Hi Alex, What are the column labels in your MTZ file? If you open your MTZ file in the "Reflection file editor" under "Reflection tools" in the GUI, you can see all the labels available in the file. You can also run phenix.mtz.dump <MTZ file> at the command line. We try to recognize standard label names, like "I(+)," "I(-)," "SIGI(+)," and "SIGI(-)," so maybe you have labels that we do not expect. Thanks! -- Billy K. Poon Research Scientist, Molecular Biophysics and Integrated Bioimaging Lawrence Berkeley National Laboratory 1 Cyclotron Road, M/S 33R0345 Berkeley, CA 94720 Tel: (510) 486-5709 Fax: (510) 486-5909 Web: https://phenix-online.org On Tue, Apr 12, 2016 at 11:53 AM, rspencer wrote:

Hi Alex,

You need to look at you anomalous signal in the aimless.log output to see if there was a detectable anomalous signal. You can also force iMosflm to treat the dataset as having an anomalous signal so that it won’t merge your intensities.

Soaking does not guarantee that you will have an anomalous signal in your crystal and you may need to do a multiple crystals with increasing soak time to get an anomalous signal. You can easily do this by soaking the crystal and checking a few frames on your in-house source. Process the frames and see if an anomalous signal is detected.

Good luck!

Ryan

*From:* [email protected] [mailto: [email protected]] *On Behalf Of *Alex Lee *Sent:* Tuesday, April 12, 2016 11:09 AM *To:* [email protected] *Subject:* [phenixbb] Phenix Xtriage label error

Dear Phenixbb members,

I have a data-set of a 8 kDa protein crystal around 2.5A resolution. The protein crystal was soaked in potassium iodine before collecting data using in-house beam (1.54A wavelength). As I expected some anomalous signal from the iodine ion from the crystal, after I use Imosflm to index, integrate and scale the data (space group P3). I got four output .mtz files: pointless_XXX.mtz; aimless_xxx.mtz; ctruncate_xxx.mtz; ctruncate_xxx-unique.mtz. After I check with viewHKL, I found the only unmerged mtz data is pointless_XXX.mtz, the other three mtz files are merged.

The next step I tried to use Phenix Xtriage (Linux version 1.10.1) to check my mtz data for anomalous completeness, I thought in this step my mtz should be unmerged type to see the anomalous signal, so I chose "pointless_xxx.mtz" as Xtriage input, but for the data labels in the Xtriage GUI panel, I can only have two choices of "I, SIGI, Merged" and "IPR, SIGIPR, Merged", it seems I do not have a choice of an unmerged mtz label. I decide to leave this choice blank by choosing data labels"---". After I click "run", Xtriage gave an error "please select labels for input data".

Any input on this issue?

Thanks in advance.

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Hi Alex, Aimless is showing that there is a strong anomalous signal : Minimum and maximum SD correction factors: Fulls 1.07 104.68 Partials 1.18 134.50 Anomalous flag switched ON in input, strong anomalous signal found And also giving you a resolution cutoff for looking for the anomalous signal : Estimate of maximum resolution for significant anomalous signal = 3.97A, from CCanom > 0.15 You have a couple of options – 1) Put the dataset in HySS and search for the Iodine (assume 1-2 iodine per molecules in your ASU) and limit the high-resolution search to 3.97. 2) go directly to Autosol and do the same resolution cutoff for searching for the anomoalous signal. I’m sure others can also give some more detailed advice on the next steps. Good luck! Ryan From: Alex Lee [mailto:[email protected]] Sent: Tuesday, April 12, 2016 1:49 PM To: rspencer Cc: [email protected] Subject: Re: [phenixbb] Phenix Xtriage label error Hi Ryan, Thank you very much! Below are the summary of my pointless log after "quick scale" from Imosflm (this time I force imosflm to choose P1 in the integration and scale but pointless chose P31): $TEXT:Result: $$ $$ Summary data for Project: New Crystal: New Dataset: New Overall InnerShell OuterShell Low resolution limit 82.13 82.13 2.49 High resolution limit 2.40 8.98 2.40 Rmerge (within I+/I-) 0.075 0.033 0.851 Rmerge (all I+ and I-) 0.086 0.048 0.919 Rmeas (within I+/I-) 0.089 0.039 1.007 Rmeas (all I+ & I-) 0.093 0.052 0.996 Rpim (within I+/I-) 0.047 0.021 0.534 Rpim (all I+ & I-) 0.035 0.021 0.380 Rmerge in top intensity bin 0.031 - - Total number of observations 214291 4072 22139 Total number unique 31247 617 3299 Mean((I)/sd(I)) 14.4 29.4 2.9 Mn(I) half-set correlation CC(1/2) 0.998 0.995 0.785 Completeness 100.0 99.9 99.8 Multiplicity 6.9 6.6 6.7 Anomalous completeness 99.7 98.1 98.8 Anomalous multiplicity 3.4 3.4 3.3 DelAnom correlation between half-sets 0.303 0.473 0.046 Mid-Slope of Anom Normal Probability 1.161 - - Estimate of maximum resolution for significant anomalous signal = 3.97A, from CCanom > 0.15 Estimates of resolution limits: overall from half-dataset correlation CC(1/2) > 0.30: limit = 2.40A == maximum resolution from Mn(I/sd) > 1.50: limit = 2.40A == maximum resolution from Mn(I/sd) > 2.00: limit = 2.40A == maximum resolution Estimates of resolution limits in reciprocal lattice directions: Along h k plane from half-dataset correlation CC(1/2) > 0.30: limit = 2.40A == maximum resolution from Mn(I/sd) > 1.50: limit = 2.40A == maximum resolution Along l axis from half-dataset correlation CC(1/2) > 0.30: limit = 2.40A == maximum resolution from Mn(I/sd) > 1.50: limit = 2.40A == maximum resolution Anisotropic deltaB (i.e. range of principal components), A^2: 12.33 Average unit cell: 65.87 65.87 164.25 90.00 90.00 120.00 Space group: P 31 Average mosaicity: 0.58 Minimum and maximum SD correction factors: Fulls 1.07 104.68 Partials 1.18 134.50 Anomalous flag switched ON in input, strong anomalous signal found $$ ============================================================== I do not know if this data set with such anomalous completeness and multiplicity is enough for me to get phase information to locate the Iodine, I do not have native dataset, and only have iodine soaking dataset. On Tue, Apr 12, 2016 at 11:53 AM, rspencer mailto:[email protected] > wrote: Hi Alex, You need to look at you anomalous signal in the aimless.log output to see if there was a detectable anomalous signal. You can also force iMosflm to treat the dataset as having an anomalous signal so that it won’t merge your intensities. Soaking does not guarantee that you will have an anomalous signal in your crystal and you may need to do a multiple crystals with increasing soak time to get an anomalous signal. You can easily do this by soaking the crystal and checking a few frames on your in-house source. Process the frames and see if an anomalous signal is detected. Good luck! Ryan From: [email protected] mailto:[email protected] [mailto:[email protected] mailto:[email protected] ] On Behalf Of Alex Lee Sent: Tuesday, April 12, 2016 11:09 AM To: [email protected] mailto:[email protected] Subject: [phenixbb] Phenix Xtriage label error Dear Phenixbb members, I have a data-set of a 8 kDa protein crystal around 2.5A resolution. The protein crystal was soaked in potassium iodine before collecting data using in-house beam (1.54A wavelength). As I expected some anomalous signal from the iodine ion from the crystal, after I use Imosflm to index, integrate and scale the data (space group P3). I got four output .mtz files: pointless_XXX.mtz; aimless_xxx.mtz; ctruncate_xxx.mtz; ctruncate_xxx-unique.mtz. After I check with viewHKL, I found the only unmerged mtz data is pointless_XXX.mtz, the other three mtz files are merged. The next step I tried to use Phenix Xtriage (Linux version 1.10.1) to check my mtz data for anomalous completeness, I thought in this step my mtz should be unmerged type to see the anomalous signal, so I chose "pointless_xxx.mtz" as Xtriage input, but for the data labels in the Xtriage GUI panel, I can only have two choices of "I, SIGI, Merged" and "IPR, SIGIPR, Merged", it seems I do not have a choice of an unmerged mtz label. I decide to leave this choice blank by choosing data labels"---". After I click "run", Xtriage gave an error "please select labels for input data". Any input on this issue? Thanks in advance.