You say *:I ran Phaser in Phenix, it placed 18 molecules, but finds 3 MR solutions (top LLG 7962, TFZ 40). When I open the map in Coot it fits the model well, but there is a fair amount of residual density in between the different molecules (due to tNCS?). However, I can see positive density where my ligand should be. After first refinement R factors drop to ~34 and 40.*
R factors like that mean you have almost certainly got a correct solution. But with 18 molecules in the asymmetric unit you may well have underestimated the crystal symmetry . hard to tell without more information.. Have you done a self rotation? (MOLREP does this well) Can you run integration with that cell and then check the symmetry - I like the output of pointless which lists the CC for possible symmetry elements then AMLESS Rmerges give you a good feel for which images can be used.. On Mon, 28 Apr 2025 at 16:39, Andrea Smith < 00010d84b4ba0dbf-dmarc-requ...@jiscmail.ac.uk> wrote: > Hi Kay, others that replied privately and all, > > > > For the non-integerness I could see numbers such as 2.91, 3.04 but also > one -0.69. > > > > I now used only 200 frames in the spot range for indexing. I used the > frames 1000-1200 that looked the best to me based on diffraction pattern. I > do not see multiple lattices on these frames (see an example in the > attachment), while some other frames are hard to judge because they don't > have that many spots. > > Now the cluster indices are close to integer (such as 1.98 or 1.01), space > group is number 5, unit cell constants 198, 138, 186, 90, 115, 90. Based on > the statistics I then deleted a lot of frames, kept only 550 - 1600 and got > ISa 7.86 and completeness 55%. > > > > When I use frames 500-1000 for indexing, I get the space group number 1. > > > When I try to Show frame with predicted spots, I get an error message > “cannot open image file” while in the terminal it says !!! ERROR !!! > USELESS DATA SET (which is a bit rude :D - see below). > > > > > IMAGE IER SCALE NBKG NOVL NEWALD NSTRONG NREJ SIGMAB SIGMAR > 1100 0 0.966 33961 0 5804 180 0 0.02985 0.24124 > > 175 OUT OF 180 REFLECTIONS ACCEPTED FOR REFINEMENT > REFINED PARAMETERS: POSITION BEAM ORIENTATION > > STANDARD DEVIATION OF SPOT POSITION (PIXELS) 1.51 > STANDARD DEVIATION OF SPINDLE POSITION (DEGREES) 0.54 > SPACE GROUP NUMBER 5 > UNIT CELL PARAMETERS 197.842 138.494 186.493 90.000 115.096 90.000 > REC. CELL PARAMETERS 0.005581 0.007221 0.005921 90.000 64.904 90.000 > COORDINATES OF UNIT CELL A-AXIS 94.889 -163.674 57.864 > COORDINATES OF UNIT CELL B-AXIS -1.739 45.261 130.878 > COORDINATES OF UNIT CELL C-AXIS -186.117 -11.730 1.584 > CRYSTAL ROTATION OFF FROM INITIAL ORIENTATION -0.093 0.067 0.386 > shown as x,y,z components of rotation axis X angle (degrees) > CRYSTAL MOSAICITY (DEGREES) 0.241 > LAB COORDINATES OF ROTATION AXIS 0.999965 0.005821 -0.006052 > DIRECT BEAM COORDINATES (REC. ANGSTROEM) -0.002390 -0.009083 1.088810 > DETECTOR COORDINATES (PIXELS) OF DIRECT BEAM 1228.99 1248.28 > DETECTOR ORIGIN (PIXELS) AT 1234.39 1268.80 > CRYSTAL TO DETECTOR DISTANCE (mm) 423.05 > LAB COORDINATES OF DETECTOR X-AXIS 1.000000 0.000000 0.000000 > LAB COORDINATES OF DETECTOR Y-AXIS 0.000000 1.000000 0.000000 > > > STANDARD DEVIATIONS OF BEAM DIVERGENCE AND REFLECTING RANGE OBTAINED > FROM 175 REFLECTION PROFILES AT 9 POSITIONS ON THE DETECTOR SURFACE. > POSITION NUMBER 1 2 3 4 5 6 7 8 > 9 > X-COORDINATE (pixel) 1231.0 2148.9 1880.1 1231.0 582.0 313.1 581.9 > 1231.0 1880.0 > Y-COORDINATE (pixel) 1263.0 1263.0 1928.9 2204.8 1928.9 1263.0 597.1 > 321.2 597.0 > NUMBER 75 14 12 10 9 9 11 > 16 16 > SIGMAB (degree) 0.046 0.045 0.048 0.048 0.047 0.047 0.047 > 0.047 0.045 > SIGMAR (degree) 0.210 0.216 0.176 0.178 0.213 0.213 0.177 > 0.141 0.211 > > > 5804 REFLECTION INTENSITIES INTEGRATED > > 0 REFLECTIONS SAVED ON FILE "INTEGRATE.HKL" > > !!! ERROR !!! USELESS DATA SET > mv: cannot stat 'FRAME.cbf': No such file or directory > <checking format="CBF"> > CBF::read image: > >> Error: Cannot open image file > > > > > > Pondělí, Duben 28, 2025 12:00 CEST, Kay Diederichs < > kay.diederi...@uni-konstanz.de> napsal: > > > > Hi Andrea, > > if the indexing is incorrect, anything downstream, including integration, > spacegroup determination and structure solution will fail. > > You say "in IDXREF the cluster indices in the third column are not integer > numbers". The question is how far away from integers are they? A bit of > non-integerness (between say -0.15 and +0.15 or so) is normal if the > crystals have high mosaicity since then the spots' centers of gravity do > not always coincide with their theoretical positions. You should adapt > MINIMUM_NUMBER_OF_PIXELS_IN_A_SPOT to the actual reflections of your > dataset; if what you tried so far was 3 (maybe the default in your setup?), > then try at least 6 unless your reflections are really small. > > Other reasons for non-integerness are a split crystal, or multiple > lattices, or wrong geometry specification (beam center, but also rotation > axis, wavelength ...) in XDS.INP. > > One needs to look at the raw data; the first two pathologies should be > visible. Wrong beam center is not an issue since your ISa is reasonable. > Does the pattern of predictions with P1 and the given cell agree with the > pattern of visible reflections? XDSGUI shows this easily; check with > several frames. > > HTH, > Kay > > > On Mon, 28 Apr 2025 10:22:52 +0200, Andrea Smith < > andrea.sm...@uochb.cas.cz> wrote: > > > > >Dear all, > > > >I used XDS with default settings to process my dataset. I cut the data at > 2.5A based on CC1/2 values with *. I got space group number 1, unit cell > constants 120.61, 120.73, 179.39, 105.799, 95.254, 110.186 and ISa 15.54. > However, in IDXREF the cluster indices in the third column are not integer > numbers. When I run pointless, I get a warning that the data may be twinned. > > > >I can see that mosaicity is higher for the first ~600 and last ~400 > frames (up to around 0.4 compared to +- 0.15). > >After running the statistics, I see higher delta CC1/2 for the frames > 1200-2400, while first and last frames have delta CC1/2 lower. > >Cutting off a couple of hundred of first and last frames didn't lead to > assignment of a different space group so at the end I left all the frames > in (also because I excluded some resolution ranges for ice-rings which > already decreased the completeness of the data). > > > >Xtriage detected tNCS, ice rings and a large number of outliers. > >Matthews suggests 17 molecules present in the ASU. > > > >I ran Phaser in ccp4Cloud with different settings, but none of the > results were good. Maps didn't fit the model and R factors were above 50 > after refinement. > >I ran Phaser in Phenix, it placed 18 molecules, but finds 3 MR solutions > (top LLG 7962, TFZ 40). When I open the map in Coot it fits the model well, > but there is a fair amount of residual density in between the different > molecules (due to tNCS?). However, I can see positive density where my > ligand should be. After first refinement R factors drop to ~34 and 40. > > > >Is there something I should do to proceed with such problematic dataset? > It is the best we have :) > > > >Thank you for suggestions, > >Andrea Smith > > > >P.S. Same protein in the same condition but without inhibitor > crystallized in P61 with unit cell constants 86.7 86.7 146 90 90 120. > > > >######################################################################## > > > >To unsubscribe from the CCP4BB list, click the following link: > >https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > > >This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a > mailing list hosted by www.jiscmail.ac.uk, terms & conditions are > available at https://www.jiscmail.ac.uk/policyandsecurity/ > > > > > > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
