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

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