Re: [ccp4bb] Residue positional validation
Hej, In thread to my previous mail, i calculated the minimum and maximum errors as well as DPI for the structure. But want to ask what are the allowed limits for the above values to decide for the correctness of the positional movements within the structure ? Thanks On Mon, Apr 27, 2015 at 10:54 AM, Monica Mittal monica.mitta...@gmail.com wrote: Hi everyone, Can anyone guide me to calculate the diffraction component precision index (DPI) for validating the positional error of certain critical residues in my structure ? Thanks in advance Monica
[ccp4bb] Residue positional validation
Hi everyone, Can anyone guide me to calculate the diffraction component precision index (DPI) for validating the positional error of certain critical residues in my structure ? Thanks in advance Monica
[ccp4bb] Protein-Ligand Crystallization
Hi all, I am working to crystallize a protein-ligand complex. I did a preliminary melting curve analysis for the protein in the absence and presence of 2 ligands (dissolved in protein buffer). I kept the other controls as buffer an a known standard to confirm instrument performance. All expts done in triplicates. Now the results are like : Tm of protein alone is 56 deg, Tm in the presence of Lig1 conc. of 0.05mM, 0.1mM, 1.0mM and 10.0mM is 56.2, 56.1, 56.0 and 53.5 respectively !! Tm in the presence of Lig 2 conc. of 0.05mM, 0.1mM, 1.0mM, 4.0mM and 10.0mM is 56.2, 56.1, 54.9, 52.3 and 50.6 respectively !! Although the effective delta Tm for both is different at higher concentration, but both are kind of making protein less stable. So i was wondering, will it be difficult to co-crystallize them !! Any suggestions in this regard are highly appreciated !! Thanks Monica
Re: [ccp4bb] Crystallization problem
Hi everyone, Many Thanks for your suggestions !! I checked for the everything related to the components i am using : No contamination, not very old, no PEGs etc. And then i followed the way Markov and Prem suggested !! For a trial set up i just et up 10 different concentration variations of my previous conditions (having Tacsimate) and stored at my bench !! I checked after 2 days and cud see some needles back !! But only in 1 out of 10 conditions tested !! The number of needles has also decreased. And the last thing noticeable was that this 1 condition is at lower side (in terms of concentration of precipitant solution) of tested condition as compared to the previous hit solution. So i was wondering is this 1 hit condition (although with poor needles) is just a stochastic hit or should i screen the whole range of precipitant solution again ?? Secondly any suggestion to increase the number of wells having crystals ?? Or the quality of needles ?? Actually i am little concerned with the reproducibility issue also because as i proceed for the optimization, i get confused that is it because of batch variation i m not getting crystals or the tried approach didn't work out ?? Its been long time that i am optimizing these crystals so any suggestions on this matter is highly appreciated !! Thanks Monica On Mon, Jan 26, 2015 at 10:08 PM, Emilia C. Arturo (Emily) ec...@drexel.edu wrote: On Mon, Jan 26, 2015 at 8:22 PM, Monica Mittal monica.mitta...@gmail.com wrote: Hi everyone, I need an advice on some strange thing happening to one of the protein i am working on. I used to purify it and set up trays and get some needle shaped crystals and trying seeding and other methods to optimise them. But recently, it stopped giving crystals even small needles. I am still following the same protocol with same buffer stocks. By 'buffer stocks', do you also mean your stock of precipitating reagent? It's happened to me that a hit I pursued and optimized vanished when I opened a new bottle of PEG. It took too long to realize that the old PEG could have been at a higher concentration than I'd assumed, given its shelf-age. I increased the PEG % and got back the crystals. Good luck, Emily. And not just once but since last three times it is happening. The purified protein in gel filtration is perfectly fine eluting at same position with symmetrical distribution. However when i am setting up trays under previous conditions, i am not getting the crystals. Instead the drops are quite clear. So i increased the concentration of the protein also from 8 to 11mg/ml, but still the same. Infact i tried adding ligand also but again no crystals. So i would be really grateful if anyone can give a valuable suggestion regarding this problem !! Thanks BR Monica
[ccp4bb] Crystallization problem
Hi everyone, I need an advice on some strange thing happening to one of the protein i am working on. I used to purify it and set up trays and get some needle shaped crystals and trying seeding and other methods to optimise them. But recently, it stopped giving crystals even small needles. I am still following the same protocol with same buffer stocks. And not just once but since last three times it is happening. The purified protein in gel filtration is perfectly fine eluting at same position with symmetrical distribution. However when i am setting up trays under previous conditions, i am not getting the crystals. Instead the drops are quite clear. So i increased the concentration of the protein also from 8 to 11mg/ml, but still the same. Infact i tried adding ligand also but again no crystals. So i would be really grateful if anyone can give a valuable suggestion regarding this problem !! Thanks BR Monica
[ccp4bb] Presence of Fe3+ or Fe2+
Hi everyone !! I am working on a protein which binds to its ligand in a particular state i.e. Fe3+ state (active) and reduces it to Fe2+ state (inactive). I am trying to set up crystallisation trials with the ligand but seems like difficult to get them. However i just needed an advice here, is there any way to know whether the protein is in Fe2+ state or Fe3+ state or how can i make the protein to exist in more of Fe3+ state so that i increase the probability of ligand binding to the protein. Your advice is highly appreciated. Thanks in advance !! Cheers Monica
[ccp4bb] crystallization with hydrophobic ligands
Dear All Can anyone give suggestions for handling the solubility problem of highly hydrophobic compounds, during co-crystallization or inhibition assays? The ligands I am using are almost insoluble in aquous medium. In DMSO or 95% Ethanol, the solubility is higher. Besides crystallization, this solubility is also a hindrance for in-vitro or in-vivo assays requiring higher conc. of ligand. Thanks in advance ! Monica
[ccp4bb] Protein_Ligand Suggestion
Dear all I have solved a structure of protein with ligand at 2.7 and 2.8 A. Both have ligand bound in it as i soaked it. For the first structure i soaked for lesser time and lesser concentration of ligand and solved it at 2.7 A resolution. Although the ligand has good density (2Fo-Fc at 0.9 sigma) but it is little unconnected from its middle portion. Its RSCC value is 0.76 and B-factor of 70.0. Overall completeness of structure is 91%. So If i decrease the occupancy of ligand here, will it improve the ligand statistics esp. the Real space R-value?? However for the second structure, i soaked for longer time and higher ligand concentration, so the ligand is perfect with clear 2Fo-Fc map at 1.2 sigma and Fo-Fc map at 2.8 sigma along with well defined simulated annealing and composite omit maps. The RSCC value is 0.81 with B-factor of 40.1.The only issue is the overall completeness of the structure is 88%. But if i get evrything OK at this completeness, den can i consider the second structure with ligand as the correct one?? Thanks in advance. Monica
[ccp4bb] Arp/warp error
Dear all I am getting this error while running arp/warp to build DNA/RNA: PHIB is not assigned to an mtz label Input was merged data (.mtz) However no such problem while using arp/warp classic as i chose for automated model building from existing model and not experimental phases. Kindly help how to give PHIB and FOM labels here?? Thanx in advance Monica
[ccp4bb] Help in Cell content analysis
Dear all i have a small query to ask and seek your suggestions: I have collected a data for a protein with 324 residues and processed at its best in P212121. So Matthews suggest 1 mol in ASU with expected Mol. weight of 43 kDa with sovent content of 58% and 2 mol./ASU with 18% solvent content. However the data suggest possibility of translational NCS so i think i should ask for two molecules so that both get corrected for NCS. However for 2 mol./ASU, Matthewssuggests a total mol. weight of 52 kDa. So how to decide which way to proceed for MR? Thanks Monica
[ccp4bb] MIND in shelxd
Dear all How to decide for minimum distance between heavy atoms in shelxd esp w.r.t. S-SAD. Thanx in advance Monica
[ccp4bb] shelxd
Dear all I am naive in phasing experiments. CAn anyone please guide how to do the following: In a S-SAD for *SHELXD* searches, try various high-resolution cutoffs for example 2.5 to 5 Å in steps of 0.1 Å. Based on these attempts, use a high-resolution cutoff at for example 3.8 Å for substructure determination with an *E* min value of for example 1.6 to search for X no. of protein sulfur sites. Thanx in advance Monica
[ccp4bb] absorption correction during anomalous scaling
Hi, everyone I have a question about HKL2000. During scaling, there is a check option call absorption correction. I processed the data with and without it checked. With absorption correction checked, my chisq. anomalous is more than 2.0 for all resolution shells. When I leave it out, chisq. anomalous goes below 2.0. I read through the HKL2000 online manual and only find direction cosines produce information that can be read by an outside absorption correction program, such as Shelx. The need for it will disappear as the HKL-2000 absorption correction routines are implemented. So do I need to use the absorption correction routinely or just for some trouble dataset. Obviously I don't want to incorporate bad spots. Thanks for your input. Monica
[ccp4bb] scaling HKL2000
Dear all I need an advice at the part of scale.log file that i am attaching herewith this mail that do i have to compromise the resolution as I/sig and Rsymm seem to be bad in resolution shell 2.3 to 2.18. Kindly suggest Thanx Monica Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 5.70 9015.9 157.865.3 9.801 0.052 0.060 5.70 4.52 7052.3 122.654.8 8.818 0.057 0.068 4.52 3.95 5642.499.745.9 8.667 0.060 0.068 3.95 3.59 3592.280.450.3 13.981 0.088 0.090 3.59 3.33 2111.747.931.7 9.035 0.095 0.101 3.33 3.14 1499.236.525.5 6.179 0.090 0.095 3.14 2.98 904.428.722.8 4.657 0.100 0.099 2.98 2.85 641.625.622.0 3.632 0.112 0.105 2.85 2.74 460.824.021.5 2.986 0.126 0.110 2.74 2.65 340.823.021.4 2.710 0.151 0.131 2.65 2.56 254.522.521.4 2.060 0.168 0.126 2.56 2.49 201.622.621.9 2.371 0.213 0.644 2.49 2.42 181.223.422.7 1.851 0.235 0.173 2.42 2.37 126.723.723.3 1.517 0.305 0.224 2.37 2.31 126.725.224.8 1.488 0.323 0.203 2.31 2.26-7.233.132.9 1.725 0.000 0.218 2.26 2.22 1648.671.362.6 43.073 0.234 0.350 2.22 2.18 -59.734.934.9 1.599 0.000 0.376 2.18 2.1465.929.429.2 0.986 0.607 0.324 2.14 2.1058.730.029.9 0.904 0.660 0.413 All reflections 1700.447.432.3 5.069 0.080 0.074
[ccp4bb] ligand occupancy
Dear all I have a protein which is dimer having one ligand binding site in each monomer. I refined the crystal structure with ligand in both sites finally. I refined will full occupancy of 1 for ligands (same in both). But now i want to see is there any difference in the occupancy of both ligands in ligand binding sites of monomer A and B. Is there any way i can get any information about the occupancy of ligands in two monomers like one is binding more tightly than another so that i can get an idea about their differential binding contacts also. Thank you very much in advance. Regards Monica
[ccp4bb] hkl2map
Hi all I am new to phasing experiments. I have downloaded hkl2map-0.3.i-beta.tgz and i need help to install it on linux. I would be thankful if anyone can guide me. Thanx Monica
[ccp4bb] shelxc/hkl2map
Hi all I installed shelxc/d/e on linux by unpacking and then making executable by chmod +x. I got the executables for all files. Now when i run as ./shelxc i get the infromation about the program. I guess i need to define the input file also along with shelxc. But instead of that i want to use GUI for that. How can i do that. Additionally i have installed hkl2map also and while i run it it asks for shelxc executable so how can i sort it out. Many thanx in advance Monica
[ccp4bb] Shelx GUI usage with hkl2map
Hi all I am new to use the programs for anamolous data processing. I installed hkl2map as well as shelxc/d/e. I am abl to run shelx on command line but not through GUI. Kindly guide me how to use the GUI for shelx. Thanx in advance. Monica
[ccp4bb] Coot error
Hi all I am facing a problem while installing coot in CentOS. After configuring its showing No package'glib-2.0' found. Either it asks me to adjust PKG_CONFIG_PATH env variable if i installed in non standard prefix or i can set set env variables GLIB_CFLAGS and GLIB_LIBS to avoid need to call pkg-config. But how to do any of the above. I dont have idea. So guidance on this will be highly appreciated. Thanx Monica
Re: [ccp4bb] AW: [ccp4bb] regarding Fo-Fc map in coot
Dear Sir May i know how to refine the group occupancy for a ligand??? Thanx in advance On Mon, Mar 10, 2014 at 2:11 PM, herman.schreu...@sanofi.com wrote: Dear Amlan, The sigma of an Fo-Fc map map depends on the residual noise in your map. In a well-refined structure, the sigma will be low, so at 3 sigma it will show very weak features. My guess is that your ligand is present in partial occupancy and that you will find it in your 2Fo-Fc map when you scroll down your contour level. If you see convincing Fo-Fc density without a ligand being fitted, the presence of the ligand must be real and you can fit it. However, I would refine a group occupancy for your ligand. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Amlan Roychowdhury Gesendet: Montag, 10. März 2014 09:09 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] regarding Fo-Fc map in coot Dear All, Some times during model building in coot we have found that at the position of ligand molecules and water, there is a good Fo-Fc map (above 3 sigma), devoid of any 2Fo-Fc map. 1.What does it physically mean and why the 2Fo-Fc map was not generated properly? 2. Can we fit ligand molecule there? Thanks in advance. Best Wishes Amlan. -- Amlan Roychowdhury. Senior Research Fellow. Protein Crystallography Lab. Dept. of Biotechnology, IIT Kharagpur. Kharagpur 721302 West Bengal. India.
Re: [ccp4bb] size of a flexible pdb structure
Hi rajan I guess calculating radius of gyration for your whole protein with time will tell you about the size variation at difeerent time points or you canm say different frames. This is how you can cluster your whole trajectory PDBs into a few clusters and then get the representative PDBs from each cluster to finally select what you want. Best Monica On Wed, Mar 5, 2014 at 8:42 PM, rajan kumar rajugunju...@gmail.com wrote: Dear all, sorry for asking an off topic question. My protein is composed of two domains connected by a flexible linker (15aa). after 50ns simulation i came across the fact that one domain is flexible. so my question is that how could i be able to calculate the size of molecule or radius of my molecule and which conformation i should prefer to calculate the size of molecule. your any suggestion will be very helpful. thank you all in advance with best regards -- Rajan kumar choudhary Senior Research Fellow Department of Atomic Energy(Govt.Of India) ACTREC TATA Memorial Center Kharghar Navi-Mumbai Mumbai-410210 India
Re: [ccp4bb] SELF-ROTATION FUNCTION FROM MOLREP
Dear CCP4 Users, In Phenix.xtriage and phaser, the analyses of the Patterson function reveals a significant off-origin peak that is 23.25 % of the origin peak, indicating pseudo translational symmetry at frac. cord. vector 0.0000.5000.022 . It did not indicate any twinng. Data is good with resolution upto 2.8A. The cell dimension in P21 SG is 57.877, 63.321, 108.028, 90, 89.987, 90 and in P222 SG is 57.805, 63.266, 108.053, 90, 90, 90. We can say cell dimension align well in in P21 and P222 SG. For MR template, about 75% of domain1, we have solved in our lab and remaining 25% is available from pdb with 30% sequence identity. It is a transcription factor with ligand bindng domain and DNA binding domain. I tried finding MR solution but does not gave satisfaction result. The Rwork and Rfree stalls around 47% and 53% respectively. Is there any possiblity to guess the no of molecules present in ASU from self-rotation function? If we can then we will be sure of whether it is full length of truncated one. Thank you Monica On 11/18/13, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: First Q - how good is your data - is there no possibility of twinning or any other distraction? Second Q - To compare those results properly we need to know how the P2 and the P222 cell align - are the cell dimensions more or less the same? But the 2 plots you attach (and the list above) show both very strong 222 symmetry so the most likely assumption is that the pointgroup is P222. The next peak down the list is only 0.13 for one and 0.17 for the other pointgroup, which only borders on significance.. But this doesnt really prove anything - for example, if there is a flexible linker the 2 domains of each molecule may be in different relative orientations . Do you have any MR search model for the 2 domains? I would search with them and see what they predict - on the whole self rotation functions are most comprehensible AFTER the structure is solved! Eleanor On 18 November 2013 09:58, Monica Mittal monica.mitta...@gmail.com wrote: Dear CCp4 users, May anyone help me in interpreting the self-rotation function from molrep. Data can be indexed,scaled equally well in P21 and P222. This protein has two domain linked by a long flexible linker and get cleaved during crystallization. After crystallizing it in many condition, i believed that i found new condition where it may be full length. May anyone please suggest me by looking at the self-rotation function, how many molecule exist in ASU. Matthews coefficient suggest that there would be 2 copy of full length protein or four of truncated protein in P21 space group. For your interpretation, please find attached images of rotation function around K=180 Molrep self rotation peaks are P21 space group theta phi chi P(i)/P(0)| +--+ | 1 0.000.000.001.00 | | 290.00 -0.00 180.000.79 | | 335.78 -0.00 180.000.17 | | 490.00 -170.05 180.000.17 | | 558.71 180.00 180.000.14 | | 6 108.34 180.00 180.000.14 | | 7 117.39 180.00 180.000.13 | | 890.00 90.00 108.540.13 | | 990.00 -90.00 108.540.13 | | 10 144.50 -0.00 180.000.13 | | 1137.57 25.59 179.980.12 | | 12 148.100.00 180.000.12 | | 13 121.736.36 179.620.11 | | 1463.31 -42.27 180.000.11 | | 1590.00 90.00 162.290.11 | | 1690.00 -90.00 162.290.11 | | 1771.12 136.88 180.000.10 | | 1899.17 -180.00 180.000.10 | | 19 144.80 -161.87 180.000.10 | | 2090.00 -138.24 180.000.10 | | 2182.27 -76.45 180.000.10 | | 2290.00 90.00 115.940.10 | | 2390.00 -90.00 115.940.10 | | 24 159.16 27.07 180.000.10 | | 2595.08 -35.92 179.840.10 | | 26 113.86 -139.44 179.540.10 | | 2790.23 -0.00 89.320.10 | | 2820.18 -156.63 179.980.10 | | 29 116.90 -40.40 179.500.10 | | 3063.12 139.60 179.500.10 P222 space group theta phi chi P(i)/P(0)| +--+ | 1 0.000.000.001.00 | | 290.00 -21.41 180.000.13 | | 3 125.320.00 180.000.13 | | 4 151.18 -90.00 180.000.12 | | 590.00 -180.00 90.000.12 | | 6 141.32 26.82 180.000.12 | | 790.00 -42.59 180.000.11 | | 8 127.22 -25.21 180.000.11 | | 937.04 -14.48 180.000.11 | | 1059.17 -129.14 180.000.10 | | 1156.66 -174.09 180.000.09
[ccp4bb] Data Fitting for protein-ligand interaction.
Dear All, Firstly sorry for asking a non-crystallography question, but i want help in understanding the data analysis for fitting a protein-ligand binding data. Actually i have a protein which is a tetramer in solution and i have done its flourescence binding with a ligand. I am trying to fit the data to a 4-site binding model in scientist. But i donot have a correct model to fit in the data for identical or non-identical, co-operative or sequential binding. Can anyone help me in analysing the binding data. Any help will be highly appreciated. Thankyou !
[ccp4bb] RSCC
Dear all I am new to crystallography. I recently solved a structure with a ligand. Can anyone please help me to find how to calculate Real space correlation coefficient for the ligand. I will highly grateful. I tried CCP4 but the values it gave I could not understand as it ranges from negative to more than 1 also. Thankyou Monica
