Re: [ccp4bb] Noisy difference maps with high solvent content?
You've probably done it already, but solvent flattening/flipping/massaging at 80% solvent should provide a cheap thrill with regard to phase improvement. = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Fri, 28 Jan 2011 14:29:07 -0600 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Todd Geders ged...@strayxray.com) Subject: [ccp4bb] Noisy difference maps with high solvent content? To: CCP4BB@JISCMAIL.AC.UK Greetings CCP4bb, Short version: Very noisy difference maps from a crystal with extremely high solvent content, seeking advice on how best to handle such high solvent content to eliminate noise in difference maps. http://strayxray.com/images/coot.jpg Long version: I'm having trouble with a 3.0Å dataset from a crystal with 80% solvent content. The space group is P4132 and I'm quite confident the high solvent content is real (there is a species-specific set of helices extending into the solvent channels that appears to prevent tighter packing). I was able to get a MR solution using a structurally related enzyme, but the difference maps are terribly noisy (see link). There are lots of negative density in empty spaces between well-defined 2Fo-Fc electron density. http://strayxray.com/images/coot.jpg The 2Fo-Fc density actually looks fairly good. The initial MR maps had clear density correlating to the sequence differences between the MR model and the crystallized protein. After fixing the model as best I could, the refinement statistics are R/Rfree of 27.5/30.3 with a data/parameters ratio of 1.7. The mosaicity ranges from 0.15-0.3, data were collected with 0.5° oscillations and 180° of data were collected. http://strayxray.com/images/diffraction.jpg Since the crystals appeared to suffer from radiation decay (based on scaling statistics), I only use the first 40° of data (which still gives around 8-fold redundancy). Using more minimal wedges of data or more data does not noticeably make better or noisier maps. Any advice on improving the maps? Could the noisy maps be due to the extraordinarily high solvent content? I'd appreciate any advice or comments. ~Todd Geders
Re: [ccp4bb] Noisy difference maps with high solvent content?
Hi, This sort of problem can occur if you are missing your lowest resolution data and/or your model for the bulk solvent is inappropriate. You might want to double check these issues. With 80% solvent you have to be careful when choosing your contour level. If you are a fan of normalized maps and contouring based on sigma (and it's not a sigma by the way) you should be aware that the normalization factor is calculated over the protein and all that empty space and will be smaller than one calculated for equivalent protein density in a low solvent crystal. Plus/minus 3 contours will be lower and the significance of features will be inflated. One way to calibrate a contour level would be to leave out a known good bit of model and calculate your difference map. Then select a contour level that shows the understood omission well. Other peaks that show up at that level are errors as significant as the one you created. Dale Tronrud On 01/28/11 12:29, Todd Geders wrote: Greetings CCP4bb, *Short version: Very noisy difference maps from a crystal with extremely high solvent content, seeking advice on how best to handle such high solvent content to eliminate noise in difference maps. * *http://strayxray.com/images/coot.jpg* Long version: I'm having trouble with a 3.0Å dataset from a crystal with 80% solvent content. The space group is P4132 and I'm quite confident the high solvent content is real (there is a species-specific set of helices extending into the solvent channels that appears to prevent tighter packing). I was able to get a MR solution using a structurally related enzyme, but the difference maps are terribly noisy (see link). There are lots of negative density in empty spaces between well-defined 2Fo-Fc electron density. http://strayxray.com/images/coot.jpg The 2Fo-Fc density actually looks fairly good. The initial MR maps had clear density correlating to the sequence differences between the MR model and the crystallized protein. After fixing the model as best I could, the refinement statistics are R/Rfree of 27.5/30.3 with a data/parameters ratio of 1.7. The mosaicity ranges from 0.15-0.3, data were collected with 0.5° oscillations and 180° of data were collected. http://strayxray.com/images/diffraction.jpg Since the crystals appeared to suffer from radiation decay (based on scaling statistics), I only use the first 40° of data (which still gives around 8-fold redundancy). Using more minimal wedges of data or more data does not noticeably make better or noisier maps. Any advice on improving the maps? Could the noisy maps be due to the extraordinarily high solvent content? I'd appreciate any advice or comments. ~Todd Geders
Re: [ccp4bb] Noisy difference maps with high solvent content?
Are you sure that the space group is right? P4132 is cubic, so the spots should have equal spacing on the detector, i.e., squares. Don't you have rectangles? Even the least distorted spots towards the beamstop (bottom right?) seem to be non-square rectangles. JPK On Fri, Jan 28, 2011 at 3:07 PM, Dale Tronrud det...@uoxray.uoregon.edu wrote: Hi, This sort of problem can occur if you are missing your lowest resolution data and/or your model for the bulk solvent is inappropriate. You might want to double check these issues. With 80% solvent you have to be careful when choosing your contour level. If you are a fan of normalized maps and contouring based on sigma (and it's not a sigma by the way) you should be aware that the normalization factor is calculated over the protein and all that empty space and will be smaller than one calculated for equivalent protein density in a low solvent crystal. Plus/minus 3 contours will be lower and the significance of features will be inflated. One way to calibrate a contour level would be to leave out a known good bit of model and calculate your difference map. Then select a contour level that shows the understood omission well. Other peaks that show up at that level are errors as significant as the one you created. Dale Tronrud On 01/28/11 12:29, Todd Geders wrote: Greetings CCP4bb, *Short version: Very noisy difference maps from a crystal with extremely high solvent content, seeking advice on how best to handle such high solvent content to eliminate noise in difference maps. * *http://strayxray.com/images/coot.jpg* Long version: I'm having trouble with a 3.0Å dataset from a crystal with 80% solvent content. The space group is P4132 and I'm quite confident the high solvent content is real (there is a species-specific set of helices extending into the solvent channels that appears to prevent tighter packing). I was able to get a MR solution using a structurally related enzyme, but the difference maps are terribly noisy (see link). There are lots of negative density in empty spaces between well-defined 2Fo-Fc electron density. http://strayxray.com/images/coot.jpg The 2Fo-Fc density actually looks fairly good. The initial MR maps had clear density correlating to the sequence differences between the MR model and the crystallized protein. After fixing the model as best I could, the refinement statistics are R/Rfree of 27.5/30.3 with a data/parameters ratio of 1.7. The mosaicity ranges from 0.15-0.3, data were collected with 0.5° oscillations and 180° of data were collected. http://strayxray.com/images/diffraction.jpg Since the crystals appeared to suffer from radiation decay (based on scaling statistics), I only use the first 40° of data (which still gives around 8-fold redundancy). Using more minimal wedges of data or more data does not noticeably make better or noisier maps. Any advice on improving the maps? Could the noisy maps be due to the extraordinarily high solvent content? I'd appreciate any advice or comments. ~Todd Geders -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Noisy difference maps with high solvent content?
Hi Todd, in addition to all other suggestions, here is some basic sanity check that you can do in no time: - how the R-factor vs Resolution plot looks like? - did the bulk-solvent correction was done right (what are k_sol and B_sol values)? - plot the scatterer graph Fobs vs Fmodel and see if there are severe outliers? Did removing them changes the map? - how the Data completeness vs Resolution plot looks like? - how POLYGON plot looks like? The answers will give some food for thought, and probably will invite some more more specific suggestions. All the best! Pavel. On Fri, Jan 28, 2011 at 12:29 PM, Todd Geders ged...@strayxray.com wrote: Greetings CCP4bb, *Short version: Very noisy difference maps from a crystal with extremely high solvent content, seeking advice on how best to handle such high solvent content to eliminate noise in difference maps. * *http://strayxray.com/images/coot.jpg* Long version: I'm having trouble with a 3.0Å dataset from a crystal with 80% solvent content. The space group is P4132 and I'm quite confident the high solvent content is real (there is a species-specific set of helices extending into the solvent channels that appears to prevent tighter packing). I was able to get a MR solution using a structurally related enzyme, but the difference maps are terribly noisy (see link). There are lots of negative density in empty spaces between well-defined 2Fo-Fc electron density. http://strayxray.com/images/coot.jpg The 2Fo-Fc density actually looks fairly good. The initial MR maps had clear density correlating to the sequence differences between the MR model and the crystallized protein. After fixing the model as best I could, the refinement statistics are R/Rfree of 27.5/30.3 with a data/parameters ratio of 1.7. The mosaicity ranges from 0.15-0.3, data were collected with 0.5° oscillations and 180° of data were collected. http://strayxray.com/images/diffraction.jpg Since the crystals appeared to suffer from radiation decay (based on scaling statistics), I only use the first 40° of data (which still gives around 8-fold redundancy). Using more minimal wedges of data or more data does not noticeably make better or noisier maps. Any advice on improving the maps? Could the noisy maps be due to the extraordinarily high solvent content? I'd appreciate any advice or comments. ~Todd Geders
