Re: [ccp4bb] ligand bonds (AlF3) breaking up after refinement in refmac
Dear Ansuman, I agree with Matthew that the refinement seems to be OK - the reason the bonds are longer is because what you actually have bound is MgF3- and not AlF3. MgF3- is a much better transition state analogue as it is isosteric and isoelectronic with a transferring phosphoryl group. At pH values above ~7.5 Al precipitates out as AlOH and this leaves Mg to fill the gap. AlF3 does exist as a species but it is octahedral and not trigonal bipyramidal and has a water molecule occupying the missing F. There are plenty of MgF structures in the PDB and the MgF bond length is around 1.9A so I am pretty sure this is the species that you have bound. Hope this helps, best wishes, Matt. On 2015-01-16 20:07, ansuman biswas wrote: Dear users, I have a data at 2.2 A resolution. I am able to model AlF3 into the electron density (fig attached). However after one cycle of refinement the AlF3 molecule is exploding and the atoms move apart (fig2). AlF3 is already present in refmac library. First, I used that. But it broke up after refinement. Then, I extracted AlF3 coordinate from already published PDB and prepared the cif file. But, it also failed. I modified the cif file by changing the bond lengths according to the broken AlF3 structure but it was of no help. Kindly suggest how to carry out the refinement. regards, Ansuman
[ccp4bb] Redundancy vs no of frames
Dear all, Can anyone tell me how to calculate number of frames from redundancy or vica versa Thank you
Re: [ccp4bb] additional density on cysteine residue
Dear Sreetama, I would consider the possibility that this active site cysteine is involved in a mixed-disulfide with beta-mercaptoethanol, which is present at a considerable concentration in your protein buffer. The fact that the residual density in both the Fo-Fc and 2Fo-Fc maps actually increased beyond the modeled S-OH group after refinement and the features thereof, provide evidence for the likelihood of a mixed-disulfide with betaME. best wishes Savvas Savvas Savvides Unit for Structural Biology, L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html http://www.lprobe.ugent.be/xray.html On 17 Jan 2015, at 19:22, sreetama das somon_...@yahoo.co.in wrote: Dear Users, I am solving a structure from x-ray diffraction data (1.62A resolution). The protein has a single cysteine residue (which is also the catalytic residue), and it has a positive density on it (fig 1; R/Rfree = 16.88/19.94). The positive density is retained upto 11.5 sigma level. Modelling with water retains the positive density (fig 2; R/Rfree = 16.85/19.94) upto 5.2 sigma level. Modelling with CSO (S-hydroxycysteine, fig 3, R/Rfree = 16.82/ 19.81) produces partial positive and negative densities, which are retained upto 5 sigma. Moreover, after real-space refinement in coot followed by refinement in refmac, the N-terminus of CSO is not bonded to the preceding residue, nor is its C-terminus bonded to the succedding residue. All maps are contoured at 1sigma (2Fo-Fc map) and 3sigma (fo-fc map). The protein preparation contained Tris buffer at pH 7.2, NaCl, glycerol and beta-mercaptoethanol (2mM), while the crystallization condition contained citric acid (pH 3.5) and ammonium sulfate. Please suggest how to interpret the data. thanking in advance, sreetama coot_Cys-job12.pngcoot_Cys+H2O_job10.pngcoot_Cso-job11.png
Re: [ccp4bb] additional density on cysteine residue
Hi Sreetama, The water S-gamma distance made me think that it might be a cysteine beta-mercaptoethanol adduct. Try building CME instead of CSO. Cheers, Robbie -Oorspronkelijk bericht- Van: sreetama das somon_...@yahoo.co.in Verzonden: 17-1-2015 19:27 Aan: CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UK Onderwerp: [ccp4bb] additional density on cysteine residue Dear Users, I am solving a structure from x-ray diffraction data (1.62A resolution). The protein has a single cysteine residue (which is also the catalytic residue), and it has a positive density on it (fig 1; R/Rfree = 16.88/19.94). The positive density is retained upto 11.5 sigma level. Modelling with water retains the positive density (fig 2; R/Rfree = 16.85/19.94) upto 5.2 sigma level. Modelling with CSO (S-hydroxycysteine, fig 3, R/Rfree = 16.82/ 19.81) produces partial positive and negative densities, which are retained upto 5 sigma. Moreover, after real-space refinement in coot followed by refinement in refmac, the N-terminus of CSO is not bonded to the preceding residue, nor is its C-terminus bonded to the succedding residue. All maps are contoured at 1sigma (2Fo-Fc map) and 3sigma (fo-fc map). The protein preparation contained Tris buffer at pH 7.2, NaCl, glycerol and beta-mercaptoethanol (2mM), while the crystallization condition contained citric acid (pH 3.5) and ammonium sulfate. Please suggest how to interpret the data. thanking in advance, sreetama
Re: [ccp4bb] additional density on cysteine residue
Maybe somehow do partial cys partial cme, refine occupancies—is this possible in refmac? JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of sreetama das Sent: Saturday, January 17, 2015 3:11 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] additional density on cysteine residue Hi, Thanks for the quick replies. I modelled in CME and refined. However, I get a blob of negative density around the S-S bond, which is retained up to 5 sigma. Does it mean it is not CME ? Thanks regards, sreetama On Sunday, 18 January 2015 12:41 AM, Roger Rowlett rrowl...@colgate.edumailto:rrowl...@colgate.edu wrote: If CSO does not account for the density -- the SO bond should be about 1.8 A IIRC -- a possibility is an adventitious metal ion. Roger Rowlett On Jan 17, 2015 1:25 PM, sreetama das somon_...@yahoo.co.inmailto:somon_...@yahoo.co.in wrote: Dear Users, I am solving a structure from x-ray diffraction data (1.62A resolution). The protein has a single cysteine residue (which is also the catalytic residue), and it has a positive density on it (fig 1; R/Rfree = 16.88/19.94). The positive density is retained upto 11.5 sigma level. Modelling with water retains the positive density (fig 2; R/Rfree = 16.85/19.94) upto 5.2 sigma level. Modelling with CSO (S-hydroxycysteine, fig 3, R/Rfree = 16.82/ 19.81) produces partial positive and negative densities, which are retained upto 5 sigma. Moreover, after real-space refinement in coot followed by refinement in refmac, the N-terminus of CSO is not bonded to the preceding residue, nor is its C-terminus bonded to the succedding residue. All maps are contoured at 1sigma (2Fo-Fc map) and 3sigma (fo-fc map). The protein preparation contained Tris buffer at pH 7.2, NaCl, glycerol and beta-mercaptoethanol (2mM), while the crystallization condition contained citric acid (pH 3.5) and ammonium sulfate. Please suggest how to interpret the data. thanking in advance, sreetama
Re: [ccp4bb] additional density on cysteine residue
I suspect it may be a reaction with your reducing agent. What did you use either in the preparation, or in the crystallization. If you didn’t have reducing agent it probably oxidized to sulfuric acid. You should figure it out with difference maps and maybe mass spec also. bob On Jan 17, 2015, at 4:10 PM, sreetama das somon_...@yahoo.co.in wrote: Hi, Thanks for the quick replies. I modelled in CME and refined. However, I get a blob of negative density around the S-S bond, which is retained up to 5 sigma. Does it mean it is not CME ? Thanks regards, sreetama On Sunday, 18 January 2015 12:41 AM, Roger Rowlett rrowl...@colgate.edu wrote: If CSO does not account for the density -- the SO bond should be about 1.8 A IIRC -- a possibility is an adventitious metal ion. Roger Rowlett On Jan 17, 2015 1:25 PM, sreetama das somon_...@yahoo.co.in mailto:somon_...@yahoo.co.in wrote: Dear Users, I am solving a structure from x-ray diffraction data (1.62A resolution). The protein has a single cysteine residue (which is also the catalytic residue), and it has a positive density on it (fig 1; R/Rfree = 16.88/19.94). The positive density is retained upto 11.5 sigma level. Modelling with water retains the positive density (fig 2; R/Rfree = 16.85/19.94) upto 5.2 sigma level. Modelling with CSO (S-hydroxycysteine, fig 3, R/Rfree = 16.82/ 19.81) produces partial positive and negative densities, which are retained upto 5 sigma. Moreover, after real-space refinement in coot followed by refinement in refmac, the N-terminus of CSO is not bonded to the preceding residue, nor is its C-terminus bonded to the succedding residue. All maps are contoured at 1sigma (2Fo-Fc map) and 3sigma (fo-fc map). The protein preparation contained Tris buffer at pH 7.2, NaCl, glycerol and beta-mercaptoethanol (2mM), while the crystallization condition contained citric acid (pH 3.5) and ammonium sulfate. Please suggest how to interpret the data. thanking in advance, sreetama coot_CME.png all the best! Bob
Re: [ccp4bb] X-ray Source Differences (WAS: RE: [ccp4bb] How far does rad dam travel?)
If there are no other noise sources, then then final signal to noise of measuring the photons you describe is always EXACTLY the same. This is why photon-counting is such a useful error currency: doesn't matter how you slice them up or lump them together. Photons are photons, and the square root of their count is the error. Think of a detector that not only counts them one at a time, but stores them in individual files. You get a lot of files, but if you add them all together into one or into a million the signal-to-noise is the same. In reality, however, there is ALWAYS another source of error, and if that error changes up with each acquisition, then yes, you do average over the extra error with multiplicity. Examples of such error are shutter jitter, beam flicker, sample vibration, and read-out noise. Detector calibration is also one of these errors, provided you never use the same pixel twice. That's the nature of systematic errors, you can turn them into random errors if you can find a way to keep changing their source. But if you use the same pixels over and over again to measure the same thing you might be only fooling yourself into thinking you are reducing your total error. The number of photons sets a lower limit on the total error. You can't do anything about that. Profile fitting allows you to reduce the error incurred from not clearly knowing the boundary of a spot, but there is no way to get around shot noise (aka photon counting error). But yes, in reality multiplicity is definitely your friend. The trick is making it true multiplicity, where all sources of error have been changed up. Personally, since there is so much contention about using the term multiplicity or redundancy, I think it should be called multiplicity when you are actually averaging over errors, but redundancy when you are not. -James Holton MAD Scientist On 1/15/2015 4:14 PM, Keller, Jacob wrote: I think a summary is that: Background levels and errors thereof can be estimated very precisely as a percentage of the level, but as an absolute number, it nevertheless swamps out the signal contained in a Bragg peak. For example, if background = 10^6 photons +/- 1000 (very good estimate as a percentage--0.1%), that still adds an absolute number of +/- 1000 photons to a Bragg peak which might represent 1000 photons or so, so this noise is a big problem. One would, however, gain a lot by having many pixels in each spot and profile-fitting them, as you mentioned, as this would improve sampling and reduce error. What about this comparison, though: either measure a photon count of 10^6 once on a background of 10^7 or measure the same reflection 1000 times independently at 10^3 photons on a background of 10^4. Assuming no readout noise or other noise source, wouldn't the latter obviously be better? In a sense, each pixel in the Bragg peak in the first case would really be 1000 pixels in the second, and the noise from the background could be cancelled much more effectively? Admittedly this is an orthogonal issue to the background subtraction one, since obviously as you demonstrated the background makes I/sig worse, but this does make background subtraction markedly better, maybe even enough to warrant always erring on the side of too much non-crystal stuff? And definitely always arguing to measure data at low intensities multiple times rather than once at high intensity, for a given total dose! Jacob -Original Message- From: James Holton [mailto:jmhol...@lbl.gov] Sent: Thursday, January 15, 2015 12:00 PM To: Keller, Jacob;CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] X-ray Source Differences (WAS: RE: [ccp4bb] How far does rad dam travel?) Jacob, Background subtraction is unfortunately not as forgiving as you think it is. You can subtract the background, but you can't subtract the noise. This is because noise (by definition) is never the same twice. Yes, the average or true background under a spot may be flat, but any given observation of it will be noisy, and there is no way to separate the noise that came from the Bragg-scattered photons from the background-scattered photons that hit the same pixel. Each photon is an independent event, after all. Consider an example: if you see 4 photons in an isolated spot after 1 second and there is zero background then sigma(I) = sqrt(I) = sqrt(4) = 2, and your I/sigma is most likely 2. I write most likely because the true photon arrival rate (the thing that is proportional to F^2) doesn't have to be 4 photons/s just because you counted four in one observation. The long-term average could easily be something else, such as 3 photons/s, 5 photons/s or even 3.2 photons/s (on average). Observing 4 photons is not unlikely in all these scenarios. However, if you consider all possible true rates, simulate millions of trials and isolate all the instances where you counted 4 photons you will find that
[ccp4bb] Visualizing Stereo view
Dear all, First of all sorry to put this off topic and silly question on bb. Can anybody suggest me, how to create a stereo image and how it is different from the normal. How can I visualize it, if anybody has answer for this please suggest me its significance in analysis. Thank you very much in advance. Thanks Jeorge
