Hi Yarrow, of course the data quality will suffer but ultimately it depends on the amount of splitting - as long as the integration profile covers both parts then the total intensity is conserved, and recovered. I have seen many datasets with split reflections that were usable for MR and refinement. But I doubt that many of these would be suitable for experimental phasing. best, Kay Am 18.04.14 23:21, schrieb Yarrow Madrona:

Hi Kay,

Looking at the data I can see that many of the spots are actually split. I guess this may be causing problems in refinement if split spots are integrated as a single spot?

On Fri, Apr 18, 2014 at 2:17 PM, Kay Diederichs mailto:kay.diederichs@uni-konstanz.de> wrote:

It is a good idea to try several data processing programs in difficult cases.

BTW the latest MOSFLM can also integrate two (or more) lattices.

Pls report your results.

Kay

Am 18.04.14 22:59, schrieb Yarrow Madrona:

Thanks Kay,

XDS kicked out a lot of reflections. There were about 23,000 rejected reflections out of ~ 190,000 collected. I can clearly see another minor lattice in many frames and I presume that the rejections are coming from the minor lattice that was not selected. I was thinking of processing with EVAL15 to see if I get better results. Thanks for your help. Maybe respond off-line as this is a discussion maybe more suited for another mailing list (CCP4?).

-Yarrow

On Fri, Apr 18, 2014 at 7:11 AM, Kay Diederichs mailto:kay.diederichs@uni-konstanz.de mailto:kay.diederichs@uni-konstanz.de>>

wrote:

Hi Yarrow,

the problem is that during structure solution, many wrong paths may have to be followed until finally identifying the correct path.

So the general answer to this kind of problem is: in some way, your parameterization of the experiment is wrong or incomplete.

From what you write, data quality does not seem to be the problem. But: did XDS really integrate _all_ the reflections, or only a subset (say, every second reflection)?

Check out http://strucbio.biologie.uni-____konstanz.de/ccp4wiki/index.____php/Refineme... http://strucbio.biologie.uni-__konstanz.de/ccp4wiki/index.__php/Refinement#w...

<http://strucbio.biologie.uni-__konstanz.de/ccp4wiki/index.__php/Refinement#w... http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Refinement#what_...>

If, after thorough attempts, you fail to find the solution, upload your current model, sequence and raw data frames to a Dropbox folder and post the link here - there may be people who succeed in processing the data nicely, or otherwise can identify the problem based on the data (rather than based on your description only).

HTH,

Kay

Am 18.04.14 01:25, schrieb phenixbb-request@phenix-____online.org mailto:phenixbb-request@phenix-__online.org mailto:phenixbb-request@phenix-online.org>:

Date: Thu, 17 Apr 2014 16:25:00 -0700 From: Yarrow Madronamailto:amadrona@uci.edu mailto:amadrona@uci.edu>> To: PHENIX user mailing listmailto:phenixbb@phenix-online.org>>

Subject: [phenixbb] Stalled refinement Message-ID:

<__CAMHjG6bPE4q1xWidpE2VwFMJ9qLS____qjtLRuLMM1ef9vWBHBfZKg@mail.____gmail.com http://gmail.com

mailto:CAMHjG6bPE4q1xWidpE2VwFMJ9qLSqjtLRuLMM1ef9vWBHBfZKg@mail.gmail.com>>

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Hello,

I using the latest stable build of phenx.refine (1.8.4) I recently collected data, processed and obtained an MR solution using phaser. I am stuck trying to refine with an Rfree sitting at 40%

I really want to know if the high Rfree is due to poor data quality or if non-crystallographic symmetry involving a near perfect two fold rotation between the two molecules in the ASU could somehow impede refinement. Stats and other information is below. Thank you for any help you can give.

-Yarrow

Visually, the quality of the data is marginal at best (streaky/ice rings in many frames) despite good processing stats from XDS. Processing with mosflm or HKL2000 managed to index but failed pretty bad in integration and scaling.

Phaser gave high TFZ scores for 2 molecules in the asu (see below).

Density for a cholesterol like ligand shows up even though not present in the search model.

MolRep Self rotation shows rotational symmetry. https://www.dropbox.com/s/____2zsajl5o091k50r/CYP142A2-____032814_21_rf%20co... https://www.dropbox.com/s/__2zsajl5o091k50r/CYP142A2-__032814_21_rf%20copy.p...

<https://www.dropbox.com/s/__2zsajl5o091k50r/CYP142A2-__032814_21_rf%20copy.p... https://www.dropbox.com/s/2zsajl5o091k50r/CYP142A2-032814_21_rf%20copy.pdf>

The 2 molecules in the ASU are related by almost a 2 fold rotation:

Rotation matrix for chain A to chain B:

new_ncs_group rota_matrix 1.0000 0.0000 0.0000 rota_matrix 0.0000 1.0000 0.0000 rota_matrix 0.0000 0.0000 1.0000 tran_orth 0.0000 0.0000 0.0000

center_orth 15.2016 0.5245 33.7070

rota_matrix -0.9860 -0.1636 -0.0309 rota_matrix -0.1659 0.9511 0.2605 rota_matrix -0.0132 0.2620 -0.9650 tran_orth 34.3310 -24.0033 107.0457

center_orth 15.7607 7.2426 77.7512

RMSD, B onto A = 0.0007 after phaser RMSD, B onto A = 0.347 after one round of refinement in phenix

Refinement using aniostropically corrected data (ucla web server: Services.mbi.ucla.edu/____anisoscale http://Services.mbi.ucla.edu/__anisoscale <http://Services.mbi.ucla.edu/__anisoscale http://Services.mbi.ucla.edu/anisoscale>) did not improve the

Rfree in refinement.

Statistics are listed below:

UNIT CELL: 51.487 88.923 89.592 90 97.15 90 P21

RESOLUTION NUMBER OF REFLECTIONS COMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr

5.99 8280 1927 2087 92.3% 3.1% 3.3% 8246 35.09 3.5% 99.8* 20* 0.909 1296 4.30 14606 3401 3487 97.5% 3.3% 3.5% 14580 33.37 3.8% 99.9* 11* 0.843 2273 3.53 17961 4244 4445 95.5% 3.8% 3.9% 17944 31.11 4.4% 99.8* -2 0.789 2721 3.06 21954 5068 5221 97.1% 4.9% 5.1% 21933 24.81 5.6% 99.7* -2 0.780 3455 2.74 25741 5830 5933 98.3% 7.6% 7.6% 25713 18.88 8.6% 99.5* -2 0.782 4165 2.51 27859 6311 6483 97.3% 10.8% 10.8% 27824 14.06 12.3% 99.1* -2 0.774 4385 2.32 31336 6979 7084 98.5% 14.9% 15.3% 31296 10.49 16.8% 98.5* -4 0.748 5095 2.17 32396 7347 7567 97.1% 22.3% 22.7% 32341 7.46 25.4% 97.3* -7 0.728 5055 2.05 32254 7339 8047 91.2% 33.1% 33.5% 32075 5.06 37.5% 94.8* -6 0.724 5155 total 212387 48446 50354 96.2% 7.8% 7.9% 211952 16.57 8.8% 99.7* -3 0.768 33600

Processing with mosflm or HKL2000 managed to index but failed pretty bad in integration and scaling.

Phaser:

SOLU SET RFZ=27.5 TFZ=24.2 PAK=0 LLG=1711 RF++ TFZ=64.6 PAK=0 LLG=3610 LLG=4865