Dear all,

Thank you very much for all of your constructive comments.
Based on all of your comments, I have done several treatment on my data sets, and here are some questions related with both your comments and my results.

First of all, I would like to make a short note summarizing all of your comments for the people who will have similar problem.

1) Twinning and tNCS has opposite effects to each other, and one should carefully analyze the data if one of them present.
2) Simple tNCS can be automatically analyzed and corrected during Phaser, but if it does not work, you may try to lower symmetry, decrease the size of the Uni cell, or turning off the tNCS option during phaser.
3) If one think there is twinning in the crystal, to make sure whether your data is twinned, it is better to take a look twinning test than seeing the R-values from refinement with twin operators. Applying twin operators always give you better R- values.

Then, here are some questions related to my current analysis.
Q1. As many of you concerned and I also had speculation on my C2 cell refinement with twin operators, I tried to analyze twinning of my datasets. (ATTACHED)
As you can see the log file from truncate, I guess my crystal is twinned. In addition to this, I also followed the discussion between Randy and Lan. I wanted to make sure whether my refinement with twin operators is correct or not.  As Randy recommend and based on the paper from Garib N. Murshudov (http://www.ysbl.york.ac.uk/refmac/papers/Rfactor.pdf), I could see the drop of R-values with twin operators is always possible, however, in this paper, I could see the gap between refinements w/ or w/o twin operators is quite smaller than my case. (0.49-> 0.41 or 0.58 -> 0.52 vs my case 0.39 -> 0.23). Together, based on both twinning-test and good R-values, I now believe C2 refinement with twin operators are true. What do you think?  

Q2-1. Because some of you asked me about the original spots and re-indexing the data sets with high symmetry SG,  I went back to mosflm, and here are some images from my analysis. As you can clearly see there are strong/weak spots in the frames, and if you increase the threshold you can pick the strong spots (ATTACHED). Based on this, initially, I assumed that my crystal has tNCS, because I thought this strong - weak pattern is caused by tNCS. or Am I wrong? 

Q2-2. During new indexing, I choose two unit cells (large and small) and  integrated them into 2 SG (P31 1 2 or P31 2 1) as Lijun commented. (four data sets)
All of these data sets indicated twinning, while smaller one showed higher twinning fractions. Then, I ran phaser, and found that phaser only found the solution from P31 2 1SG from both large and small cells. However, I could see crash of molecules from large cell, and I decided to stick with small cell again. Then, during refinement with twin operators the R-values drops from (0.46/0.49) to (0.37/0.42), even the map looks already good. I assume that applying high symmetry SG is not possible for this case, and again C2 refinement might be correct.

Q2-3. Related to Q1 and 2-1, Now, I think C2 (small cell) refinement with twin operator is right solution. Then, here I have another question.
Based on the observations from frames, I guessed my crystal has tNCS, and I only followed strong spots. (Maybe I am wrong ?)
Then, data sets with only strong spots suggests my crystal is twinned. 
Together, I am thinking my crystal has both tNCS and twinning, and I could solve the structure by following strong spots with twin operators.

Again, I would like to express my special thanks to you all for giving me valuable comments. 
I hope this post will also useful for others in the future.

Donghyuk


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