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Dear All, I have refined these atoms as water with half occupancy. I have a ligand in the solution and most of the half occupied water molecules form a cluster close to this ligand which is bound to my protein but not at full occupancy. The B-values are between 9 and 15 for 0.5 occupied water but my guess is that it is not water but ligand with an alternative conformation I see. I will do new experiments increasing the ligand concentration of my crystals to see if I could increase the occupancy and see it more clearly. Best regards Maria 22 Sep 2008 kl. 14:19 skrev Eleanor Dodson: > First check - look for anom peaks. > If your S atoms are showing up in the Dano map, them you can check > whether this is a compound with S in it. crystallisation and cro- > protectants contain a wealth of small molecules which often bind > Eleanor > > > > 9 sigma peaks are rarely due to multiple site waters.. > Eleanor > > Borhani, David wrote: >> Maria, 1.7 A is too long for common diatomic molecules (N2, 1.1 A; >> O2, 1.2 A; CO, 1.1 A) or molecules like hydrogen peroxide (~1.48 >> A), methanol (ditto), etc. >> >> Does the "water" have unusually low temp. factors (i.e., it's >> really a much heavier atom, and the peak you see is a Fourier >> ripple)? >> >> It may be indeed that you have (one) water at two alternate >> positions, but I think you need to reset to nothing modeled to be >> certain of density interpretaion moving forward. >> >> Thus, try removing the water you have already built, any >> surrounding waters or other ligands, and also the protein atoms to >> which all these are hydrogen bonded (set occ = 0), refine a few >> cycles, and then look at the diff. maps. >> >> Dave >> David Borhani, Ph.D. >> D. E. Shaw Research, LLC >> 120 West Forty-Fifth Street, 39th Floor >> New York, NY 10036 >> [log in to unmask] >> 212-478-0698 >> http://www.deshawresearch.com >> >> >>> -----Original Message----- >>> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf >>> Of Maria Håkansson >>> Sent: Friday, September 19, 2008 8:00 AM >>> To: [log in to unmask] >>> Subject: Re: [ccp4bb] Unexplained electron density >>> >>> Hello David and others, >>> >>> Thanks for yur comments. >>> >>> I guess it might be as simple as water molecules, present in the >>> structure but not at the same time. >>> >>> The density looks like a rod with uneven distribution. Both ends >>> of the rods (1.7-1.8 Å in between) >>> make hydrogen bonds to protein or other water molecules - normal >>> distances (2.3-3.3 Å). Could it be >>> strong water molecules but with partial occupancy meaning that >>> both sites are occupied but not at the same time? >>> >>> I guess refmac automatically refines the molecules that way >>> although I have not specified it in my file. So after >>> refinement as too close water molecules there is no clash just >>> nice density. However I assume it is appropriate to >>> specify these water molecules as the same water but with an >>> alternative conformation in the pdb file. >>> >>> Best Regards, >>> >>> Maria >>> >>> >>> >>> >>> 19 Sep 2008 kl. 11:10 skrev David Briggs: >>> >>> >>>> Hi Maria, >>>> >>>> Initial questions: >>>> 1) What's present in crystallisation/purification buffers? >>>> 2) Are any other ligand visible for the 9sigma peak? >>>> 3) Does the 9 sigma peak also have a peak in an anomalous >>> difference >>>> map? >>>> >>>> Assuming 1.7A is accurate (and with 1.5A resolution, you'd hope it >>>> would be!) a metal-ion - water interaction is looking less likely. >>>> Take a look at Marjorie Harding's papers and website for >>> target metal >>> >>>> ion - ligand distances, and the closest you'll see is water:Mg2+, >>>> at >>>> 2.07. >>>> >>>> http://tanna.bch.ed.ac.uk/newtargs_06.html >>>> >>>> So one would assume it is a covalently bonded compound. >>>> So back to question 1. >>>> >>>> What's in your buffers? >>>> A quick search for bond length tables suggests Carbon-Sulphur (1.8) >>>> and Carbon-Chlorine at 1.7. >>>> >>>> hope this helps >>>> >>>> David >>>> >>>> 2008/9/19 Maria Håkansson <[log in to unmask]>: >>>> >>>>> Hello All, >>>>> >>>>> I have a problem with a 9 sigma positive peak 1.7 Å away >>> from a water >>> >>>>> molecule (or what I believe is >>>>> a water molecule). There are several similar peaks in my >>> map though >>>>> only one >>>>> is as high as 9 sigma. >>>>> >>>>> My first thought was to exclude these too close waters. >>> However the >>>>> R-values >>>>> increased by more than 0.5 %. Could it >>>>> be carbonmonoxide or oxygen atoms? By the way, It is 1.5 Å >>>>> resolution data. >>>>> Any suggestions? >>>>> >>>>> Best Regards, >>>>> >>>>> Maria Håkansson >>>>> >>>>> >>>>> >>>>>> Maria Håkansson, Ph.D. >>>>>> tel: +46 (0) 76 8585 706 >>>>>> Senior Scientist, Max-lab, Lund University >>>>>> fax: +46 46 222 47 10 >>>>>> Ole Römers väg 1 (P.O. Box 188) >>>>>> www.maxlab.lu.se >>>>>> SE-221 00 Lund, Sweden >>> [log in to unmask] >>> >>>> >>>> -- >>>> ============================ >>>> David C. Briggs PhD >>>> Father & Crystallographer >>>> http://drdavidcbriggs.googlepages.com/home >>>> AIM ID: dbassophile >>>> ============================ >>>> Maria Håkansson, Ph.D. >>>> tel: +46 (0) 76 8585 706 >>>> Senior Scientist, SARomics Biostructures AB fax: +46 46 19 12 77 >>>> Scheelevägen 22 (P.O. Box 724) >>> www.saromicsbiostructures.com >>> >>>> SE-220 07 Lund, Sweden >>> [log in to unmask] >>> >> >> >> >> > Maria Håkansson, Ph.D. > tel: +46 (0) 76 8585 706 > Senior Scientist, SARomics Biostructures AB fax: +46 46 19 12 77 > Scheelevägen 22 (P.O. Box 724) www.saromicsbiostructures.com > SE-220 07 Lund, Sweden [log in to unmask] >