I've seen this before in some structures and have verified in those cases that correction of the bulk solvent mask fixes the negative density. Those were refined with CNS, and I had a script running model_mask.inp, followed by mama to remove the internal cavities (invert it, do island_erase, invert it back). I then supplied CNS minimization scripts with this corrected bulk solvent mask and the negative density magically disappeared. It was quite convincing that it's indeed empty cavities large enough to be filled with artifact bulk solvent.
Some time ago I bothered Garib with this issue, and his recommendation was to > 1) Put water or some atoms inside the cavity > 2) then use the instructions > > refinement exclude all from [residue] [chain] to [residue] [chain] I apologize to Garib for the lack of follow up on my side - I was supposed to verify it to provide more evidence to support the inclusion of internal cavity removal into future version of refmac. Which I didn't do (too late to say "yet" since this was almost 2 yrs ago). In general, my experience was that although the negative density disappears, effect on R/Rfree is negligible (less than 0.1%). They seem to be cavities made exclusively out of hydrophobic sidechains (i.e. carbon-only), but may set of observations is, of course, limited. On Tue, 2009-06-02 at 10:34 +0100, Ian Tickle wrote: > The other possibility of course is that the data is good, that this is > an accurate experimental result and there really is a void, or at least > a cavity where the mean bulk density is lower than in bulk water. One > way to test the void theory would be to fill the cavity with O atoms of > zero (or very small, say 0.01) occupancy. Hopefully (!) that will > prevent Refmac filling the cavity with bulk solvent. One could then try > giving these O atoms large B factors, say 200, to smear them out, and > then increase the occupancies to titrate the actual bulk density. > > There was a paper by Brian Mathews group (sorry I don't have the > reference) on a high pressure form of lysozyme where they found a large > hydrophobic void (I think sufficient to contain something like 5 > waters). Bulk water could only be compelled to enter the void by > application of very high external pressure. > > Interesting! > > Cheers > > -- Ian > > > -----Original Message----- > > From: [email protected] [mailto:[email protected]] > On > > Behalf Of Dirk Kostrewa > > Sent: 02 June 2009 10:13 > > To: CCP4BB > > Subject: Re: [ccp4bb] solvent mask with refmac > > > > Dear Ana-Maria, > > > > in my experience, mask bulk solvent artifacts can only occur at narrow > > channels, where the mask radius is too big to define the channel as > > belonging to the bulk solvent region, leaving it "empty" and thus > > resulting in positive (!) difference density. Changing from simple > scaling > > to Babinet scaling is an important check to exclude mask bulk solvent > > artifacts, but there, you have to uncheck the "calculate contribution > from > > the solvent region", because this is done by the Babinet scaling, > already. > > However, in your case, you have negative difference density in a, as > you > > say, big cavity. This will be certainly filled with bulk solvent > density. > > One possible reason for a negative difference density are > underestimated > > magnitudes of |Fobs| at very low resolution, either because they are > > weakened by the beam-stop (half-)shadow, or because they are overloads > > that have been poorly extrapolated. A simple check for wrongly > determined > > low-resolution |Fobs| is to cut your low resolution data during > refinement > > at a somewhat higher resolution, say 20 A instead of 80 A, and see > whether > > the negative difference density disappears. If, yes, you should check > your > > data processing again. > > > > Best regards, > > > > Dirk. > > > > Am 02.06.2009 um 10:43 schrieb Ana-Maria Goncalves: > > > > > > Dear BB, > > > > I'm refining a structure which presents a big hydrophobic cavity > > where a strong residual difference negative density can be seen. I > presume > > that this is an effect of not having a proper solvent mask determined > and > > I was wondering if there is a way to provide a better mask description > to > > REFMAC. The resolution of the data is 1.6 A and I have already tried > > changing from simple to Babinet scaling, without any improvement. Any > > advice/suggestion would be very welcome. > > > > Many thanks, > > Ana-Maria > > > > > > > > -- > > Ana-Maria Goncalves > > Macromolecular Crystallography Group > > European Synchrotron Radiation Facility > > B.P. 220, 6 rue Jules Horowitz > > F-38043 GRENOBLE CEDEX > > FRANCE > > > > Tel: +33 (0) 4.76.88.19.12 > > Fax: +33 (0) 4.76.88.29.04 > > > > > > > > > > ******************************************************* > > Dirk Kostrewa > > Gene Center, A 5.07 > > Ludwig-Maximilians-University > > Feodor-Lynen-Str. 25 > > 81377 Munich > > Germany > > Phone: +49-89-2180-76845 > > Fax: +49-89-2180-76999 > > E-mail: [email protected] > > ******************************************************* > > > > > > Disclaimer > This communication is confidential and may contain privileged information > intended solely for the named addressee(s). It may not be used or disclosed > except for the purpose for which it has been sent. 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