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Hi Nicholas, > This is my first experimental phasing experiment, so I would like some advice or guidance about either SAD or MAD phasing procedures in general. > For example, what programs do people typically use, and why choose one program over another? Which is most reliable? I had expected the experimental phases to give me a map with good enough quality to at least > build a rough model, but so far, this isn't the case. So what steps can be taken now to improve the quality of the maps or phases to at least get > to a map with some discernable features? It sounds like you're lumping 3 steps into one (heavy atom site location, phasing/refinement, and solvent flattening), so I'd recommend troubleshooting each step seperately. For site finding, with no prior phase info you're more or less limited to anomalous/dispersive difference patterson or direct-methods based techniques. Once you've got a few sites, you can phase off of them for use in anomalous/dispersive difference fouriers (I'm a big fan of these, but obviously they require some phases. You'll probably have to try both hands here). I've heard good things about likelihood residual maps, but haven't had a chance to try them out yet. As Ana Gonzalez said, it's better to use fewer sites than include sites that you're unsure about. Sometimes people will do difference amplitude or patterson refinement of the sites (gref or vecref), but this may not be necessary. For phasing and phase refinment, there are quite a few programs to choose from, and which to choose seems to be a mix of which program you feel more comfortable with, and which is happier with your data. Personally I like phasit, but cns or mlphare should give similar results. Sharp and bp3 (based on my understanding) use different refinement targets, which may or may not make a difference for your data. Solvent flattening (or flipping, which people sometimes refer to as solvent flattening) can be sensitive to the percentage solvent in your crystal, so you may want to try a range of values. Solvent flattening is very dependent on getting the correct mask, so if it doesn't work well you could try reducing the resolution (and later doing phase extension). One thing I found helpful when starting out with experiment phasing was to run through one of the tutorials, or use a test dataset, to make it easier to make sure I was doing the procedure correctly (as opposed to the procedure not working on my experimental data). If you do have to recollect, I'll second Ana Gonzalez's comments about collecting in wedges (or inverse beam, for anomalous). I've also had good experience integrating anomalous wedges in chronological order (instead of rotation order), but this may be overkill. Good luck, Pete Pete Meyer Fu Lab BMCB grad student Cornell University
