1.    Is it really twinned since including twin-law in refinment gave much better numbers? I would say, it likely is twinned. But as Jürgen pointed out, if the twin domain is close to 50%, you are likely in too low of symmetry. Compare the maps between both by eye and use your best judgement. Both set of stats (R and Rfree) are pretty good, so I'm leaning towards the higher symmetry model as being correct. Question: Do you have 1mol/ASU in the I422 model? (You mention 2 mol/ASU in I4).

2.    When refining with twin-law, Are the maps by phenix.refine output already “corrected” mtz/map?

YES.

3.    If I use xray_data.r_free_flags.generate=True, can I write the data mtz out with the generated r-free-flags so that each round of refinement uses the same CV set?

NO! You presumably already have an Rfree set. When you run your first round of phenix.refine, it will generate an mtz file labelled "refine_data.mtz" or something similar. This file contains your experimental data (and rfree). Use the same file for each round of phenix.refine. You do not want to select a new set of Rfree during each refinement, which I believe is what the xray_data.r_free_flags.generate=True command will do, as that can increase bias (by including reflections in rfree that have been used to refine the model) Kelly ******************************************************* Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 ******************************************************* On Tue, May 8, 2012 at 11:11 AM, Zhou, Tongqing (NIH/VRC) [E] wrote:

Hi Jürgen, Kelly and Nat,

I tried XDS and MosFilm, they simply cannot index the data.

I reprocessed the data yesterday with HKL2000 starting from  frame #30 (splitting 1-29, 30-360), I did get it indexed to I4 and refined to very nice x/y chi^2 and covering most spots, I then used the index/refinement numbers for the whole data set. it seems that other spots were from a “nicely” positioned secondary crystal which fooled the program to pick P4 space group.

I scaled the data as I4 and I422 to see which one is better. The I4 data set, ~95% complete overall, 9.2% R-sym. Xtriage suggested it should have higher symmetry I422 or twin (-k, -h, -l, almost 50%). MR sent well and best SG is I4, 2 mol/ASU, r  and r-free went to about 27% and 32%, but if I used the twin-law in phenix.refine and ask phenix to generate r-free-flags with twinning in mind, the numbers went to ~22%  and 27%.

Shell Lower Upper Average      Average     Norm. Linear Square

limit    Angstrom       I   error   stat. Chi**2  R-fac  R-fac

      50.00   7.32   440.7     8.8     5.1  1.481  0.027  0.030

       7.32   5.81   167.2     4.2     3.2  1.868  0.045  0.048

       5.81   5.08   196.3     5.1     3.9  2.130  0.053  0.058

       5.08   4.62   243.1     6.2     4.7  2.264  0.056  0.061

       4.62   4.29   236.7     6.4     4.9  2.057  0.055  0.055

       4.29   4.03   167.6     5.6     4.7  1.819  0.067  0.066

       4.03   3.83   139.5     5.6     5.0  1.740  0.083  0.075

       3.83   3.66   110.2     5.5     5.1  1.645  0.102  0.091

       3.66   3.52    92.5     5.6     5.3  1.459  0.117  0.101

       3.52   3.40    76.5     5.7     5.4  1.372  0.143  0.124

       3.40   3.30    59.3     5.7     5.5  1.278  0.179  0.146

       3.30   3.20    47.3     5.7     5.6  1.161  0.212  0.179

       3.20   3.12    40.7     5.8     5.7  1.103  0.241  0.201

       3.12   3.04    31.6     5.8     5.8  1.057  0.298  0.254

       3.04   2.97    28.9     5.9     5.9  1.031  0.331  0.271

       2.97   2.91    22.6     5.9     5.9  0.985  0.400  0.340

       2.91   2.85    19.0     6.0     6.0  0.991  0.484  0.405

       2.85   2.80    17.1     6.1     6.1  0.957  0.518  0.437

       2.80   2.75    14.6     6.2     6.2  0.908  0.577  0.485

       2.75   2.70    13.0     6.4     6.3  0.931  0.645  0.550

  All reflections    112.9     5.9     5.3  1.455  0.092  0.066

The I422 data (below), 1 mol/ASU, r and r-free were about 27% and 32%.

Shell Lower Upper Average      Average     Norm. Linear Square

limit    Angstrom       I   error   stat. Chi**2  R-fac  R-fac

      50.00   7.32   430.0     8.4     5.0  1.344  0.025  0.028

       7.32   5.81   163.4     4.0     3.0  1.489  0.040  0.042

       5.81   5.08   192.4     4.8     3.6  1.694  0.046  0.052

       5.08   4.62   231.6     5.6     4.3  1.790  0.049  0.053

       4.62   4.29   227.8     5.8     4.5  1.534  0.045  0.043

       4.29   4.03   161.3     5.0     4.2  1.401  0.055  0.052

       4.03   3.83   135.3     5.0     4.4  1.367  0.066  0.059

       3.83   3.66   106.2     4.9     4.4  1.302  0.082  0.071

       3.66   3.52    88.1     4.8     4.5  1.253  0.097  0.082

       3.52   3.40    75.0     4.9     4.6  1.193  0.113  0.096

       3.40   3.30    56.7     4.8     4.6  1.121  0.144  0.115

       3.30   3.20    44.8     4.8     4.7  1.061  0.172  0.141

       3.20   3.12    38.0     4.8     4.7  1.011  0.203  0.166

       3.12   3.04    29.1     4.7     4.7  1.029  0.253  0.209

       3.04   2.97    27.7     4.8     4.8  0.985  0.261  0.209

       2.97   2.91    21.4     4.9     4.8  0.917  0.320  0.266

       2.91   2.85    18.3     4.9     4.8  0.949  0.380  0.316

       2.85   2.80    16.1     4.9     4.9  0.955  0.431  0.358

       2.80   2.75    14.2     5.0     5.0  0.940  0.483  0.399

       2.75   2.70    12.6     5.2     5.2  0.886  0.536  0.462

  All reflections    110.9     5.1     4.5  1.236  0.076  0.054

So my questions are:

1.    Is it really twinned since including twin-law in refinment gave much better numbers?

2.    When refining with twin-law, Are the maps by phenix.refine output already “corrected” mtz/map?

3.    If I use xray_data.r_free_flags.generate=True, can I write the data mtz out with the generated r-free-flags so that each round of refinement uses the same CV set?

Thnaks,

Tongqing

Tongqing Zhou, Ph.D.

Staff Scientist

Structural Biology Section

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Building 40, Room 4609B

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From: Bosch, Juergen [mailto:[email protected]] Sent: Monday, May 07, 2012 1:34 PM To: PHENIX user mailing list Subject: Re: [phenixbb] Difficult dataset and refinement--P422? I422?Twinning?

Why don't you index over the whole dataset (XDS) ?

Or at least over a couple of images (Mosflm)

Jürgen

On May 7, 2012, at 12:50 PM, Kelly Daughtry wrote:

I agree.

The fact that two "crystals forms" appear, with related unit cells: P422 data:      unit_cell = 191.6999969 191.6999969 311.0539856 90 90 90      space_group = "P 4 2 2" I422 data: unit_cell = 191.783 191.783 103.775 90 90 90      space_group = "I 4 2 2"

indicated to me you have mis-indexed the P422 data. It is likely I422. Kelly ******************************************************* Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 *******************************************************

On Mon, May 7, 2012 at 12:18 PM, Nathaniel Echols wrote:

On Mon, May 7, 2012 at 8:46 AM, Kelly Daughtry wrote:

Looking at the P422 data, it look like you have pseudo translational symmetry.

Did you try processing this data as I422?

From the P422_xtriage.log:

The full list of Patterson peaks is:

  x      y      z            height   p-value(height)

( 0.500, 0.500, 0.165 ) :   78.635   (6.634e-07)

( 0.000, 0.000, 0.330 ) :   56.292   (2.795e-05)

( 0.500, 0.500, 0.497 ) :   30.920   (1.207e-03)

One word of caution: an exceptionally high off-origin peak can mean

that the unit cell was measured too large, and you've integrated extra

reflections that are really non-existent.  (I'm not sure what the

threshold for this is, but 80% seems pretty large.)  Splitting and

various indexing artifacts can sometimes lead to this.  I'd recommend

running labelit.index on the images and seeing what it thinks the

lattice should be.

-Nat

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