Re: [ccp4bb] Low resolution refinement
Dear Joane, we had a case, where we had five molecules in the assymmetric unit where the biological functional unit was a homotrimer. So we had one non-crystallographic trimer and two monomers, which were located along the 3-fold symmetry axis of space group I213. One of the monomers also showed electron density of considerably lower quality, obviously going along with higher B-factors. By a careful analysis of the crystal packing we could see that this chain has only very few crystal contacts. If you want to have a closer look, see: Schuldt L, Weyand S, Kefala G, Weiss MS J. Mol. Biol. (2009), 863-879. The Section Crystal Packing and structural variation describes this in more detail. Best wishes, Linda Joane Kathelen Rustiguel schrieb: Dear all I am refining a structure at 3.4 A resolution that contains 3 molecules in the a.u. The chain A sits on a 2-fold crystallographic axis forming the dimeric functional structure expected for this class of proteins. The other two chains B and C, which also form the functional dimer, seem to be, somehow, a lot more flexible than chain A. As a result, whereas the electron density map, b-factor and geometry for chain A is pretty reasonable for a 3.4 A resolution structure, the refinement for the other two chains (B and C) does not behave well. Even playing with different weights for geometry, analysing different levels of 2Fo-Fc/Fo-Fc maps, using NCS, TLS, etc..., nothing works. The map for the helical regions is ok, but the electron density map for strands and loops of chains B and C are broken along the main chain, B-factors are really high, and the geometry keeps being distorted. Right now, the R-factor and R-free are 24.2 and 28.6, respectively. Any suggestions in how to proceed the refinement? And even a more difficult question, how do we report this type of structure? How do we deposit those coordinates? We can certainly use chain A as a model to perform interesting studies of structure-function relationship, but we know that chain B and chain C have problems. Any help will be greatly appreciated. Regards Joane -- Joane Kathelen Rustiguel Bonalumi Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP Laboratório de Cristalografia de Proteínas Departamento de Física e Química Fone: +55.16.3602.4193 *** Dr. Linda Schuldt Department of Molecular Biology University of Aarhus Science Park Gustav Wieds Vej 10c DK-8000 Århus C Denmark
[ccp4bb] About protein stability
Dear All, I am working on enzyme which is expressed in Mycobacterium smegmatis. After affinity chromatography and gel filtration, it is observed that enzyme stays in dimeric form for 3hour approx. by the time it goes oligomeric form. i want it to stay in dimeric form to get homogeneity of solution to set up crystallization. Required experts suggestions. Very thankful to you in advance *Elution Buffer of affinity chromatography* 50mm Phosphate pH7.4 200mm NaCl 1mM DTT 300 immidazole *Gel filtration chromatography buffer* 20Mm phosphate pH7.4 100mM Nacl 2mM DTT -- Yogesh Khandokar SRF Protein Crystallography Lab., National Institute of Immunology, New Delhi. India
[ccp4bb] crystallization of a weird protein
Dear All I have a difficulty to crystallize my membrane protein(his tagged). I got the salt crystals from many different screening conditions. The protein is in PBS buffer and 130mM salt with 0.1% SDS. I store It in the 4 degree although it forms the milk solution. The solution is getting clear in the room temperature within 5 mins when I set up the trays. I would like to take any advice. I would appreciate your input. Best Min
Re: [ccp4bb] crystallization of a weird protein
The milk solution is a suspension of lots of micro-crystals which form when temperature goes down. When you take the solution out, it worms up and the crystals dissolve. If you take a look at them under high magnification microscope in the cold room, chances are you'll see those crystals. I cannot give any recommendations on chemical makeup of your solution but you could try using the temperature gradient as the driving force for crystallization. Good luck, Nukri Ruslan Sanishvili (Nukri), Ph.D. GM/CA-CAT Biosciences Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 rsanishv...@anl.gov From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of WEI MIN Sent: Friday, May 20, 2011 9:36 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystallization of a weird protein Dear All I have a difficulty to crystallize my membrane protein(his tagged). I got the salt crystals from many different screening conditions. The protein is in PBS buffer and 130mM salt with 0.1% SDS. I store It in the 4 degree although it forms the milk solution. The solution is getting clear in the room temperature within 5 mins when I set up the trays. I would like to take any advice. I would appreciate your input. Best Min
Re: [ccp4bb] Twinning, Wilson scaling and B factor
Thanks Ian, I tried to do this: I took the file containing hkl I and sigI and generated a new file containing hkl I/2 and sigI because I know, from the refined structure that the twin fraction is nearly 0.5. Now, using this new file the wilson plot give me a more reliable estimated B factor. Do you think this procedure was correct? Fulvio Il giorno gio, 19/05/2011 alle 14.14 +0100, Ian Tickle ha scritto: Hi Fulvio There are 2 different issues here: the Wilson plot scale B factor on the one hand and Wilson statistics on the other. The first are not affected by twinning since they depend only on the intensity averages in shells. The second refers to the distribution of intensities (i.e. the proportion of reflections with intensity less than a specified value) within a shell, or to the distribution of normalised intensities (Z = I/I ignoring symmetry issues for now) over the whole dataset. This distribution is different for a twin because averaging the components which contribute to the intensity of a twinned reflection tends to shift the distribution towards the mean, so you get fewer extreme values. The Wilson B factor is not a 'statistic' in the strict sense, merely a derived parameter. I suspect the low value you get has more to do with the fact that the resolution is only 3 A, than the fact it's twinned. See here for more mathematically-oriented info: http://www.ccp4.ac.uk/dist/html/pxmaths/bmg10.html Cheers -- Ian On Thu, May 19, 2011 at 1:45 PM, fulvio saccoccia fulvio.saccoc...@uniroma1.it wrote: Dear ccp4 users, I have a data set arising from a nearly-perfect pseudo-merohedrally twinned cystal, diffracting up to 3 A. I solved the structure and ready for deposition, but there is still a trouble. The Wilson scaling from raw data gave a B of 3A^2. Initially, I did not seemed too alarming. But I do not know why I have these statistics. Does anyone know why Wilson scaling falls when treating that kind of twinned data? I read that twinned data do not obey twe Wilson statistics but I don't know why. Here the presentation I read: http://bstr521.biostr.washington.edu/PDF/Twinning_2007.pdf Do you know any articles, reviews or book in which this particular aspect of of twinned data is treated in depth, possibly in mathematical manner? Thanks to all Fulvio Saccoccia, PhD student Biochemical Sciences Dept. Sapienza University of Rome
Re: [ccp4bb] crystallization of a weird protein
Hi Wei, this milk is precipitated/microcrystals of SDS probably with some phosphate since you are in PBS buffer. I would have two suggestions. 1-Can't you find a better detergent than SDS for your membrane protein? Have you run a detergent screen for this protein to find a milder and more crystallization friendly detergent for the reconstiution/purification and handling of your sample. SDS is very very rarely used for membrane protein crystallization. 2- I would try to avoid preparing a protein for crystallization in a phosphate containing buffer (unless you have no choice). Phosphates tend to yield more salt crystals at the screening stage. Hope this helps. -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 356 Boyer Hall office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] crystallization of a weird protein
It might be SDS precipitation. Although 0.1% SDS is generally considered not high enough to precipitate at 4 degree, it might interact with other components in your solution to form even less soluble material. Nian On Fri, May 20, 2011 at 9:36 AM, WEI MIN butiany...@gmail.com wrote: Dear All I have a difficulty to crystallize my membrane protein(his tagged). I got the salt crystals from many different screening conditions. The protein is in PBS buffer and 130mM salt with 0.1% SDS. I store It in the 4 degree although it forms the milk solution. The solution is getting clear in the room temperature within 5 mins when I set up the trays. I would like to take any advice. I would appreciate your input. Best Min
Re: [ccp4bb] Twinning, Wilson scaling and B factor
No, simply applying a single overall scale factor to the intensities can't possibly make any difference to the Wilson B since the fall-off with resolution will remain unchanged. The Wilson plot is a plot of ln(mean(I')/S) in shells of constant d* vs d*^2, where I' is I corrected for symmetry and S is a function of the scattering factors for the known unit cell content. Changing the overall scale factor shifts the plot up or down but doesn't change the gradient, and the Wilson B factor depends on the gradient (actually B = -2*gradient). In any case detwinning is impossible if as you say the twin fraction is near 0.5. Your procedure doesn't perform detwinning. For example, suppose the true intensities of the components of the twin are (say) 90 and 110. For tf = 0.5 you will observe the mean value (i.e. half from each component), so I(twin) = 100. Taking I(twin)/2 = 50 doesn't give you back the true intensity (in fact in this case I(twin) is actually a better estimate of I(true)); in any case any attempt at detwinning must give you 2 values, one for each component of the twin. Cheers -- Ian On Fri, May 20, 2011 at 3:43 PM, fulvio saccoccia fulvio.saccoc...@uniroma1.it wrote: Thanks Ian, I tried to do this: I took the file containing hkl I and sigI and generated a new file containing hkl I/2 and sigI because I know, from the refined structure that the twin fraction is nearly 0.5. Now, using this new file the wilson plot give me a more reliable estimated B factor. Do you think this procedure was correct? Fulvio Il giorno gio, 19/05/2011 alle 14.14 +0100, Ian Tickle ha scritto: Hi Fulvio There are 2 different issues here: the Wilson plot scale B factor on the one hand and Wilson statistics on the other. The first are not affected by twinning since they depend only on the intensity averages in shells. The second refers to the distribution of intensities (i.e. the proportion of reflections with intensity less than a specified value) within a shell, or to the distribution of normalised intensities (Z = I/I ignoring symmetry issues for now) over the whole dataset. This distribution is different for a twin because averaging the components which contribute to the intensity of a twinned reflection tends to shift the distribution towards the mean, so you get fewer extreme values. The Wilson B factor is not a 'statistic' in the strict sense, merely a derived parameter. I suspect the low value you get has more to do with the fact that the resolution is only 3 A, than the fact it's twinned. See here for more mathematically-oriented info: http://www.ccp4.ac.uk/dist/html/pxmaths/bmg10.html Cheers -- Ian On Thu, May 19, 2011 at 1:45 PM, fulvio saccoccia fulvio.saccoc...@uniroma1.it wrote: Dear ccp4 users, I have a data set arising from a nearly-perfect pseudo-merohedrally twinned cystal, diffracting up to 3 A. I solved the structure and ready for deposition, but there is still a trouble. The Wilson scaling from raw data gave a B of 3A^2. Initially, I did not seemed too alarming. But I do not know why I have these statistics. Does anyone know why Wilson scaling falls when treating that kind of twinned data? I read that twinned data do not obey twe Wilson statistics but I don't know why. Here the presentation I read: http://bstr521.biostr.washington.edu/PDF/Twinning_2007.pdf Do you know any articles, reviews or book in which this particular aspect of of twinned data is treated in depth, possibly in mathematical manner? Thanks to all Fulvio Saccoccia, PhD student Biochemical Sciences Dept. Sapienza University of Rome
Re: [ccp4bb] Twinning, Wilson scaling and B factor
Thanks Ian, but your reply confused me a little. I hope you can explain me where I was wrong. I know that I(twin)=tf*I(h1)+(1-tf)*I(h2) I supposed that having tf=0.5 I could take the I(twin), dividing by 2 I will get both I(h1) and I(h2), that are the two component (that are equal in this case). Rather I thought that a possible mistake could be the sigI associated to every intensities ( and I don't know how I can take it into account for Wilson B). Just to tell you and review the procedure I followed: I took the .sca, I operated in order to halve the Intensities column (I used octave to calculate them), saved the new file in .txt and than I applied label FP and SIGFP using F2mtz (ccp4i). After this, I run wilson (ccp4) within 30-3,0 A resolution and obtain a more reliable B factor with respect that obtained from raw data that was of 3A^2. Next, I tried changing the resolution 30-4.5 and 30-4.4 and the results are all similar (28, 31 and 38 A^2). The SCALE were 186 204 and 194 and I considered them quite similar one to another. I did not made this procedure in order to detwin data just to understand how play with raw data affected by perfect twin and to clarify me how these data affect statistics. Thank you for your attention and for all the good advice. Cheers Fulvio Il giorno ven, 20/05/2011 alle 16.26 +0100, Ian Tickle ha scritto: No, simply applying a single overall scale factor to the intensities can't possibly make any difference to the Wilson B since the fall-off with resolution will remain unchanged. The Wilson plot is a plot of ln(mean(I')/S) in shells of constant d* vs d*^2, where I' is I corrected for symmetry and S is a function of the scattering factors for the known unit cell content. Changing the overall scale factor shifts the plot up or down but doesn't change the gradient, and the Wilson B factor depends on the gradient (actually B = -2*gradient). In any case detwinning is impossible if as you say the twin fraction is near 0.5. Your procedure doesn't perform detwinning. For example, suppose the true intensities of the components of the twin are (say) 90 and 110. For tf = 0.5 you will observe the mean value (i.e. half from each component), so I(twin) = 100. Taking I(twin)/2 = 50 doesn't give you back the true intensity (in fact in this case I(twin) is actually a better estimate of I(true)); in any case any attempt at detwinning must give you 2 values, one for each component of the twin. Cheers -- Ian On Fri, May 20, 2011 at 3:43 PM, fulvio saccoccia fulvio.saccoc...@uniroma1.it wrote: Thanks Ian, I tried to do this: I took the file containing hkl I and sigI and generated a new file containing hkl I/2 and sigI because I know, from the refined structure that the twin fraction is nearly 0.5. Now, using this new file the wilson plot give me a more reliable estimated B factor. Do you think this procedure was correct? Fulvio Il giorno gio, 19/05/2011 alle 14.14 +0100, Ian Tickle ha scritto: Hi Fulvio There are 2 different issues here: the Wilson plot scale B factor on the one hand and Wilson statistics on the other. The first are not affected by twinning since they depend only on the intensity averages in shells. The second refers to the distribution of intensities (i.e. the proportion of reflections with intensity less than a specified value) within a shell, or to the distribution of normalised intensities (Z = I/I ignoring symmetry issues for now) over the whole dataset. This distribution is different for a twin because averaging the components which contribute to the intensity of a twinned reflection tends to shift the distribution towards the mean, so you get fewer extreme values. The Wilson B factor is not a 'statistic' in the strict sense, merely a derived parameter. I suspect the low value you get has more to do with the fact that the resolution is only 3 A, than the fact it's twinned. See here for more mathematically-oriented info: http://www.ccp4.ac.uk/dist/html/pxmaths/bmg10.html Cheers -- Ian On Thu, May 19, 2011 at 1:45 PM, fulvio saccoccia fulvio.saccoc...@uniroma1.it wrote: Dear ccp4 users, I have a data set arising from a nearly-perfect pseudo-merohedrally twinned cystal, diffracting up to 3 A. I solved the structure and ready for deposition, but there is still a trouble. The Wilson scaling from raw data gave a B of 3A^2. Initially, I did not seemed too alarming. But I do not know why I have these statistics. Does anyone know why Wilson scaling falls when treating that kind of twinned data? I read that twinned data do not obey twe Wilson statistics but I don't know why. Here the presentation I read: http://bstr521.biostr.washington.edu/PDF/Twinning_2007.pdf Do you know any articles, reviews or book in which this particular aspect of of twinned data is treated in
Re: [ccp4bb] Twinning, Wilson scaling and B factor
The true values of the components of the twin can't in general be equal since they come from _different_ reflections that are unrelated by the true crystal symmetry (they are only related by the pseudo-symmetry of the twin). Let's say: Itwin(h1)=tf*I(h1)+(1-tf)*I(h2) where I(h1) and I(h2) are the true intensities of the reflections h1 and h2 related by the twin operator. h1 and h2 are reflections that are _un_related by the true symmetry operators, i.e. they are different reflections so do not in general have the same intensity. and Itwin(h2)=tf*I(h2)+(1-tf)*I(h1) is the twinned intensity of the reflection related to Itwin(h1) by the twin operator. If and only if tf = 0.5 then we have: Itwin(h1)=0.5*(I(h1)+I(h2)) Itwin(h2)=0.5*(I(h2)+I(h1)) which are obviously equal. So it's the intensities in the _twin_ that become equal when tf=0.5, NOT the true intensities (these obviously remain the same whether it's twinned or not). Multiplying all the intensities by the same scale factor cannot possibly have any effect on the Wilson B factor, since the intensities are in any case on an arbitrary scale. For example the diffracted intensity is proportional to the incident intensity, so the intensities will be on a different scale depending on where you collected the data (also the size of the collimator, thickness of crystal, thickness of attenuator, type of mirror focusing, beam current etc etc). Clearly none of these factors can possibly influence the B factor, which is an inherent property of the crystal structure, Strictly if you multiply I by a factor you must multiply sigma(I) by the same factor (since I/sigma(I) must stay the same), maybe this is the problem. A simpler one-step way to halve I and sigma(I) is to use sftools, that's how I would do it. Maybe something went wrong in your procedure. Cheers -- Ian On Fri, May 20, 2011 at 5:43 PM, fulvio.saccoc...@uniroma1.it fulvio.saccoc...@uniroma1.it wrote: Thanks Ian, but your reply confused me a little. I hope you can explain me where I was wrong. I know that I(twin)=tf*I(h1)+(1-tf)*I(h2) I supposed that having tf=0.5 I could take the I(twin), dividing by 2 I will get both I(h1) and I(h2), that are the two component (that are equal in this case). Rather I thought that a possible mistake could be the sigI associated to every intensities ( and I don't know how I can take it into account for Wilson B). Just to tell you and review the procedure I followed: I took the .sca, I operated in order to halve the Intensities column (I used octave to calculate them), saved the new file in .txt and than I applied label FP and SIGFP using F2mtz (ccp4i). After this, I run wilson (ccp4) within 30-3,0 A resolution and obtain a more reliable B factor with respect that obtained from raw data that was of 3A^2. Next, I tried changing the resolution 30-4.5 and 30-4.4 and the results are all similar (28, 31 and 38 A^2). The SCALE were 186 204 and 194 and I considered them quite similar one to another. I did not made this procedure in order to detwin data just to understand how play with raw data affected by perfect twin and to clarify me how these data affect statistics. Thank you for your attention and for all the good advice. Cheers Fulvio Il giorno ven, 20/05/2011 alle 16.26 +0100, Ian Tickle ha scritto: No, simply applying a single overall scale factor to the intensities can't possibly make any difference to the Wilson B since the fall-off with resolution will remain unchanged. The Wilson plot is a plot of ln(mean(I')/S) in shells of constant d* vs d*^2, where I' is I corrected for symmetry and S is a function of the scattering factors for the known unit cell content. Changing the overall scale factor shifts the plot up or down but doesn't change the gradient, and the Wilson B factor depends on the gradient (actually B = -2*gradient). In any case detwinning is impossible if as you say the twin fraction is near 0.5. Your procedure doesn't perform detwinning. For example, suppose the true intensities of the components of the twin are (say) 90 and 110. For tf = 0.5 you will observe the mean value (i.e. half from each component), so I(twin) = 100. Taking I(twin)/2 = 50 doesn't give you back the true intensity (in fact in this case I(twin) is actually a better estimate of I(true)); in any case any attempt at detwinning must give you 2 values, one for each component of the twin. Cheers -- Ian On Fri, May 20, 2011 at 3:43 PM, fulvio saccoccia fulvio.saccoc...@uniroma1.it wrote: Thanks Ian, I tried to do this: I took the file containing hkl I and sigI and generated a new file containing hkl I/2 and sigI because I know, from the refined structure that the twin fraction is nearly 0.5. Now, using this new file the wilson plot give me a more reliable estimated B factor. Do you think this procedure was correct? Fulvio Il giorno gio, 19/05/2011 alle 14.14 +0100,
[ccp4bb] problem with LIBCHECK
Greetings fellow Crystallographers, I'm working on a structure at 1.8-A resolution that contains an acetone crosslink between 2 cysteines (crosslink was incorporated by adding 1,3-dichloroacetone). I figured that the easiest way to model this is to mutate one of the cysteines to S-acetonylcysteine (CSA in the PDB) and then link it to the other cys. I've seen how to do this in COOT using the mutate-by-overlap function; however, CSA is of course not in the CCP4 library that is installed on our system. I've built restraints for CSA using phenix.elbow and tried importing the residue into COOT that way, still to no avail. So I figured the way to go now is to import the cif file directly into the LIBCHECK library in our system and then I should (in theory) be able to use mutate-by-overlap to place the residue. However, this is where I'm stuck. I can't seem to figure out the notation for importing the cif file into LIBCHECK. I tried using FILE_CIF CSA.cif and I get an error reading title of input file. What am I doing wrong? Is there another approach I should consider? Any help or advice would be greatly appreciated. Cheers, Geoff -- Geoffrey K. Feld Department of Chemistry 492 Stanley Hall University of California, Berkeley Vigilia pretium libertatis
[ccp4bb] how to remove part of data with bad signal to noise ratio
Hi All, I am currently working on a 3A resolution dataset. The scaled file shows the following statistics (scroll down to the end of this email). It is P212121 space group with R merge of 8.8%. My question is : Is there a way to selectively use only the data with I/Sigma value of 2 and more for refinement? And how do i achieve this using refmac? I am aware that this would come at the cost of compromising data completeness. Any suggestions/help would be greatly appreciated. Thanks much, Seema Mittal Department of Biochemistry Molecular Pharmacology 970L Lazare Research Building University of Massachusetts Medical School 364 Plantation Street Worcester, MA 01605 Shell I/Sigma in resolution shells: LowerUpper % of of reflections with I / Sigma less than limit limit 0 1 2 3 5 10 2020 total 50.00 6.46 2.0 3.8 5.3 6.27.6 12.5 34.3 65.0 99.3 6.46 5.13 0.7 2.2 3.9 5.38.2 15.7 36.6 63.4 100.0 5.13 4.48 1.3 2.8 4.0 5.89.3 13.8 27.3 72.7 100.0 4.48 4.07 0.7 1.7 4.0 5.47.9 13.9 35.4 64.1 99.5 4.07 3.78 1.8 3.6 5.1 6.9 11.8 20.8 49.6 47.3 96.9 3.78 3.56 1.5 3.8 6.7 8.7 13.3 26.7 65.4 30.8 96.2 3.56 3.38 0.8 3.2 7.1 8.9 12.9 31.1 76.6 20.0 96.6 3.38 3.23 2.0 4.8 8.1 14.8 23.4 44.8 84.7 12.7 97.5 3.23 3.11 4.1 9.2 13.8 18.4 29.6 51.0 86.0 11.0 96.9 3.11 3.00 2.4 8.6 13.9 18.8 30.6 53.9 92.44.596.9 All hkl1.7 4.3 7.19.8 15.3 28.0 58.1 39.9 98.0 Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 6.46 511.720.0 8.8 1.098 0.065 0.073 6.46 5.13 284.610.1 6.3 1.047 0.062 0.064 5.13 4.48 500.917.0 8.8 1.007 0.062 0.069 4.48 4.07 446.117.4 9.2 1.032 0.069 0.070 4.07 3.78 307.114.5 8.4 1.065 0.089 0.092 3.78 3.56 243.413.8 7.9 1.033 0.108 0.112 3.56 3.38 182.312.0 8.3 1.083 0.132 0.134 3.38 3.23 136.510.4 7.7 1.048 0.155 0.151 3.23 3.11 107.4 9.2 7.3 1.096 0.184 0.163 3.11 3.0091.0 8.7 7.3 1.0440.215 0.201 All reflections287.713.5 8.0 1.055 0.088 0.082
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
On Friday, May 20, 2011 02:28:26 pm Mittal, Seema wrote: Hi All, I am currently working on a 3A resolution dataset. The scaled file shows the following statistics (scroll down to the end of this email). It is P212121 space group with R merge of 8.8%. Your data statistics look fine. In fact, it looks to me that your crystal is probably yielding good data to considerably better resolution than 3A. Why did you choose to cut it there? My question is : Is there a way to selectively use only the data with I/Sigma value of 2 and more for refinement? That is a bad idea. By removing data you are throwing away information. Noisy data is still better than no data. good luck with your [probably better than 3A] structure, Ethan And how do i achieve this using refmac? I am aware that this would come at the cost of compromising data completeness. Any suggestions/help would be greatly appreciated. Thanks much, Seema Mittal Department of Biochemistry Molecular Pharmacology 970L Lazare Research Building University of Massachusetts Medical School 364 Plantation Street Worcester, MA 01605 Shell I/Sigma in resolution shells: LowerUpper % of of reflections with I / Sigma less than limit limit 0 1 2 3 5 10 20 20 total 50.00 6.46 2.0 3.8 5.3 6.27.6 12.5 34.3 65.0 99.3 6.46 5.13 0.7 2.2 3.9 5.38.2 15.7 36.6 63.4 100.0 5.13 4.48 1.3 2.8 4.0 5.89.3 13.8 27.3 72.7 100.0 4.48 4.07 0.7 1.7 4.0 5.47.9 13.9 35.4 64.1 99.5 4.07 3.78 1.8 3.6 5.1 6.9 11.8 20.8 49.6 47.3 96.9 3.78 3.56 1.5 3.8 6.7 8.7 13.3 26.7 65.4 30.8 96.2 3.56 3.38 0.8 3.2 7.1 8.9 12.9 31.1 76.6 20.0 96.6 3.38 3.23 2.0 4.8 8.1 14.8 23.4 44.8 84.7 12.7 97.5 3.23 3.11 4.1 9.2 13.8 18.4 29.6 51.0 86.0 11.0 96.9 3.11 3.00 2.4 8.6 13.9 18.8 30.6 53.9 92.44.596.9 All hkl1.7 4.3 7.19.8 15.3 28.0 58.1 39.9 98.0 Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 6.46 511.720.0 8.8 1.098 0.065 0.073 6.46 5.13 284.610.1 6.3 1.047 0.062 0.064 5.13 4.48 500.917.0 8.8 1.007 0.062 0.069 4.48 4.07 446.117.4 9.2 1.032 0.069 0.070 4.07 3.78 307.114.5 8.4 1.065 0.089 0.092 3.78 3.56 243.413.8 7.9 1.033 0.108 0.112 3.56 3.38 182.312.0 8.3 1.083 0.132 0.134 3.38 3.23 136.510.4 7.7 1.048 0.155 0.151 3.23 3.11 107.4 9.2 7.3 1.096 0.184 0.163 3.11 3.0091.0 8.7 7.3 1.0440.215 0.201 All reflections287.713.5 8.0 1.055 0.088 0.082 -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
Hi Ethan, You are absolutely right. As a matter of fact, I had initially processed the data to 2.7A and it looked pretty decent with R symm less than 10%. The maps looked good too. The problem arose during second round of refinement. The Rfree got stuck at around 29-30 while the Rfactor kept decreasing to about 20-21. The bond length and angle values are fine too. I cut down the resolution to 3A hoping to improve the data quality by removing some noise. But, it did not work. i also tried to put restrains on the backbone B factors with limited success. Any thoughts on how i can resolve this Rfree issue? Thanks much, Seema On May 20, 2011, at 5:38 PM, Ethan Merritt wrote: On Friday, May 20, 2011 02:28:26 pm Mittal, Seema wrote: Hi All, I am currently working on a 3A resolution dataset. The scaled file shows the following statistics (scroll down to the end of this email). It is P212121 space group with R merge of 8.8%. Your data statistics look fine. In fact, it looks to me that your crystal is probably yielding good data to considerably better resolution than 3A. Why did you choose to cut it there? My question is : Is there a way to selectively use only the data with I/Sigma value of 2 and more for refinement? That is a bad idea. By removing data you are throwing away information. Noisy data is still better than no data. good luck with your [probably better than 3A] structure, Ethan And how do i achieve this using refmac? I am aware that this would come at the cost of compromising data completeness. Any suggestions/help would be greatly appreciated. Thanks much, Seema Mittal Department of Biochemistry Molecular Pharmacology 970L Lazare Research Building University of Massachusetts Medical School 364 Plantation Street Worcester, MA 01605 Shell I/Sigma in resolution shells: LowerUpper % of of reflections with I / Sigma less than limit limit 0 1 2 3 5 10 2020 total 50.00 6.46 2.0 3.8 5.3 6.27.6 12.5 34.3 65.0 99.3 6.46 5.13 0.7 2.2 3.9 5.38.2 15.7 36.6 63.4 100.0 5.13 4.48 1.3 2.8 4.0 5.89.3 13.8 27.3 72.7 100.0 4.48 4.07 0.7 1.7 4.0 5.47.9 13.9 35.4 64.1 99.5 4.07 3.78 1.8 3.6 5.1 6.9 11.8 20.8 49.6 47.3 96.9 3.78 3.56 1.5 3.8 6.7 8.7 13.3 26.7 65.4 30.8 96.2 3.56 3.38 0.8 3.2 7.1 8.9 12.9 31.1 76.6 20.0 96.6 3.38 3.23 2.0 4.8 8.1 14.8 23.4 44.8 84.7 12.7 97.5 3.23 3.11 4.1 9.2 13.8 18.4 29.6 51.0 86.0 11.0 96.9 3.11 3.00 2.4 8.6 13.9 18.8 30.6 53.9 92.4 4.596.9 All hkl1.7 4.3 7.19.8 15.3 28.0 58.1 39.9 98.0 Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 6.46 511.720.0 8.8 1.098 0.065 0.073 6.46 5.13 284.610.1 6.3 1.047 0.062 0.064 5.13 4.48 500.917.0 8.8 1.007 0.062 0.069 4.48 4.07 446.117.4 9.2 1.032 0.069 0.070 4.07 3.78 307.114.5 8.4 1.065 0.089 0.092 3.78 3.56 243.413.8 7.9 1.033 0.108 0.112 3.56 3.38 182.312.0 8.3 1.083 0.132 0.134 3.38 3.23 136.510.4 7.7 1.048 0.155 0.151 3.23 3.11 107.4 9.2 7.3 1.096 0.184 0.163 3.11 3.0091.0 8.7 7.3 1.0440.215 0.201 All reflections287.713.5 8.0 1.055 0.088 0.082 -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] How to generate mask file represented by envelope function
Hi Hailiang, I guess you can store the map in CCP4 map binary format or convert it into corresponding Fourier map coefficients and store them in MTZ format (note: in this case if you convert them back into a mask it will not be a binary function anymore) - both shouldn't take a huge amount of space. Pavel. On Fri, May 20, 2011 at 8:14 PM, Hailiang Zhang zhan...@umbc.edu wrote: Hi, As I understand, the general molecular mask generated by CCP4 (eg sfall+mapmask) are binary mask file which needs lots of memory space. I just wonder whether we can generate some small mask files represented by, say, envelope function (F(sita,psi)) (http://journals.iucr.org/d/issues/2001/10/00/ba5001/ba5001.pdf). This will save lots of disc space and lots of efforts for my problem. Thanks! Hailiang
Re: [ccp4bb] How to generate mask file represented by envelope function
Thanks Pavel. Its not huge, but I need to process massive cases. Svergun's envelope function or spherical harmonics expansion provides some concise mask description, but just not sure whether ccp4 or other facilities can generate it handy (from a pdb file to an atomic mask!) Thanks again! Hailiang Hi Hailiang, I guess you can store the map in CCP4 map binary format or convert it into corresponding Fourier map coefficients and store them in MTZ format (note: in this case if you convert them back into a mask it will not be a binary function anymore) - both shouldn't take a huge amount of space. Pavel. On Fri, May 20, 2011 at 8:14 PM, Hailiang Zhang zhan...@umbc.edu wrote: Hi, As I understand, the general molecular mask generated by CCP4 (eg sfall+mapmask) are binary mask file which needs lots of memory space. I just wonder whether we can generate some small mask files represented by, say, envelope function (F(sita,psi)) (http://journals.iucr.org/d/issues/2001/10/00/ba5001/ba5001.pdf). This will save lots of disc space and lots of efforts for my problem. Thanks! Hailiang
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
The only thing you can do is to move the detector in and collect higher resolution. Then you can determine what the I/sigI ratio is for each of your resolution shells and manually set the resolution limit once you run HKL2000. As far as the R/Rfree values go, you will need to manually set the weighting factor if you are using refmac. I have found that during the initial rigid body refinement and the first few rounds of restrained refinement, it works well if you keep the weighting factor low (0.01-0.9), then as you go through a few rounds, gradually increase the weighting factor. But keep in mind that as you increase the weighting factor, the difference between R/Rfree will also increase. -- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 -- -- From: Mittal, Seema seema.mit...@umassmed.edu Sent: Friday, May 20, 2011 5:28 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] how to remove part of data with bad signal to noise ratio Hi All, I am currently working on a 3A resolution dataset. The scaled file shows the following statistics (scroll down to the end of this email). It is P212121 space group with R merge of 8.8%. My question is : Is there a way to selectively use only the data with I/Sigma value of 2 and more for refinement? And how do i achieve this using refmac? I am aware that this would come at the cost of compromising data completeness. Any suggestions/help would be greatly appreciated. Thanks much, Seema Mittal Department of Biochemistry Molecular Pharmacology 970L Lazare Research Building University of Massachusetts Medical School 364 Plantation Street Worcester, MA 01605 Shell I/Sigma in resolution shells: LowerUpper % of of reflections with I / Sigma less than limit limit 0 1 2 3 5 10 20 20 total 50.00 6.46 2.0 3.8 5.3 6.27.6 12.5 34.3 65.0 99.3 6.46 5.13 0.7 2.2 3.9 5.38.2 15.7 36.6 63.4 100.0 5.13 4.48 1.3 2.8 4.0 5.89.3 13.8 27.3 72.7 100.0 4.48 4.07 0.7 1.7 4.0 5.47.9 13.9 35.4 64.1 99.5 4.07 3.78 1.8 3.6 5.1 6.9 11.8 20.8 49.6 47.3 96.9 3.78 3.56 1.5 3.8 6.7 8.7 13.3 26.7 65.4 30.8 96.2 3.56 3.38 0.8 3.2 7.1 8.9 12.9 31.1 76.6 20.0 96.6 3.38 3.23 2.0 4.8 8.1 14.8 23.4 44.8 84.7 12.7 97.5 3.23 3.11 4.1 9.2 13.8 18.4 29.6 51.0 86.0 11.0 96.9 3.11 3.00 2.4 8.6 13.9 18.8 30.6 53.9 92.44.596.9 All hkl1.7 4.3 7.19.8 15.3 28.0 58.1 39.9 98.0 Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 6.46 511.720.0 8.8 1.098 0.065 0.073 6.46 5.13 284.610.1 6.3 1.047 0.062 0.064 5.13 4.48 500.917.0 8.8 1.007 0.062 0.069 4.48 4.07 446.117.4 9.2 1.032 0.069 0.070 4.07 3.78 307.114.5 8.4 1.065 0.089 0.092 3.78 3.56 243.413.8 7.9 1.033 0.108 0.112 3.56 3.38 182.312.0 8.3 1.083 0.132 0.134 3.38 3.23 136.510.4 7.7 1.048 0.155 0.151 3.23 3.11 107.4 9.2 7.3 1.096 0.184 0.163 3.11 3.0091.0 8.7 7.3 1.0440.215 0.201 All reflections287.713.5 8.0 1.055 0.088 0.082
