The current Rfree selection is done in the highest possible Laue group - eg trigonal uses P6/mmm - then the selection is proogated to the chosen Laue group - eg P3. So IF the ncs reflects a higher Laue symmetry as it often does the FreeR is sort of buffered against the ncs- effect..
That wont always be true of course but it does help avoid NCS bias. Eleanor On Sat, 1 Jun 2019 at 22:57, Jonathan Cooper < 00000c2488af9525-dmarc-requ...@jiscmail.ac.uk> wrote: > I have done some more tests with different programs for choosing the > R-free set in shells or at random and the results are at the same link: > > https://www.ucl.ac.uk/~rmhajc0/rfreetests.pdf > > There still seems to be no significant difference between the normal > R-free and the R-free in shells, with up to 20-fold NCS present. I can't > comment on twinning, but with NCS it would seem that the normal CCP4 way of > picking the R-free set is as good as anything else! > On Sunday, 26 May 2019, 14:02:50 BST, dusan turk <dusan.t...@ijs.si> > wrote: > > > Dear colleagues, > > > > Does ncs bias R-free? And if so, can it be avoided by special selection > of > the free set? > > It occurs to me that we tend to forget that the objective of structure > determination is not the model with the lowest model bias, but the model > which is closest to the true structure. The structure without model bias is > the structure without a model - which is not really helpful. > > An angle on the NCS issue is provided by the work of Silva & Rossmann > (1985, Acta Cryst B41, 147-157), who discarded most of data almost > proportionally to the level of NCS redundancy (using 1/7th for WORK set and > 6/7 for TEST set in the case of 10-fold NCS). They did it in 1990s in order > to make refinement of their large structure computationally feasible: > “Despite the reduction in the number of variables imposed by the > non-crystallographic constraints, the problem remained a formidable one if > all 298615 crystallographically independent reflections were to be used in > the refinement. However, the reduction of size of the asymmetric unit in > real space should be equivalent to a corresponding reduction in reciprocal > space. Hence, one-tenth of refinement of the independent data might suffice > for refinement.” In conclusion they stated that “This is the first time > that the structure of a complete virus has been refined by a > reciprocal-space method.” To conclude, to select an independent data set to > refined against, one should take an n-th fraction of reflections from the > data set containing the n-fold NCS. > > Now on the bias of the concept of R-free itself. As we known, each term in > the Fourier series is orthogonal to all other terms, hence the projection > of any two terms on each other is zero. We also know that diffraction > pattern of a crystal structure is composed of Iobs which reflect Fobs. Fobs > are a Fourier series of terms . From measured set of Iobs we can directly > calculate |Fobs|, but not their phase. To calculate the phase in refinement > we use Fmodel structure factors, of which the most significant part are > Fcalc calculated from atomic model. However, the model is changed during > model building and refinement (atomic positions, B-factors and > occupancies), all Fmodel structure factors change in size and in phase > angle. > > During refinement using a cross validated maximum likelihood target > function atomic model is fitted against the selected subset of |Fobs|, > called WORK set, using a corresponding subset of Fmodel. The remaining part > of structure factors of Fmodel, called the TEST set is used to calculate > the weighted terms used in refinement and is based on phase error > estimates. This Fmodel fraction equally depends on attributes of all atoms > of the model. As consequence, the TEST fraction of Fmodel structure factors > is model dependent. Now comes the catch, if the TEST fraction of structure > factors (Fobs) was truly independent from the model, then it should remain > so also during the refinement. As consequence and simultaneous proof of > this independency, the R-free should not be affected by refinement. As we > know this holds only for the incorrect structure solutions. Their atoms are > refined in direction that do not lead towards the true structure. As soon > as a structure solution is correct, its improvements will lower R-free > because the model is related to the true crystal structure. This is in my > opinion the only true value of the R-free gap criterion. The problems are > that use of the WORK subset makes refinement to aim off the true target and > that the use of TEST fraction for estimating phase error correctness is an > approximation not justified by the claim of independency of the TEST set. I > do not want to undermine the historical importance of the TEST set use for > refinement and structure validation, however we need and can do better. > > As shown by Silva & Rossman in 1985 the concept of independency of a TEST > subset fraction of Fobs structure factors is not true for the structures > composed of equal copies of molecules present in asymmetric unit of a > crystal (crystals with NCS) . The same reasoning can be applied to the > twinned data sets. However, de-twining is model dependent, hence the claim > of independency of TEST and WORK subsets of Fobs structure factors actually > fail due to dependency of the Fmodel WORK and TEST subsets. > > The significant part of model bias originates from the use of chemical > restraints in refinement that effect positions of intermediate bonding and > non-bonding partners and propagate through crystallographic terms to all > atoms. To overcome this problem we replaced the calculation of phase error > estimates, which is based on the TEST subset of structure factors, by > calculation of phase error estimates which is using WORK subset or all data > and Fmodel structure factors calculated from kicked model generated by > randomly displacing atomic positions. In the Figures 6 and 7 there is a > poor or non-existing correlation between R-free gaps and phase errors. For > details please read ( Praznikar, J. & Turk, D. (2014) Free kick instead of > cross-validation in maximum-likelihood refinement of macromolecular crystal > structures. Acta Cryst. D70, 3124-3134. > http://journals.iucr.org/d/issues/2014/12/00/lv5072/lv5072.pd) We > concluded “Since the ML FK approach allows the use of all data in > refinement with a gain in structure accuracy and thereby delivers lower > model bias, this work encourages the use of all data in the refinement of > macromolecular structures.” > > Just to add, it appears that the R-free discussions keep resurfacing, > because the use of the R-free concept in refinement and structure > validation persistently raises doubts about its validity. The discussions > that follow try to strengthen the beliefs. In my opinion, however, »the > persistent use of R-free as an indicator of structure correctness is a > result of the desire to simplify the reality by wishful thinking.” (Turk > (2017), Boxes of Model Building and Visualization, Protein Crystallography, > Methods in MolecularBiology 1607, Springer protocols). > > I hope this helps to clarify a few issues. > > dusan turk > > > On 25 May 2019, at 01:00, CCP4BB automatic digest system < > lists...@jiscmail.ac.uk> wrote: > > > > Date: Fri, 24 May 2019 22:27:28 +0000 > > From: Jonathan Cooper <bogba...@yahoo.co.uk> > > Subject: Re: Does ncs bias R-free? And if so, can it be avoided by > special selection of the free set? > > > > Having been fond of the idea discussed above i.e. that when NCS is > present, one should have the R-free set chosen in shells, I did some simple > tests. Many others must have done the same, but here's how it went: > > 1) Choose a few familiar structures, both with and without NCS and get > the data. > > 2) Since there was some difficulty in remembering if the original R-free > sets were in shells or not, I ditched any existing test set (shock, but see > 3 below) and generated new ones, both at random and in shells (using > SFTOOLS and I repeated some with an old copy of SHELXPRO). Some of the > reflection files lacked original R-free sets since they weredeposited > before the R-free was invented. > > 3) Reduce the bias of each model to the reflections that are now in the > new test setsand tease out over-fitting by rattling the structures a bit, > i.e. add a random +/-0.1 Angstroms to x, y and z of each atom (0.17 > Angstroms net shift) and reset all the B-factors to 30 A^2. > > 4) Refine the rattled structures with the new R-free sets, i.e. random > and in shells (no NCS restraints). > > 5) If anyone is really interested, the results are here: > > https://www.ucl.ac.uk/~rmhajc0/rfreetests.pdf > > but to summarise, assuming the programs have picked the test sets in > shells or otherwise correctly (!), there seems to be no significant > difference between the R-free in shells and the normal one, whether NCS is > present or not. If anything, the R-free in shells tends to be a tiny bit > lower than the normal R-free when NCS is present, although this is probably > by chance due to the small number of tests done! > > I am sure this is a well known fact, but haven't had the chance to test > it till now! On Sunday, 19 May 2019, 13:22:00 BST, Ian Tickle < > ianj...@gmail.com> wrote: > > > > > > Hi Ed > > Yes, Rfree: my favourite topic, I'll take this one on! First off, we > all need to be ultra-careful and precise about the terminology here, for > fear of creating even more confusion. For example what on earth is meant > by "reflections ... are uncorrelated"? A reflection can be regarded as a > object that possesses a set of attributes (indices, d spacing, setting > angles, position on detector, LP correction, intensity, amplitude, phase, > errors in those, etc. etc.). An object as such is not associated with any > kind of value (it is rather an instance of a class of objects possessing > the same set of attributes but with different values for those attributes), > so it's totally meaningless to talk about the correlation, or lack thereof, > of two sets of objects (what's the correlation of a bag of apples and a bag > of oranges?). You can only talk about the correlation of the values of the > objects' attributes (e.g. the apples' and oranges' size or weight). > Perhaps you'll say that it was clear from the context that you meant the > correlation of the reflection's measured intensities (or amplitudes). If > that is what you meant then you would be wrong! The fact that it's not > about NCS-related intensities or amplitudes does rather throw a spanner in > the works of those who claim that's it's the correlation of these > quantities that obliges one to choose the test set in a certain way. > > Before I say why, I would also point out that R factors are not the > quantities minimised in refinement: for one thing the conventional Rwork > and Rfree are unweighted so all reflections whether poorly or well-measured > contribute equally, which makes no sense. In ML refinement it's the > negative log-likelihood gain (-LLG) that's minimised so that is the > quantity you should be using. This means that one cannot expect Rwork to > be a minimum at convergence since it's not directly related to LLGwork. In > addition one has no idea what is the confidence interval of an R factor so > it's impossible to say whether a given decrease in R is significant or > not. So R factors are entirely unsuited for any kind of quantitative > analysis of model errors, and I despair when I read papers that do just > that. The R factor was devised in the 50's before calculators or computers > became readily available and crystallographic computations were performed > with pencil & paper! So the form of the R factor, i.e. using an unweighted > absolute value instead of a weighted square as would have been appropriate > for least squares refinement, was specifically designed as a > rough-and-ready guide of refinement progress, not a quantitative measure. > > To see why it's not about intensities or amplitudes, it's important to > understand the purpose and operation of cross-validation (a.k.a. > 'jack-knife test') with a test set set aside for this purpose and using a > statistic such as LLGfree (or Rfree if you must), in order to quantify the > agreement of the model with the test set. In any scientific experiment the > measuring apparatus is never perfect so never reports the true values of > the quantities being measured: measurement errors are an inevitable fact of > life. Cross-validation flags up the impact of these errors on the model > that is used to explain the measurements by some process of best-fitting to > them. Note that by 'model' I mean the mathematical model, i.e. in this > case the structure-factor equation that relates the atomic model to the > measurements. The adjustments in the model's variable parameters (x, y, z, > B etc.) during refinement may give a closer fit between the true and > calculated amplitudes in which case both -LLGwork and -LLGfree will both > decrease (as indicated above Rwork and Rfree may go up or down > unpredictably). > > Unfortunately we have only the measured amplitudes, not the true ones, > so in this process of fitting one may go too far and fit to the measurement > errors ('overfitting'), which will obviously introduce errors in the > model. If one only considers the refinement target function (LLG) or > Rwork, it will always appear that the model is improving even when it isn't > (i.e. agreeing better with the measured values but not necessarily with the > true values due to the errors in the measured values). This generally > happens because in the attempt to extract more detail in the model from the > data one has set up a model with more variables (or fewer/too loose > restraints) than the data can support. > > Since the changes in the model on overfitting will not be related to > changes required to obtain the true model values but to completely > arbitrary random numbers unrelated to the truth, and provided the > measurement errors in the test set are uncorrelated with those in the > working set, the test-set statistic will most likely go on its own sweet > way (i.e. up) indicating overfitting. If for any reason the measurement > errors of working and test-set reflections are correlated, then the > test-set statistic will be biased towards the working-set value and so will > not be a reliable diagnostic of overfitting. Note that the overfitting > fate is decided at the point where we choose the starting set of parameters > and restraints, though it doesn't become apparent until after the > subsequent refinement run has completed. Then one should redesign the > model with fewer variables and/or more/tighter restraints, and repeat the > last run, rather than proceed further with the faulty model. If > overfitting is diagnosed by the cross-validation test, try something else! > > So there you have it: what matters is that the _errors_ in the > NCS-related amplitudes are uncorrelated, or at least no more correlated > than the errors in the non-NCS-related amplitudes, NOT the amplitudes > themselves. This is like when talking about the standard deviation of a > quantity, do you mean the quantity itself (e.g. the electron density in the > map), or the _error_ in that quantity (the practice of calling the latter > the 'standard deviation in the error' or 'standard error' to avoid this > confusion is to be commended). > > Finally let's examine this: are the _errors_ in the NCS-related > amplitudes expected to be more correlated than errors of non-NCS-related > amplitudes, giving test-set statistic bias if the NCS-related working-set > reflection is selected to be in the test-set. as opposed to having both in > the same set? Clearly counting errors are totally random and uncorrelated > with anything so they will contribute zero correlation to both NCS and > non-NCS-related errors in amplitudes. What other sources of measurement > error are there? - most likely errors in image scale factors, errors due > to variability in the illuminated volume of the crystal and errors due to > radiation damage. Is there any reason to believe that any of these effects > could introduce more correlation of errors of NCS-related intensities > compared with non-NCS-related? I would suggest that this could happen only > by a complete fluke! > > Cheers > > -- Ian > > > > On Sun, 19 May 2019 at 04:34, Edward A. Berry <ber...@upstate.edu> > wrote: > > > > Revisiting (and testing) an old question: > > > > On 08/12/2003 02:38 PM, wgsc...@chemistry.ucsc.edu wrote: > >> *** For details on how to be removed from this list visit the *** > >> *** CCP4 home page http://www.ccp4.ac.uk *** > > > >> On 08/12/2003 06:43 AM, Dirk Kostrewa wrote: > >>> > >>> (1) you only need to take special care for choosing a test set if you > _apply_ > >>> the NCS in your refinement, either as restraints or as constraints. If > you > >>> refine your NCS protomers without any NCS restraints/constraints, both > your > >>> protomers and your reflections will be independent, and thus no > special care > >>> for choosing a test set has to be taken > >> > >> If your space group is P6 with only one molecule in the asymmetric unit > but you instead choose the subgroup P3 in which to refine it, and you now > have two molecules per asymmetric unit related by "local" symmetry to one > another, but you don't apply it, does that mean that reflections that are > the same (by symmetry) in P6 are uncorrelated in P3 unless you apply the > "NCS"? > > > > =================================================== > > The experiment described below seems to show that Dirk's initial > > statement was correct: even in the case where the "ncs" is actually > > crystallographic, and the free set is chosen randomly, R-free is not > > affected by how you pick the free set. A structure is refined with > > artificially low symmetry, so that a 2-fold crystallographic operator > > becomes "NCS". Free reflections are picked either randomly (in which > > case the great majority of free reflections are related by the NCS to > > working reflections), or taking the lattice symmetry into account so > > that symm-related pairs are either both free or both working. The final > > R-factors are not significantly different, even with repeating each mode > > 10 times with independently selected free sets. They are also not > > significantly different from the values obtained refining in the correct > > space group, where there is no ncs. > > > > Maybe this is not really surprising. Since symmetry-related reflections > > have the same resolution, picking free reflections this way is one way > > of picking them in (very) thin shells, and this has been reported not to > > avoid bias: See Table 2 of Kleywegt and Brunger Structure 1996, Vol 4, > > 897-904. Also results of Chapman et al.(Acta Cryst. D62, 227–238). And > see: > > http://www.phenix-online.org/pipermail/phenixbb/2012-January/018259.html > > > > But this is more significant: in cases of lattice symmetry like this, > > the ncs takes working reflections directly onto free reflections. In the > > case of true ncs the operator takes the reflection to a point between > > neighboring reflections, which are closely coupled to that point by the > > Rossmann G function. Some of these neighbors are outside the thin shell > > (if the original reflection was inside; or vice versa), and thus defeat > > the thin-shells strategy. In our case the symm-related free reflection > > is directly coupled to the working reflection by the ncs operator, and > > its neighbors are no closer than the neighbors of the original > > reflection, so if there is bias due to NCS it should be principally > > through the sym-related reflection and not through its neighbors. And so > > most of the bias should be eliminated by picking the free set in thin > > shells or by lattice symmetry. > > > > Also, since the "ncs" is really crystallographic, we have the control of > > refining in the correct space group where there is no ncs. The R-factors > > were not significantly different when the structure was refined in the > > correct space group. (Although it could be argued that that leads to a > > better structure, and the only reason the R-factors were the same is > > that bias in the lower symmetry refinement resulted in lowering Rfree > > to the same level.) > > > > Just one example, but it is the first I tried- no cherry-picking. I > > would be interested to know if anyone has an example where taking > > lattice symmetry into account did make a difference. > > > > For me the lack of effect is most simply explained by saying that, while > > of course ncs-related reflections are correlated in their Fo's and Fc's, > > and perhaps in in their |Fo-Fc|'s, I see no reason to expect that the > > _changes_ in |Fo-Fc| produced by a step of refinement will be correlated > > (I can expound on this). Therefore whatever refinement is doing to > > improve the fit to working reflections is equally likely to improve or > > worsen the fit to sym-related free reflections. In that case it is hard > > to see how refinement against working reflections could bias their > > symm-related free reflections. (Then how does R-free work? Why does > > R-free come down at all when you refine? Because of coupling to > > neighboring working reflections by the G-function?) > > > > Summary of results (details below): > > 0. structure 2CHR, I422, as reported in PDB, with 2-Sigma cutoff) > > R: 0.189 Rfree: 0.264 Nfree:442(5%) Nrefl: 9087 > > > > 1. The deposited 2chr (I422) was refined in that space group with the > > original free set. No Sigma cutoff, 10 macrocycles. > > R: 0.1767 Rfree: 0.2403 Nfree:442(5%) Nrefl: 9087 > > > > 2. The deposited structure was refined in I422 10 times, 50 macrocycles > > each, with randomly picked 10% free reflections > > R: 0.1725±0.0013 Rfree: 0.2507±0.0062 Nfree: 908.9± Nrefl: 9087 > > > > 3. The structure was expanded to an I4 dimer related by the unused I422 > > crystallographic operator, matching the dimer of 1chr. This dimer was > > refined against the original (I4) data of 1chr, picking free reflections > > in symmetry related pairs. This was repeated 10 times with different > > random seed for picking reflections. > > R: 0.1666±0.0012 **Rfree:0.2523±0.0077 Nfree: 1601.4 Nrefl:16011 > > > > 4. same as 3 but picking free reflections randomly without regard for > > lattice symmetry. > > On average 15 free reflections were in pairs, 212 were invariant under > > the operator (no sym-mate) and 1374 (86%) were paired with working > > reflections. > > R: 0.1674±0.0017 **Rfree:0.2523±0.0050 Nfree: 1600.9 Nrefl:16011 > > > > (**-Average Rfree almost identical by coincidence- the individual > > results were all different) > > > > Detailed results from the individual refinement runs are available in > > spreadsheet in dropbox: > > https://www.dropbox.com/s/fwk6q90xbc5r8n1/NCSbias.xls?dl=0 > > Scripts used in running the tests are also there in NCSbias.tgz: > > https://www.dropbox.com/s/sul7a6hzd5krppw/NCSbias.tgz?dl=0 > > > > ======================================== > > > > Methods: > > I would like an experiment where relatively complete data is available > > in the lower symmetry. To get something that is available to everyone, I > > choose from the PDB. A good example is 2CHR, in space group I422, which > > was originally solved and the data deposited in I4 with two molecules in > > the asymmetric unit(structure 1CHR). > > > > 2CHR statistics from the PDB: > > R R-free complete (Refined 8.0 to 3.0 A > > 0.189 0.264 81.4 reported in PDB, with 2-Sig cutoff) > > Nfree=442 (4.86%) > > Further refinement in phenix with same free set, no sigma cutoff: > > 10 macrocycles bss, indiv XYZ, indiv ADP refinement; phenix default > > Resol 37.12 - 3.00 A 92.95% complete, Nrefl=9087 Nfree=442(4.86%) > > Start: r_work = 0.2097 r_free = 0.2503 bonds = 0.008 angles = 1.428 > > Final: r_work = 0.1787 r_free = 0.2403 bonds = 0.011 angles = 1.284 > > (2chr_orig_001.pdb, > > > > The number of free reflections is small, so the uncertainty > > in Rfree is large (a good case for Rcomplete) > > Instead for better statistics, use new 10% free set and repeat 10 times; > > 50 macrocycles, with different random seeds: > > R: 0.1725±0.0013 Rfree: 0.2507±0.0062 bonds:0.010 Angles:1.192 > > Nfree: 908.9±0.32 Nrefl: 9087 > > > > For artificially low symmetry, expand the I422 structure (making what I > > call 3chr for convenience although I'm sure that ID has been taken): > > > > pdbset xyzin 2CHR.pdb xyzout 3chr.pdb <<eof > > exclude header > > spacegroup I4 > > cell 111.890 111.890 148.490 90.00 90.00 90.00 > > symgen X,Y,Z > > symgen X,1-Y,1-Z > > CHAIN SYMMETRY 2 A B > > eof > > > > Get the structure factors from 1CHR: 1chr-sf.cif > > Run phenix.refine on 3chr.pdb with 1chr-sf.cif. > > This file has no free set (deposited 1993) so tell phenix to generate > > one. I don't want phenix to protect me from my own stupidity, so I use: > > generate = True > > use_lattice_symmetry = False > > use_dataman_shells = False > > (the .eff file with all non-default parameters is available as > > 3chr_rand_001.eff in the .tgz mentioned above) > > > > For more significance, use the script multirefine.csh to repeat the > refinement 10 times with different random seed.After each run, grep > significant results into a log file. > > > > > > To check this gives free reflections related to working reflections, I > > used mtz2various and a fortran prog (sortfree.f in .tgz) to separate the > > data (3chr_rand_data.mtz) into two asymmetric units: h,k,l with h>k > > (columns 4-5) and with h<k (col 6-7), listed the pairs, thusly: > > > > mtz2various hklin 3chr_rand_data.mtz hklout temp.hkl <<eof > > LABIN FP=F-obs DUM1=R-free-flags > > OUTPUT USER '(3I4,2F10.5)' > > eof > > sortfree <<eof >sort3.hkl > > > > sort3.hkl looks like: > > ______h>k______ ______h<k______ > > h k l F free F* free* > > 1 2 3 208.97 0.00 174.95 0.00 > > 1 2 5 226.85 0.00 191.65 0.00 > > 1 2 7 144.85 0.00 164.86 0.00 > > 1 2 9 251.26 0.00 261.71 0.00 > > 1 2 11 333.84 0.00 335.18 0.00 > > 1 2 13 800.37 0.00 791.77 0.00 > > 1 2 15 412.92 0.00 409.90 0.00 > > 1 2 17 306.99 0.00 317.53 0.00 > > 1 2 19 225.54 0.00 220.91 0.00 > > 1 2 21 101.20 1.00* 104.84 0.00 > > 1 2 23 156.27 0.00 156.49 0.00 > > 1 2 25 202.97 0.00 202.23 0.00 > > 1 2 27 216.10 0.00 219.28 0.00 > > 1 2 29 106.76 0.00 100.93 0.00 > > 1 2 31 157.32 0.00 154.37 1.00* > > 1 2 33 71.84 0.00 20.78 0.00 > > 1 2 35 179.05 0.00 165.67 0.00 > > 1 2 37 254.04 0.00 239.96 1.00* > > 1 2 39 69.56 0.00 30.61 0.00 > > 1 2 41 56.20 0.00 51.02 0.00 > > > > , and awked for 1 in the free columns. Out of 6922 pairs of reflections, > > in one case: > > 674 in the first asu (h>k) are in the free set, > > 703 in the second asu (h<k) are in the free set > > only 11 pairs have the reflections in both asu free. > > > > out of 16011 refl in I4, > > 6922 pairs (=13844 refl), 1049 invariant (h=k or h=0), 1118 with absent > mate. > > > > out of 1601 free reflections: > > On average 15 free reflections were in pairs, 212 were invariant under > > the operator (no sym-mate) and 1374 (86%) were paired with working > > reflections. > > > > Then do 10 more runs of 50 macrocycles with: > > use_lattice_symmetry = False > > collecting the same statistics > > (also scripted in multirefine.csh) > > > > Finally, use ref2chr.eff to refine (as previously mentined) a monomer in > I422 (2chr.pdb) 10 times with 10% free, 50 macrocycles > > (also scripted in multirefine.csh) > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
