[phenixbb] Strong anomalous signal but AutoSol fails

Diana Tomchick Diana.Tomchick at utsouthwestern.edu
Tue Aug 11 11:31:25 PDT 2015


I’m curious—how many copies of the lower resolution model did you find in the higher resolution cell? That might provide a clue for higher Laue symmetry.

Diana

**************************************************
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
Diana.Tomchick at UTSouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

> On Aug 11, 2015, at 12:42 PM, mohamed noor <mohamed.noor34 at gmail.com> wrote:
> 
> Hi Diana, Tom
> 
> I am able to obtain an MR solution with a different crystal form in P41212. Autobuild and refinement to 3.8 A gives me an Rfree of about 30 %. When I go back to my higher resolution data in the point group P321, Phaser runs with the search model that I used for the P41212 data failed. However, I can get a solution (TFZ=14, LLG=400) with Phaser.MRage using the lower resolution (P41212) model.
> 
> Autobuilding-refining this solution gives an Rfree of 48 %. So I think something is wrong somewhere but I can't figure out where. XDS selected P321 and this was more or less confirmed by Pointless and Xtriage. When I checked FRAME.cbf, all spots were predicted and the number of spots rejected per frame were quite low (5-10 rejected out of 200 or so strong reflections).
> 
> I remember having a pseudosymmetry case in a different protein recently whereby I had good anomalous signal in P321 (yes, same thing again!!) with the Rfree being stuck in 35 % (1.2 A dataset). But I reprocessed it to P3 and refinement and map was much better (Rfree around 22 %) although the anomalous signal was not there.
> 
> In short, I am currently running a few more Phaser runs in P3, P6 and P622 point groups and checking for all the possible space groups.
> 
> Thanks again.
> 
> On Mon, Aug 10, 2015 at 5:09 PM, Diana Tomchick <Diana.Tomchick at utsouthwestern.edu> wrote:
> I second the recommendation on calculating a rotation function, though you may need some assistance with interpreting the results of the calculation.
> 
> Running any phasing program (including AutoSol) at lower resolution will give you higher absolute values for the FOM but that doesn’t translate to a real solution.
> 
> At this point in the game, I would start to question whether the Laue symmetry that you chose for your data processing is correct—it is possible that you should have a higher symmetry, or you have pseudosymmetry. Either possibility could really complicate the location of MR models and heavy atom sites.
> 
> If you can use SHELX to find heavy atom sites, then it’s been my experience that 80% of the time you can put your data into PHENIX and AutoSol will give you a solution. The other 20% of the time requires more manual effort and fiddling.
> 
> Diana
> 
> **************************************************
> Diana R. Tomchick
> Professor
> Departments of Biophysics and Biochemistry
> University of Texas Southwestern Medical Center
> 5323 Harry Hines Blvd.
> Rm. ND10.214A
> Dallas, TX 75390-8816
> Diana.Tomchick at UTSouthwestern.edu
> (214) 645-6383 (phone)
> (214) 645-6353 (fax)
> 
> > On Aug 9, 2015, at 10:29 AM, Terwilliger, Thomas Charles <terwilliger at lanl.gov> wrote:
> >
> > Hi Mohamed,
> >
> > I don't know of any way to know the number of copies in the ASU exactly, no.
> >
> > It does seem that the solutions you have found so far are not likely to be correct, as you suggest.  Here is one more general approach to try:  as you have a high degree of NCS, try to use it to evaluate your solutions. I assume there is 1 Fe per molecule.  With high NCS in the crystal, it is likely (but not for sure) that the molecules in the ASU have some kind of symmetric arrangement.  So you could look at any potential substructure solutions you find and see if there is any symmetry in the sites.  You can just look at them in Coot with symmetry turned on or you can use phenix.find_ncs to look for NCS in the sites.
> >
> > Additionally you could calculate a rotation function from your native data and from your anomalous differences and see if there are NCS rotation axes that are present (for example a 13-fold rotation axis or something like that).
> >
> > All the best,
> > Tom T
> >
> > From: mohamed noor [mohamed.noor34 at gmail.com]
> > Sent: Sunday, August 09, 2015 4:39 AM
> > To: Terwilliger, Thomas Charles
> > Cc: PHENIX user mailing list
> > Subject: Re: [phenixbb] Strong anomalous signal but AutoSol fails
> >
> > One more thing, is there any way of knowing how many copies there should be in the ASU? Right now, I am doing it blindly as there is a huge range based on Matthew's coefficient calculation.
> >
> > On Sun, Aug 9, 2015 at 11:37 AM, mohamed noor <mohamed.noor34 at gmail.com> wrote:
> > Hi Tom
> >
> > When I used data in P 3(2)21, Hyss returned a CC of 0.2 for 10 sites and in P 3, I obtained a CC of 0.32 for 17 sites (out of 20 requested). From the documentation, CC 0.2 is considered possible and 0.3 is good. However, this does not translate into good models built by AutoBuild with the R factor being in the 45-50 % and low model CC (0.25). The FOM in AutoSol also seems to not reflect what it should be even if it is better than 0.4. Interestingly, when I input a dataset with low anomalous signal to 5 A (out of 3.5 A), I got an FOM of > 0.4 and Bayes-CC of 45 - but bad model.
> >
> > I also tried to run Phaser/MRage in all possible SG within both point groups without any luck.
> >
> > Mohamed
> >
> > On Fri, Aug 7, 2015 at 10:26 PM, Terwilliger, Thomas Charles <terwilliger at lanl.gov> wrote:
> > Hi Mohamed,
> >
> > This looks generally fine to me.  One thing that stands out though is the line:
> >
> > 4.0- 3.5  0.79   3.23  0.24    4001  2577
> >
> > where the half-dataset CC is 0.24, much greater than for lower resolutions.  This suggests that something is systematically wrong in this resolution shell (or elsewhere).  It may be useful to cut the data at 4 A just for this reason.  As Diana points out it may be useful for other reasons as well to cut the resolution at 4 A for finding the sites.
> >
> > I would suggest taking all of the datasets you have that are more or less isomorphous and putting them in a directory and then using scale_and_merge to put them all together (this is what it is designed for).  It should be able to down-weight the ones that are most different from the average.
> >
> > You could also try the brute-force option in phenix.hyss to try very hard to find the sites.
> >
> > All the best,
> > Tom T
> >
> > From: mohamed noor [mohamed.noor34 at gmail.com]
> > Sent: Friday, August 07, 2015 10:55 AM
> > To: Terwilliger, Thomas Charles
> > Cc: PHENIX user mailing list
> > Subject: Re: [phenixbb] Strong anomalous signal but AutoSol fails
> >
> > Hi Tom
> >
> > Just to check that I am on the right track, I have attached below the output from phenix.anomalous_signal using a single XDS_ASCII.HKL file from a single crystal. It is one of a few datasets, but based on Xtriage seems to be the best one.
> >
> > The space group is P 3(1/2) 2 1. All datasets were collected at the peak wavelength (fluorescence scan). My initial feeling that there is a strong signal comes from CORRECT.LP. Aimless and Xtriage. IIRC, the resolution difference between the optimistic and pessimistic measurability is about 0.3-0.4 A.
> >
> >
> > Estimation of anomalous signal in a dataset
> >
> > Estimating B-value for anomalous substructure as    91.2  based on
> > overall B-value of    75.4 (Note: you can set this with b_value_anomalous=xx)
> >
> > Getting scaled data and half-datasets with scale_and_merge
> > Log file will be: scale.log
> >
> > Files for half_dataset CC: ['TEMP4/XDS_ASCII_SG150_1800frames_reprocFriedel_0_1.sca']
> > Files for half_dataset CC: ['TEMP4/XDS_ASCII_SG150_1800frames_reprocFriedel_0_1.sca']
> > Files for half_dataset CC: ['TEMP4/XDS_ASCII_SG150_1800frames_reprocFriedel_0_1.sca']
> > Scaled data are in: scaled_data.mtz
> > Half-dataset A is in: half_dataset_a.mtz
> > Half-dataset B is in: half_dataset_b.mtz
> > Using scaled data in analysis
> >
> >
> > Setting up estimator for CC*
> >
> > -------------------Summary of signal in this dataset ------------------------
> >
> >        Shell
> >                        CCano   Nrefl Nrefl
> > Resolution Esqr I/sigI  half   anom   half
> > 47.8- 7.0  0.50  19.55  0.32    2389  2360
> >  7.0- 6.5  0.69  10.63  0.11     621   608
> >  6.5- 6.0  0.87   8.47  0.08     850   818
> >  6.0- 5.5  0.88   7.52  0.08    1189  1141
> >  5.5- 5.0  0.72   5.88  0.09    1709  1588
> >  5.0- 4.5  0.76   4.59  0.07    2521  2247
> >  4.5- 4.0  0.98   3.25  0.05    3881  3268
> >  4.0- 3.5  0.79   3.23  0.24    4001  2577
> >  3.5- 3.3  1.72   1.99 -0.02    3420  2118
> >
> >        Cumulative
> >
> > ----------------------Data quality-----------------    Best guess of expected
> >                                                       results of finding sites
> >                                                      ------ and phasing--------
> >
> >                      CCano   Nrefl                    P(Substr)
> > Resolution Skew Esqr  half   anom    CC* Signal  +/-     (%)       FOM*  +/-
> > 47.8- 7.0  0.02 0.47  0.32    2389  0.51  10.1   1.3      68       0.2   0.0
> > 47.8- 6.5  0.02 0.50  0.28    3010  0.48  10.7   1.7      72       0.2   0.1
> > 47.8- 6.0  0.02 0.57  0.24    3860  0.43  10.8   2.8      72       0.2   0.1
> > 47.8- 5.5  0.05 0.64  0.20    5049  0.44  12.6   2.4      78       0.2   0.1
> > 47.8- 5.0  0.01 0.66  0.17    6758  0.39  12.8   3.3      78       0.2   0.1
> > 47.8- 4.5  0.01 0.69  0.14    9279  0.36  13.4   3.4      82       0.2   0.1
> > 47.8- 4.0  0.00 0.77  0.12   13160  0.33  14.3   3.6      88       0.2   0.1
> > 47.8- 3.5  0.00 0.77  0.15   17161  0.39  18.4   3.8      98       0.3   0.1
> > 47.8- 3.3  0.00 0.88  0.14   20581  0.31  15.7   2.1      95       0.2   0.0
> >
> >
> > On Fri, Aug 7, 2015 at 2:30 PM, Terwilliger, Thomas Charles <terwilliger at lanl.gov> wrote:
> > Hi Mohamed,
> >
> > You might try running phenix.anomalous_signal on your data (may require finding your unmerged data and running phenix.scale_and_merge first).  This will give you an idea if you should be able to solve your SAD dataset.
> >
> > See:  http://www.phenix-online.org/version_docs/1.10pre-2124/reference/anomalous_signal.html
> >
> > All the best,
> > Tom T
> >
> >
> >
> >
> >
> > From: phenixbb-bounces at phenix-online.org [phenixbb-bounces at phenix-online.org] on behalf of mohamed noor [mohamed.noor34 at gmail.com]
> >
> > Sent: Thursday, August 06, 2015 3:16 PM
> >
> > To: PHENIX user mailing list
> >
> > Subject: [phenixbb] Strong anomalous signal but AutoSol fails
> >
> >
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> > Dear developers
> >
> >
> >
> >
> > I have a low resolution anomalous dataset which Aimless suggests has an effective resolution to 3.3 A and anomalous signal to 3.5 A. However, SAD phasing with AutoSol is not successful with the final R factor around 50 %.
> >
> >
> >
> >
> >
> > I also have another dataset collected at a remote wavelength without anomalous signal to 3 A but they are not isomorphous (> 2 A difference in c axis).
> >
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> > The anomalous signal comes from the ligand heme c, which is bound covalently to the protein, so its occupancy should be 1. The protein is quite small with about 120 residues. Xtriage suggests an NCS of 6 to 20 with most likely number to be 13.
> >
> >
> >
> >
> > Is there any reason why a reasonable solution cannot be found? There is no twinning.
> >
> >
> >
> >
> > I am using the latest nightly 1.10 pre2124.
> >
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> >
> > Thanks.
> >
> >
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> >
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