Hi Sabine, your reduced cell angles are exactly 90 degree. This seems unusual to me. Do you specify the unit cell constants in XDS.INP, including a space group, or do you leave the space group undefined? In the former case I am unsure if your list of clusters may be biased, although it might contain half integers in that case.
Best, Tim On Thursday, November 17, 2016 08:37:07 AM Sabine Schneider wrote: > Hello Kay, > > I'd say yes, but I might miss something. > > Here is the IDXREF.LP output: > > Sabine > > > ***** DETERMINATION OF THE RECIPROCAL LATTICE BASIS ****** > NUMBER OF DIFFERENCE VECTOR CLUSTERS USED 200 > MAXIMUM RADIUS OF DIFFERENCE VECTOR CLUSTERS (pixels) 3 > MINIMUM DISTANCE BETWEEN DIFFRACTION SPOTS (pixel) 6.0 > MINIMUM ALLOWED DISTANCE BETWEEN REC. LATTICE POINTS 0.2682E-02 > OBSERVED BASIS CELL VOLUME 0.6021E+06 > DIMENSION OF SPACE SPANNED BY DIFFERENCE VECTOR CLUSTERS 3 > > # COORDINATES OF REC. BASIS VECTOR LENGTH 1/LENGTH > > 1 0.0070481-0.0043879-0.0144998 0.0167084 59.85 > 2 0.0099022 0.0011565 0.0044633 0.0109230 91.55 > 3 -0.0001404-0.0087276 0.0025729 0.0091000 109.89 > > CLUSTER COORDINATES AND INDICES WITH RESPECT TO REC. LATTICE BASIS VECTORS > > # COORDINATES OF VECTOR CLUSTER FREQUENCY CLUSTER INDICES > 1 -0.0198775-0.0023086-0.0089462 3033. -0.00 -2.01 -0.00 > 2 0.0170296-0.0032635-0.0100896 2931. 1.01 1.00 0.00 > 3 -0.0028512-0.0055653-0.0190257 2769. 1.00 -1.00 0.00 > 4 0.0001345 0.0087600-0.0025802 2756. -0.00 -0.00 -1.00 > 5 -0.0099406-0.0011518-0.0044669 2748. -0.00 -1.00 -0.00 > 6 0.0369226-0.0009571-0.0011409 2572. 1.01 3.01 0.00 > 7 -0.0027157 0.0031989-0.0215974 2517. 1.00 -1.00 -1.00 > 8 0.0102242 0.0186700-0.0006904 2503. 0.00 1.00 -2.01 > 9 -0.0098018 0.0076072-0.0070529 2471. -0.00 -1.00 -1.00 > 10 0.0096649-0.0163634 0.0096361 2465. 0.00 1.00 2.01 > 11 -0.0171648-0.0055039 0.0126657 2460. -1.00 -1.00 1.00 > 12 -0.0070927 0.0044138 0.0145582 2456. -1.00 -0.00 -0.00 > 13 0.0072273 0.0043496-0.0171271 2446. 1.00 0.00 -1.00 > 14 0.0068016-0.0219329-0.0093878 2437. 1.00 0.00 2.01 > 15 0.0197453-0.0064538 0.0115256 2428. 0.00 2.01 1.00 > 16 0.0100947 0.0099143 0.0018904 2413. 0.00 1.00 -1.00 > 17 0.0073643 0.0131126-0.0197118 2403. 1.00 0.00 -2.01 > 18 0.0397690 0.0046107 0.0178903 2388. 0.00 4.01 0.00 > 19 0.0227406 0.0078707 0.0279747 2383. -1.00 3.01 0.00 > 20 -0.0141824 0.0088224 0.0291127 2383. -2.01 -0.00 -0.00 > 21 0.0103597 0.0274362-0.0032718 2361. 0.00 1.00 -3.01 > 22 -0.0269727 0.0021092 0.0056176 2346. -1.01 -2.01 -0.00 > 23 0.0340618-0.0065201-0.0201693 2329. 2.01 2.01 0.00 > 24 -0.0200231-0.0110674-0.0063643 2326. -0.00 -2.01 1.00 > 25 0.0125162-0.0108063 0.0286538 2325. -1.00 2.01 2.01 > 26 -0.0126463 0.0020440-0.0260664 2323. 1.00 -2.00 -1.00 > 27 0.0143145-0.0000672-0.0316916 2308. 2.01 0.00 -1.00 > 28 0.0168948-0.0120237-0.0075083 2306. 1.01 1.00 1.01 > 29 0.0066708-0.0307011-0.0068110 2284. 1.00 0.00 3.01 > 30 0.0002795 0.0175230-0.0051646 2283. -0.00 -0.00 -2.01 > 31 -0.0226058 0.0008922-0.0305478 2283. 1.00 -3.01 -1.00 > 32 0.0095181-0.0251297 0.0122100 2276. 0.00 1.00 3.01 > 33 -0.0029959-0.0143306-0.0164454 2250. 1.00 -1.00 1.00 > 34 0.0130704 0.0242365 0.0183371 2229. -1.00 2.01 -2.01 > 35 0.0267005-0.0196259-0.0004455 2214. 1.01 2.01 2.01 > 36 -0.0057125-0.0111320-0.0380540 2212. 2.01 -2.01 0.00 > 37 0.0127956 0.0067207 0.0235036 2195. -1.00 2.01 0.00 > 38 0.0272584 0.0154143-0.0107700 2194. 1.01 2.01 -2.01 > 39 0.0075127 0.0218694-0.0222930 2180. 1.00 0.00 -3.01 > 40 0.0045134 0.0075431-0.0387461 2180. 2.01 -1.00 -2.01 > 41 0.0298233 0.0034561 0.0134186 2169. 0.00 3.01 0.00 > 42 0.0271311 0.0066555-0.0081839 2159. 1.01 2.01 -1.00 > 43 -0.0242614-0.0010891 0.0272244 2138. -2.01 -1.01 1.00 > 44 0.0341972 0.0022350-0.0227552 2121. 2.01 2.01 -1.00 > 45 -0.0043753 0.0012122 0.0361620 2118. -2.01 1.00 1.00 > 46 0.0244151 0.0098478-0.0298116 2112. 2.01 1.01 -2.01 > 47 -0.0055691-0.0023701-0.0406352 2104. 2.01 -2.01 -1.00 > 48 -0.0301120-0.0209742-0.0082509 2090. -0.00 -3.01 2.01 > 49 -0.0296785 0.0052997-0.0159965 2086. -0.00 -3.01 -1.01 > 50 -0.0268292 0.0108693 0.0030314 2076. -1.01 -2.01 -1.01 > 51 -0.0004199-0.0262847 0.0077376 2066. 0.00 -0.00 3.01 > 52 0.0132169 0.0330109 0.0157589 2061. -1.00 2.01 -3.01 > 53 -0.0005534-0.0350474 0.0103260 2049. 0.00 0.00 4.02 > 54 -0.0123845 0.0195574-0.0312418 2043. 1.00 -2.01 -3.01 > 55 -0.0201672-0.0198288-0.0037871 2039. -0.00 -2.01 2.01 > 56 -0.0007071-0.0438227 0.0129006 2026. 0.00 -0.00 5.02 > 57 -0.0025795 0.0119525-0.0241882 2025. 1.00 -1.00 -2.01 > 58 0.0396384-0.0041547 0.0204763 2025. 0.00 4.02 1.01 > 59 0.0196131-0.0152142 0.0141121 2020. 0.00 2.01 2.01 > 60 -0.0241198 0.0076707 0.0246372 2014. -2.01 -1.01 -0.00 > > PARAMETERS OF THE REDUCED CELL (ANGSTROEM & DEGREES) > 59.85 91.55 109.89 90.00 90.00 90.00 > > # COORDINATES OF REC. BASIS VECTOR REDUCED CELL INDICES > > 1 0.0070481-0.0043879-0.0144998 1.00 0.00 0.00 > 2 0.0099022 0.0011565 0.0044633 0.00 1.00 0.00 > 3 -0.0001404-0.0087276 0.0025729 0.00 0.00 1.00 > > > LATTICE- BRAVAIS- QUALITY UNIT CELL CONSTANTS (ANGSTROEM & DEGREES) > CHARACTER LATTICE OF FIT a b c alpha beta gamma > > * 44 aP 0.0 59.9 91.6 110.0 90.0 90.0 90.0 > * 31 aP 0.4 59.9 91.6 110.0 90.0 90.0 90.0 > * 33 mP 0.9 59.9 91.6 110.0 90.0 90.0 90.0 > * 34 mP 1.1 59.9 110.0 91.6 90.0 90.0 90.0 > * 35 mP 1.2 91.6 59.9 110.0 90.0 90.0 90.0 > * 32 oP 1.6 59.9 91.6 110.0 90.0 90.0 90.0 > 37 mC 249.7 227.9 59.9 91.6 90.0 90.0 74.8 > 36 oC 250.1 59.9 227.9 91.6 90.0 90.0 105.2 > 39 mC 250.3 192.7 59.9 110.0 90.0 90.0 71.9 > 28 mC 250.3 59.9 227.9 91.6 90.0 90.0 74.8 > 38 oC 250.7 59.9 192.7 110.0 90.0 90.0 108.1 > 29 mC 250.7 59.9 192.7 110.0 90.0 90.0 71.9 > 27 mC 500.2 192.8 59.9 143.1 90.1 127.5 71.9 > > On 11/17/2016 08:17 AM, Kay Diederichs wrote: > > Hi Sabine, > > > > my first guess would be that you are overlooking half of the reflections > > meaning the cell has really twice the volume. Is the list of difference > > vectors in IDXREF.LP clean in this respect? > > > > Best, > > > > Kay > > > > On Wed, 16 Nov 2016 17:26:59 +0100, Sabine Schneider <sabine.schnei...@cup.uni-muenchen.de> wrote: > >> Hello everybody, > >> > >> I got a problem with solving and refining a structure of a small protein > >> of 20kDa, where we just swapped two residue at the surface by mutation > >> (AF vs FA). > >> > >> For the FA variant we got three crystal forms, where we had no problem > >> whatsoever with MR and refinement > >> - P6522 cell 63 63 184 ~2.9A > >> - P21212 cell: 53 62 109 ~3A > >> - P212121 cell 40 43 96 ~1.8A > >> > >> However with the AF-variant we only got one crystal (out of 50) that > >> diffracted reasonable well to ~2.4A at the synchrotron. And now the > >> problem starts: initially the data appear to be orthorhombic, but: > >> > >> - SG18/19: cell 59 91 109 (I noticed that the b-axis is longer by 1/2b > >> compared to the other variant?!) > >> -> running phaser checking all possible alternative SGs found initally 2 > >> mols/AUS in SG18, but since there was still space (and density!) I let > >> it search for another one (increased the clash tolerance) > >> SOLU SET RFZ=7.7 TFZ=11.8 PAK=0 LLG=141 TFZ==13.9 RFZ=3.0 TFZ=11.2 PAK=0 > >> LLG=280 TFZ==17.1 LLG=288 TFZ==17.3 RFZ=5.9 TFZ=16.0 PAK=16 LLG=979 > >> TFZ==15.3 LLG=1479 TFZ==17.4 > >> > >> - However the third molecule than clashes partly > >> > >> - running refmac: R/Rfree stays in the high 40 > >> > >> - analysing the data with Xtriage indicates tNCS > >> > >> - I also tried P212121, P21 and P1, always with the same result: space > >> as well as density between the proteins; placing more protein molecules > >> results in clashes (i.e. refinement in P1 gives R/Rfree of 38/41) > >> > >> - data were processed with XDS as well as Dials/Aimless > >> > >> - the protein is generally well folded, quite stable, without any loose > >> ends or flexible regions > >> > >> - calculation of selfrotation functions in P21 and P21221 gives peak at > >> 90dg (kappa=180dg) > >> > >> Anyone an idea what could be going on or how to solve that? > >> > >> > >> Thanks a lot in advance! > >> > >> Sabine -- -- Paul Scherrer Institut Dr. Tim Gruene - persoenlich - Principal Investigator Biology and Chemistry OFLC/102 CH-5232 Villigen PSI Phone: +41 (0)56 310 5297 GPG Key ID = A46BEE1A
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