. Fluctuations of 20% and more. Just leaving the same drop in and measuring the sample multiple times gives different values (going in both directions, so not only due to evaportations). Sure, it's easy and fast, and maybe good to have a rough idea about your protein concentration, but I would never want to use it for exact measurements such as needed for e.g. a CD or an ITC instrument. I've heard
Re: [ccp4bb] PST in refinement
Dear Maruf, I had a similar case, also to 2.4A. Those translations are often caused by a non-crystallographic 2-fold rotation which is parallel to the crystallographic 2-fold (there is an example in ruppweb as to why that creates the translation and how to interpret it: http:// www.ruppweb.org/Xray/101index.html under NCS with native Patterson maps). In your particular case, though, have you considered wether your absences match the strong-weak pattern created by your non- crystallographic translation? In that case maybe you don't have a 21 axis and your space group is P2 or C2. If your space group is right, the non crystallographic rotation will dominate your molecular replacement and your refinement and make it difficult to spot good solutions from bad ones, since as long as MR solutions recreate the non-crystallographic rotation (the axis, not necessarily the correct position of your molecules around that rotation axis), they will recreate the translation peak. I even got a series of totally different MR solutions, all of them with beautiful packing! For me it turned out that I had the correct MR solution for months, but the refinement was stalling. Running refmac for 200 cycles (not less!) with very very tight geometric restraints restraints produced a distribution of B-factors that pointed at which regions were right (low Bs) and which wrong (high Bs). Removal of the regions with high Bs followed by further refinement immediately produced good maps, showing the new regions, and refinement proceeded normally from there. Later on I could move back to normal weights, too. Only after cropping the bad regions will the difference maps really show anything (assuming the rest is right, of course). Just based on one case, but your case is s similar to mine! Good luck, Jose Antonio Cuesta-Seijo. ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRS TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, ON, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: [EMAIL PROTECTED] ** On Jul 14, 2008, at 8:42 AM, Maruf Ali wrote: Dear all I have recently collected several datasets on different crystals of a particular protein with a resolution range form 2.4 - 3.2A. All datasets seem to process well in p21 with a unit cell of 109.6 83.1 115.87 90 94.8 90, and this space group is further supported by analysis with the program pointless. The dataset have very reasonable statistics and Rmerge values, with no indication of twinning. Analysis of the self patterson indicated a 43% off origin peak at 0.3 0.5 0.47. This was further flagged by pointless and molrep as a Psuedo cell translation (PST). Looking at the systematic absences there are some unusually strong and weak peaks. Initially after some toiling with molecular replacement, there was a clear solution with four molecules in the asymmetric unit. The maps generated were good enough to build the core of the protein but do not look like maps generated from data at 2.4A-3.2. Further more the free R is stuck at around 40%, and there is no difference in free R when I apply ncs or not or any difference in the maps. After building by hand and with phenix autobuild there is still no difference in maps and Rfree. I have read papers where labs have successfully refined PST data by separating the reflections according to the PST. Oksanen et al 2006 acta D62 1369-1374: Poy et al 2001 NSMB Vol 8 no12 pg 1053: VajDos et al protein science 1997 6;2297 My specific question are Firstly how would I deal with refining PST data? (assuming this is the problem). Second with off origin peak of 0.3 0.5 0.47 how would I separate the reflections? Thirdly any comments would be valued Thank you in advance Maruf Dr Maruf Ali Section of Structural Biology, Institute of Cancer Research, 237 Fulham road, London. SW3 6JB The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network.
[ccp4bb] Binding to Nickel in the presence of SDS or arginine
Dear CCP4BBers, One of those questions regarding purification rather than crystallography: Reading the Qiagen manual for the Ni-NTA matrices, in the table of compatibility of reagents with Ni-NTA matrices, SDS is mentioned (only together with sarkosyl) as Not recommended, but up to 0.3% has been used successfully in some cases. Google is failing me to find those cases, since pretty much every paper mentioning Ni binding also mentions SDS-PAGE. Also, arginine is mentioned just as not recommended. Why are SDS and arginine not recommended, what are the physical and chemical underlying problems? Can they be used at low concentration without damaging the matrix or abolishing binding? Which are the maximal concentrations people had success with? Thanks a lot, Jose Antonio Cuesta Seijo ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRS TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, ON, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: [EMAIL PROTECTED] **
[ccp4bb] Crystallization by pH changes
Hi all! Does anybody have experience with crystallization of proteins by slow drifting of the pH? Let's say, for example, going from pH 7 to pH 5 over 1 week or 1 month. This is supposed to happen by diffusion of volatile molecules like ammonia or acetic acid but I am having a hard time finding any details or protocols. I bet that there is a lot of anecdotal experience out there. Maybe even references? Thanks, Jose. ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRS TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, ON, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: [EMAIL PROTECTED] **
[ccp4bb] Tweaking with SCBUlk and BBULk parameters in refmac
Dear All, I have a structure to 1.65A crystallized in high sulphate conditions. The R factors went smoothly to 21% and 23% (Rfree), but the difference map shows positive difference peaks in otherwise normal regions of the main chain and is more noisy than expected in general. Since the electron density in the solvent is much higher than that of water (approx. 2M sulphate) I suspected of the solvent scaling. Tweaking with SCBUlk and BBULk produced indeed a 0.5% improvement in R and a 0.3% improvement in Rfree. I have been doing this from the ccp4i interface by changing the command line through the run and view com file to scal - type BULK - reso 1.648 20.0 - LSSC FIXB SCBU 0.0 BFIX 200.0 - (those are the numbers tweaked with) ANISO I am running refmac version 5.2.2005 The SCBUlk parameter has a minimum for both R and Rfree at 0.0. Variations of BBULk from 200.0 at that SCBUlk of 0.0 had no effect on the R factors up to the 6th digit and I believe the refinement is exactly the same. Changing BBULk at SCBUlk values different from 0.0 does have an effect on R and Rfree. Also, the R factors behave symmetrically relative to SCBUlk, that is, SCBUlk 0.2 produces the same results as SCBULK -0.2 What is the excat meaning/use of those parameters? How are the experts going around with optimizing these? Thanks, Jose Antonio Cuesta Seijo. ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRS TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, On, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: [EMAIL PROTECTED] **
Re: [ccp4bb] advice for crystallizing hydrophobic small molecules
Hello Todd. An experienced synthetic chemist is your best source of help here, but otherwise have a look at these links. And greasy molecules are no problem. Organic chemists crystalize them like candy! http://www.xray.ncsu.edu/GrowXtal.html http://web.mit.edu/x-ray/cystallize.html http://64.233.167.104/search?q=cache:jT4WIhISjqwJ:chem-tech.ucsd.edu/ Recharges/SMXF/multimedia/xtal%2520growing%2520summer%2520-% 2520Richard%2520Staples.ppt+crystallization+small+molecule+evaporation +diffusionhl=enct=clnkcd=8 http://depts.washington.edu/eooptic/linkfiles/ Crystallisation_Techniques.doc Good luck, Jose. ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRs TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, On, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: [EMAIL PROTECTED] ** On Jul 18, 2007, at 5:56 PM, Green, Todd wrote: Hello all, I am asking this question for a colleague(a chemist not a crystallographer) who would like to crystallize a small molecule (for clarification this is just the small molecule not a protein complex). The compound is quite hydrophobic and is rather greasy. He has a free alcohol which could be a site of modification if this would help. I have only worked with proteins and was hoping that someone might be knowledgible and could point me in the direction of some help(literature, websites, etc) that might aide as a ground level tutorial on crystallization of small molecules, and if possible more specifically crystallization of hydrophobic/greasy small molecules. Thanks in advance- Todd Green University of Alabama at Birmingham
Re: [ccp4bb] advice for crystallizing hydrophobic small molecules
And here some more small molecule crystallization links. http://xray.chem.ufl.edu/growing%20tips.htm http://mic.ucla.edu/x-ray%20diffraction/tutorials_crystalgrowing.htm http://www.nottingham.ac.uk/~pczajb2/growcrys.htm Jose Antonio Cuesta-Seijo On Jul 18, 2007, at 5:56 PM, Green, Todd wrote: Hello all, I am asking this question for a colleague(a chemist not a crystallographer) who would like to crystallize a small molecule (for clarification this is just the small molecule not a protein complex). The compound is quite hydrophobic and is rather greasy. He has a free alcohol which could be a site of modification if this would help. I have only worked with proteins and was hoping that someone might be knowledgible and could point me in the direction of some help(literature, websites, etc) that might aide as a ground level tutorial on crystallization of small molecules, and if possible more specifically crystallization of hydrophobic/greasy small molecules. Thanks in advance- Todd Green University of Alabama at Birmingham
Re: [ccp4bb] Highest shell standards
I have observed something similar myself using Saint in a Bruker Smart6K detector and using denzo in lab and syncrotron detectors. First the I over sigma never really drops to zero, no mater how much over your real resolution limit you integrate. Second, if I integrate to the visual resolution limit of, say, 1.5A, I get nice dataset statistics. If I now re-integrate (and re-scale) to 1.2A, thus including mostly empty (background) pixels everywhere, then cut the dataset after scaling to the same 1.5A limit, the statistics are much worse, booth in I over sigma and Rint. (Sorry, no numbers here, I tried this sometime ago). I guess the integration is suffering at profile fitting level while the scaling suffers from general noise (those weak reflections between 1.5A and 1.2A will be half of your total data!). I would be careful to go much over the visual resolution limit. Jose. ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRs TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, On, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: [EMAIL PROTECTED] ** On Mar 22, 2007, at 10:59 AM, Sue Roberts wrote: I have a question about how the experimental sigmas are affected when one includes resolution shells containing mostly unobserved reflections. Does this vary with the data reduction software being used? One thing I've noticed when scaling data (this with d*trek (Crystal Clear) since it's the program I use most) is that I/sigma(I) of reflections can change significantly when one changes the high resolution cutoff. If I set the detector so that the edge is about where I stop seeing reflections and integrate to the corner of the detector, I'll get a dataset where I/sigma(I) is really compressed - there is a lot of high resolution data with I/sigma(I) about 1, but for the lowest resolution shell, the overall I/sigma(I) will be maybe 8-9. If the data set is cutoff at a lower resolution (where I/sigma(I) in the shell is about 2) and scaled, I/sigma(I) in the lowest resolution shell will be maybe 20 or even higher (OK, there is a different resolution cutoff for this shell, but if I look at individual reflections, the trend holds). Since the maximum likelihood refinements use sigmas for weighting this must affect the refinement. My experience is that interpretation of the maps is easier when the cut-off datasets are used. (Refinement is via refmac5 or shelx). Also, I'm mostly talking about datasets from well-diffracting crystals (better than 2 A). Sue On Mar 22, 2007, at 2:29 AM, Eleanor Dodson wrote: I feel that is rather severe for ML refinement - sometimes for instance it helps to use all the data from the images, integrating right into the corners, thus getting a very incomplete set for the highest resolution shell. But for exptl phasing it does not help to have many many weak reflections.. Is there any way of testing this though? Only way I can think of to refine against a poorer set with varying protocols, then improve crystals/data and see which protocol for the poorer data gave the best agreement for the model comparison? And even that is not decisive - presumably the data would have come from different crystals with maybe small diffs between the models.. Eleanor Shane Atwell wrote: Could someone point me to some standards for data quality, especially for publishing structures? I'm wondering in particular about highest shell completeness, multiplicity, sigma and Rmerge. A co-worker pointed me to a '97 article by Kleywegt and Jones: _http://xray.bmc.uu.se/gerard/gmrp/gmrp.html_ To decide at which shell to cut off the resolution, we nowadays tend to use the following criteria for the highest shell: completeness 80 %, multiplicity 2, more than 60 % of the reflections with I 3 sigma(I), and Rmerge 40 %. In our opinion, it is better to have a good 1.8 Å structure, than a poor 1.637 Å structure. Are these recommendations still valid with maximum likelihood methods? We tend to use more data, especially in terms of the Rmerge and sigma cuttoff. Thanks in advance, *Shane Atwell* Sue Roberts Biochemistry Biopphysics University of Arizona [EMAIL PROTECTED]
