Reforma a la Ley de creación del Servicio de Rentas Internas, para

(April 2001)

We have recently solved the structure of the PDZ1 domain of Na+/H+ exchanger regulatory factor using the dispersive signal from the LIII edge of Mercury (see Webster at al. (2001) Acta Cryst D57, 714-716 and our J.Mol.Biol.308, 963-973 (2001) paper). We were unable to obtain satisfactory expression of our protein from selenomethionine auxotrophs and only obtained a single mercury derivative in spite of an extensive heavy atom screen from which the SIR phases were insufficient to solve the structure. In the end then, we decided to try a MAD experiment using our lone Mercury derivative and obtained a beautiful anomalous signal at three different wavelengths on beamline F2

at CHESS. An analysis of our data with SOLVE yielded excellent phases and a model consisting of over 80% of the protein was built by ARP/WARP in the first electron density map calculated with the new phases.

I was wondering whether anybody had done a survey of elements other than Selenium that have been successfully used for structure determination with MAD, since it seems that a lot of time can be saved if even a single, suitable heavy-atom derivative of a protein can be obtained for such an experiment. I know that there are plenty of tables of wavelengths and dispersive differences for different elements, but I would be very interested to see if anybody had compiled statistics for which elements had actually worked for MAD structure determinations. Such a survey might beneficially bias our choice of which heavy-atoms are worth screening first, especially if the biological labelling of proteins is not an option due to time constraints or technical problems at the level of expression etc.

Summary from the enquirer: It seems that there hasn't really been a comprehensive review of this for some time now. I was pointed to an article in Synchrotron Radiation News Vol 8 No 3, pp 13-18 (1995) written by Craig Ogata and Wayne Hendrickson, and a later article from 1999 also by Wayne Hendrickson (J. Synchrotron Rad. 6, 845-851). People at Daresbury have found Xenon at high pressure to be an excellent choi ce, their results for this work on the structure of crustacyanin is Cianci et al. Acta D.57,1219-1229. Note that sulphur (sulfur if you celebrate July 4th) has a useful anomalous signal at around 2.0Å and work using this method will be published in a forthcoming paper. It is commented that 3 wavelength experiments are often unnecessary and that the anomalous signal from a single atom of e.g. iron or zinc per protein molecule can be enough for structure determination with MAD. Also advocated is the use of elements that have a significant anomalous signal close to the copper K-alpha wavelength and therefore do not require a trip to the synchrotron. Even mercury has 7.7 anomalous electrons at 1.54Å and it was suggested that we might possibly have been able to solve our PDZ structure in-house. A protein using Xenon at 1.54Å, with 4 atoms per 47 kDa molecule (another plug for Xe there), has just been solved.

A whole slew of elements (Fe, Co, Zn, Se, Br, Rb, Ta, W, Re, Os, Ir, Pt, Au, Hg, Tl, Pb, U) was listed, with which success has been had on the beamline I9ID at the APS (Argonne Il.). It was pointed out that Se-Met has become a very popular choice due to the very high success rate that it has for phasing. The number of Se atoms generally increases with the size of the protein and there is no disturbance of the crystals by soaking as is required for traditional heavy-atom labeling. My own experience with Se-Met has led me to ...

WEBSTER'S LAWS OF METHIONINE DISTRIBUTION

"The probability of a methionine residue occurring in a protein is inversely proportional to my desire to solve the structure of that protein" "The probability of finding a methionine residue at any given point in my protein is directly proportional to the conformational flexibility of my protein at that point"

Please don't flame me or bombard me with your "selenomethionine has changed my life" stories, I know it works very well, but I just haven't been very lucky with it so far! A third article was mentioned: C. Ogata (1998) "MAD phasing grows up" Nat Struct Biol Synchrotron suppl, 638-640. Somebody mentioned they did a survey of the elements used for MAD a few years ago (but did you publish the survey?) and also cited many of the elements in the list above. Another made the excellent suggestion of having specific phasing records included in the PDB database format. This would make the compilation of the kind of statistics that I was after, effectively automatic, since users would be able to compile their own surveys directly from the database itself. How about it RCSB?

It was pointed out that you can do MAD with any element that has an absorption edge within the energy range of the most commonly used beamlines (7000 - 15000 eV) and that L-edges like the one that we used in our PDZ structure determination, often give better results than K-edges. Along with mercury, gold and lead are recommended as good candidates. Reservations are expressed about using platinum which tends to yield many poorly occupied sites and a resulting poor signal. Also recommended: Lanthanides for their excellent signal with the caveat that they may be harder to get to bind to your protein (apparently they substitute for Ca very well in Ca binding proteins). Tantalum bromide has been used for very large cells (didn't they use this for the ribosome?). And again the recommendation for trying high pressure derivatization using Xe and NaBr.