[ccp4bb] ATP solution
Dear all, recently, i prepared the ATP solution used to assay ATPase activity. when the Control Well just containing assay buffer and ATP mixed with molybdate ,the colour changed to green, this mean that there is free pi in the solution.the assay buffer and water is free from pi.and the ATP stock solution has free pi ,maybe the ATP hydrolysis when prepared .any suggestion about preparing ATP stock solution is appreciative. Best regards! 2010-08-29 dengzq1987
Re: [ccp4bb] DM NCS averaging question
The commonest error with averaging is getting the mask wrong.
Check that the CCs after application of the averaging start at a
reasonable value - 0.3 at least and increase with each cycle ( by the
way why do ncycle 1?)
But in the end the density will not be identical, the Fobs are not
perfectly symmetric so there will be differences. The best idea is to
average (with correct matrices - I always find that takes several pases
before I get them all right - then build molecule A and refit it over
the others before starting refinement.
EleanorHailiang Zhang wrote:
Hi,
I am using the following DM script to perform a NCS averaging. I have a
fundemental question: after NCS averaging, are the density distrubitions
of different NCS unit being averaged supposed to be the same? I found they
are different by checking FCDM/PHICDM, and maybe I am wrong somewhere...
dm NCSIN ${PDB}.msk HKLIN ${PDBALL}.mtz HKLOUT ${PDBALL}-dm.mtz \
D
AVER REFI
NCSMASK NMER 1
ROTA MATRIX -0.90337 0.42792 0.02831 -0.42883 -0.90066 -0.07011
-0.00451 -0.07547 0.99714
TRAN 158.0331736.91842 3.25853
#A>F
AVER REFI
NCSMASK NMER 1
ROTA MATRIX -0.21272 -0.97702 0.01352 0.97675 -0.21300 -0.02424 0.02657
0.00805 0.99961
TRAN 100.69797 -81.01860-1.71365
#A>G
AVER REFI
NCSMASK NMER 1
ROTA MATRIX 0.61704 -0.78687 -0.01005 0.78590 0.61553 0.05899 -0.04023
-0.04429 0.99821
TRAN 32.50667 -65.76570 7.05504
#A>H
AVER REFI
NCSMASK NMER 1
ROTA MATRIX -0.85814 -0.51116 -0.04813 -0.51290 0.85771 0.03558
0.02310 0.05522 -0.99821
TRAN 156.1498142.2387348.93406
#A>I
AVER REFI
NCSMASK NMER 1
ROTA MATRIX -0.13703 -0.98975 -0.04032 -0.99048 0.13635 0.01902
-0.01332 0.04254 -0.99901
TRAN 95.9763082.3094852.82510
#A>J
AVER REFI
NCSMASK NMER 1
ROTA MATRIX 0.69662 -0.71695 -0.02645 -0.71716 -0.69691 0.00230
-0.02008 0.01737 -0.99965
TRAN 25.8458859.7628653.68224
#A>K
AVER REFI
NCSMASK NMER 1
ROTA MATRIX 0.99467 0.10258 -0.01072 0.10259 -0.99472 0.00088 -0.01057
-0.00197 -0.4
TRAN0.47215-8.8608252.78315
#A>L
AVER REFI
NCSMASK NMER 1
ROTA MATRIX 0.55782 0.82946 0.02875 0.82987 -0.55793 -0.00466 0.01218
0.02646 -0.99958
TRAN 36.68436 -68.6383349.21587
#A>M
AVER REFI
NCSMASK NMER 1
ROTA MATRIX -0.30892 0.95102 -0.01113 0.95109 0.30890 -0.00370
-0.8 -0.01173 -0.3
TRAN 109.48987 -79.0555052.39334
#A>N
AVER REFI
NCSMASK NMER 1
ROTA MATRIX -0.93676 0.34855 -0.03147 0.34600 0.93589 0.06627 0.05255
0.05119 -0.99731
TRAN 162.32979 -29.2680045.41564
LABIN FP = FWT PHIO = PHIC FOMO = WCMB
LABOUT FDM=FDM PHIDM=PHIDM FOMDM=FOMDM FCDM=FCDM PHICDM=PHICDM
END
dmtest
[ccp4bb] A query regarding GST tag protein purification
Hello one and all !! I have been working on cloning and purification aspects on a Leishmanial cysteine peptidase (pI= 8.2) for quite some time. I had cloned it in pGEX expression vector. Expression seemed quite okay when induced with 0.5 and 1mM IPTG, so did the solubility in 4 buffers at different pH conditions. i. e. Tris (6.8), HEPES (7.5), Bicine (pH = 9) and Glycine (pH=10). The problem is when I purify it using glutathione sepharose column I get only GST (size wise estimation; no western tried ) i.e. a prominent 25 KD band. At the same time I get the fusion protein in the load, equilibration and wash fractions. When I increased salt concentration to 400 mM I only could exclude the fusion protein band from wash. I had tried protease inhibitors like PMSF, sigma cocktail, and DTT in the lysate before sonication. I had also tried reduced glutathione upto 40mM in the elution buffer with two different pH at 8 and 9. I also read from the literature from similar proteases the behavior is unaltered even after doing mutagenesis of the Cysteine residue at its active site .Should I try ion exchange or affinity chromatography using any inhibitor of this enzyme?? Can anyone suggest me some tip?? Guys help me out. i am kind of struck here Ashok Ranjan Nayak Research Scholar Molecular and Structural Biology Division Central Drug Research Institute, Lucknow
[ccp4bb] Query regarding GST fusion protein purification
Hello one and all !! I have been working on cloning and purification aspects on a Leishmanial cysteine peptidase (pI= 8.2) for quite some time. I had cloned it in pGEX expression vector. Expression seemed quite okay when induced with 0.5 and 1mM IPTG, so did the solubility in 4 buffers at different pH conditions. i. e. Tris (6.8), HEPES (7.5), Bicine (pH = 9) and Glycine (pH=10). The problem is when I purify it using glutathione sepharose column I get only GST (size wise estimation; no western tried ) i.e. a prominent 25 KD band. At the same time I get the fusion protein in the load, equilibration and wash fractions. When I increased Nacl concentration upto 400 mM I only could exclude the fusion protein band from wash. I had tried protease inhibitors like PMSF, sigma cocktail, and DTT in the lysate before sonication. I had also tried reduced glutathione upto 40 mM in the elution buffer with two different pH at 8 and 9. I also read from literature that similar intracellular cysteine proteases behave same even after mutating the conserved cysteine residue at its active site. They all say that its not because of autocatalytic property of the enzyme its because of some proteases from E.coli. Should I try ion exchange or affinity chromatography using any inhibitor of this enzyme?? Can anyone suggest me some tip?? Guys help me out. i am kind of struck here Ashok Ranjan Nayak Research Scholar Molecular and Structural Biology Division Central Drug Research Institute, Lucknow
Re: [ccp4bb] Query regarding GST fusion protein purification
It sounds like list your GST construct is not binding to the column (or very well) when the peptidase is attached. GST needs to form a dimer to binding to the column - I suspect that your construct interferes with dimer formation - when the peptidase is present, but when not there due to stalled translation (rare codon?) it binds ok. The other possibility is that when your peptidase is present it causes aggregation - preventing binding. Maybe it depends on the concentration of your construct - possibly dilute and slow binding might work - but am not sure. Also - is there much of a linker between the GST and the peptidase? Maybe others have a suggestion... Good luck, Ezra On 8/29/2010 7:28 AM, Ashok Ranjan Nayak wrote: Hello one and all !! I have been working on cloning and purification aspects on a Leishmanial cysteine peptidase (pI= 8.2) for quite some time. I had cloned it in pGEX expression vector. Expression seemed quite okay when induced with 0.5 and 1mM IPTG, so did the solubility in 4 buffers at different pH conditions. i. e. Tris (6.8), HEPES (7.5), Bicine (pH = 9) and Glycine (pH=10). The problem is when I purify it using glutathione sepharose column I get only GST (size wise estimation; no western tried ) i.e. a prominent 25 KD band. At the same time I get the fusion protein in the load, equilibration and wash fractions. When I increased Nacl concentration upto 400 mM I only could exclude the fusion protein band from wash. I had tried protease inhibitors like PMSF, sigma cocktail, and DTT in the lysate before sonication. I had also tried reduced glutathione upto 40 mM in the elution buffer with two different pH at 8 and 9. I also read from literature that similar intracellular cysteine proteases behave same even after mutating the conserved cysteine residue at its active site. They all say that its not because of autocatalytic property of the enzyme its because of some proteases from E.coli. Should I try ion exchange or affinity chromatography using any inhibitor of this enzyme?? Can anyone suggest me some tip?? Guys help me out. i am kind of struck here Ashok Ranjan Nayak Research Scholar Molecular and Structural Biology Division Central Drug Research Institute, Lucknow
Re: [ccp4bb] Query regarding GST fusion protein purification
I worked with several viral cystene proteases. I do not recommend using basic pH for them dring purification or storage or crystallization. Keep it below 7, because the active site cysteine oxydazes very easily. The higher the pH, the easier the oxydation. PH 10 can also cause hydrolysis of some peptide bonds. Try His tag, then you don't need to use proteases for cleavage, you can use Ni column. Maia - Original Message - From: "Ashok Ranjan Nayak" To: Sent: Sunday, August 29, 2010 5:28 AM Subject: [ccp4bb] Query regarding GST fusion protein purification Hello one and all !! I have been working on cloning and purification aspects on a Leishmanial cysteine peptidase (pI= 8.2) for quite some time. I had cloned it in pGEX expression vector. Expression seemed quite okay when induced with 0.5 and 1mM IPTG, so did the solubility in 4 buffers at different pH conditions. i. e. Tris (6.8), HEPES (7.5), Bicine (pH = 9) and Glycine (pH=10). The problem is when I purify it using glutathione sepharose column I get only GST (size wise estimation; no western tried ) i.e. a prominent 25 KD band. At the same time I get the fusion protein in the load, equilibration and wash fractions. When I increased Nacl concentration upto 400 mM I only could exclude the fusion protein band from wash. I had tried protease inhibitors like PMSF, sigma cocktail, and DTT in the lysate before sonication. I had also tried reduced glutathione upto 40 mM in the elution buffer with two different pH at 8 and 9. I also read from literature that similar intracellular cysteine proteases behave same even after mutating the conserved cysteine residue at its active site. They all say that its not because of autocatalytic property of the enzyme its because of some proteases from E.coli. Should I try ion exchange or affinity chromatography using any inhibitor of this enzyme?? Can anyone suggest me some tip?? Guys help me out. i am kind of struck here Ashok Ranjan Nayak Research Scholar Molecular and Structural Biology Division Central Drug Research Institute, Lucknow
Re: [ccp4bb] ATP solution
Stability of ATP (to hydrolysis) is pH dependent, and even the disodium ATP is strongly acidic in distilled water. I used to make 100 mM ATP by dissolving disodium or Na,K-ATP in ~100 mM NaOH, getting pH ~6-7. This suggests 8.3 would be better: http://www.patentstorm.us/patents/6916616/description.html Also, the standard phosphate assay hydrolyzes ATP pretty rapidly, so people measuring ATPase use a special assay with milder conditions- names like Fiske-Subbarow and Lohman-Jendrassick come to mind, but not sure if either is the one suitable for ATP. No I think it was the Lowry-Lopez method which uses ascorbic acid at pH 4 instead of ANS as the reducing reagent. dengzq1987 wrote: Dear all, recently, i prepared the ATP solution used to assay ATPase activity. when the Control Well just containing assay buffer and ATP mixed with molybdate ,the colour changed to green, this mean that there is free pi in the solution.the assay buffer and water is free from pi.and the ATP stock solution has free pi ,maybe the ATP hydrolysis when prepared .any suggestion about preparing ATP stock solution is appreciative. Best regards! 2010-08-29 dengzq1987
[ccp4bb] error running reduce/probe in WinCoot
Hello, I'm trying to use the 'probe clashes' function in WinCoot. However, each time I try I get the following error message: Found 4580 hydrogens <0 hets> Standardized 6012 hydrogens <0 hets> Added 344 hydrogens <0 hets> Removed 0 hydrogens <0 hets> Adjusted 123 groups(s) If you publish work which uses reduce, please cite: Word, et al. (1999) J. Mol. Biol. 285, 1735-1747. For more information see http://kinemage.biochem.duke.edu BL WARNING:: reduce didn't run ok, so stop here! run_generic_script (probe, 0) I am aware that someone else had this problem and it was suggested that he solve it by changing which set of lines were commented out in the code. I have tried that, but found no difference when trying to run probe clashes afterward. Does anyone have any other suggestions? Thanks. Julie PCB, Duke University
Re: [ccp4bb] DM NCS averaging question
Hi,
Thanks for reminding me checking the mask. I think their might be
something wrong with the mask, since when DM read in the mask, it says:
Number of columns, rows, sections ... 84 74 69
Map mode 0
Start and stop points on columns, rows, sections -53 30
80 153 -4 64
Grid sampling on x, y, z 136 260 150
Cell dimensions . 135.57100
260.11200 150.2 90.0 101.14000 90.0
Fast, medium, slow axes .ZXY
Minimum density . 0.0
Maximum density . 0.0
Mean density 0.0
Rms deviation from mean density . 0.0
Space-group .4
Number of titles 1
It seems the mask is just null. However, I converted it to a map file, and
coot clearly showed the mask, so I am not sure why the null mask was found
by DM. Moreover, the NCS CCs are just 0s for the mask.
Anyway, following is my NCSMASK script I used to generate the above mask,
where XYZIN is the reorganized pdb containing only a single fixed NCS unit
(chain A). and all the operations were generated by LSQKAB with Chain A
mapped to other chains. Not sure whether there is something wrong here or
not...
ncsmask xyzin ${PDB}_A.pdb mskout ${PDB}.msk << eof
SYMM P1211
EXPAND 1.0
OVERLAP 3
AVERAGE 12
#Identical
ROTA MATRIX 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
TRAN 0.0 0.0 0.0
#A>C
ROTA MATRIX -0.22748 0.97259 0.04813 -0.97372 -0.22662 -0.02273
-0.01120 -0.05203 0.99858
TRAN 101.4683781.74413 2.89341
#A>D
ROTA MATRIX -0.90337 0.42792 0.02831 -0.42883 -0.90066 -0.07011
-0.00451 -0.07547 0.99714
TRAN 158.0331736.91842 3.25853
#A>F
ROTA MATRIX -0.21272 -0.97702 0.01352 0.97675 -0.21300 -0.02424 0.02657
0.00805 0.99961
TRAN 100.69797 -81.01860-1.71365
#A>G
ROTA MATRIX 0.61704 -0.78687 -0.01005 0.78590 0.61553 0.05899 -0.04023
-0.04429 0.99821
TRAN 32.50667 -65.76570 7.05504
#A>H
ROTA MATRIX -0.85814 -0.51116 -0.04813 -0.51290 0.85771 0.03558
0.02310 0.05522 -0.99821
TRAN 156.1498142.2387348.93406
#A>I
ROTA MATRIX -0.13703 -0.98975 -0.04032 -0.99048 0.13635 0.01902
-0.01332 0.04254 -0.99901
TRAN 95.9763082.3094852.82510
#A>J
ROTA MATRIX 0.69662 -0.71695 -0.02645 -0.71716 -0.69691 0.00230
-0.02008 0.01737 -0.99965
TRAN 25.8458859.7628653.68224
#A>K
ROTA MATRIX 0.99467 0.10258 -0.01072 0.10259 -0.99472 0.00088 -0.01057
-0.00197 -0.4
TRAN0.47215-8.8608252.78315
#A>L
ROTA MATRIX 0.55782 0.82946 0.02875 0.82987 -0.55793 -0.00466 0.01218
0.02646 -0.99958
TRAN 36.68436 -68.6383349.21587
#A>M
ROTA MATRIX -0.30892 0.95102 -0.01113 0.95109 0.30890 -0.00370
-0.8 -0.01173 -0.3
TRAN 109.48987 -79.0555052.39334
#A>N
ROTA MATRIX -0.93676 0.34855 -0.03147 0.34600 0.93589 0.06627 0.05255
0.05119 -0.99731
TRAN 162.32979 -29.2680045.41564
eof
> The commonest error with averaging is getting the mask wrong.
> Check that the CCs after application of the averaging start at a
> reasonable value - 0.3 at least and increase with each cycle ( by the
> way why do ncycle 1?)
>
> But in the end the density will not be identical, the Fobs are not
> perfectly symmetric so there will be differences. The best idea is to
> average (with correct matrices - I always find that takes several pases
> before I get them all right - then build molecule A and refit it over
> the others before starting refinement.
>
> EleanorHailiang Zhang wrote:
>> Hi,
>>
>> I am using the following DM script to perform a NCS averaging. I have a
>> fundemental question: after NCS averaging, are the density distrubitions
>> of different NCS unit being averaged supposed to be the same? I found
>> they
>> are different by checking FCDM/PHICDM, and maybe I am wrong somewhere...
>>
>>
>> dm NCSIN ${PDB}.msk HKLIN ${PDBALL}.mtz HKLOUT ${PDBALL}-dm.mtz \
>> <>mode AVER
>>ncycle 1
>>combine PERT
>>scheme ALL
>>solc 0.6213
>> #Identical
>> AVER REFI
>> NCSMASK NMER 1
>> ROTA MATRIX 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
>> TRAN 0.0 0.0 0.0
>> #A>C
>> AVER REFI
>> NCSMASK NMER 1
>> ROTA MATRIX -0.22748 0.97259 0.04813 -0.97372 -0.22662 -0.02273
>> -0.01120 -0.05203 0.99858
>> TRAN 101.4683781.74413 2.89341
>> #A>D
>> AVER REFI
>> NCSMASK NMER 1
>> ROTA MATRIX -0.90337 0.42792 0.02831 -0.42883 -0.90066 -0.07011
>> -0.00451 -0.07547 0.99714
>> TRAN 158.0331736.91842 3.25853
>> #A>F
>> AVER REFI
>> NCSMASK NMER 1
>> ROTA MATRIX -0.21272 -0.97702 0.01352 0.97675 -0.21300
Re: [ccp4bb] ATP solution
If you are using malachite green assay, there will be an issue with the solution turning green at high ATP concentrations. There is a protocol to reduce ATP hydrolysis after the addition of malachite green (http://www.ncbi.nlm.nih.gov/pubmed/16674910). An alternative to malachite green assay is MESG assay. Good luck! >>> dengzq1987 08/29/10 3:00 AM >>> Dear all, recently, i prepared the ATP solution used to assay ATPase activity. when the Control Well just containing assay buffer and ATP mixed with molybdate ,the colour changed to green, this mean that there is free pi in the solution.the assay buffer and water is free from pi.and the ATP stock solution has free pi ,maybe the ATP hydrolysis when prepared .any suggestion about preparing ATP stock solution is appreciative. Best regards! 2010-08-29 dengzq1987
