Uber Odd I reprocessed it and it seems to have resolved itself. Unfortunately though there seems to be no real significant anomalous differences in this dataset. Back to the drawing board. FR On Aug 11, 2009, at 8:54 AM, Francis E Reyes wrote:

1.4-87

FR

On Aug 11, 2009, at 8:53 AM, Peter Zwart wrote:

...

which version are you using?

P

2009/8/11 Francis E Reyes :

...

Hi all

I ran xtriage on my dataset to 4.5.here's the relevant section pertaining to anomalous signal

Analyses of anomalous differences

Table of measurability as a function of resolution

The measurability is defined as the fraction of Bijvoet related intensity differences for which |delta_I|/sigma_delta_I > 3.0 min[I(+)/sigma_I(+), I(-)/sigma_I(-)] > 3.0 holds. The measurability provides an intuitive feeling of the quality of the data, as it is related to the number of reliable Bijvoet differences. When the data are processed properly and the standard deviations have been estimated accurately, values larger than 0.05 are encouraging. Note that this analyses relies on the correctness of the estimated standard deviations of the intensities.

unused: - 90.6596 [ 0/1 ] bin 1: 90.6596 - 9.2614 [1371/1382] 0.0375 bin 2: 9.2614 - 7.3520 [1369/1376] 0.0033 bin 3: 7.3520 - 6.4230 [1380/1388] 0.0000 bin 4: 6.4230 - 5.8358 [1353/1359] 0.0016 bin 5: 5.8358 - 5.4176 [1367/1371] 0.0000 bin 6: 5.4176 - 5.0982 [1361/1369] 0.0000 bin 7: 5.0982 - 4.8429 [1378/1382] 0.0000 bin 8: 4.8429 - 4.6321 [1351/1359] 0.0000 bin 9: 4.6321 - 4.4538 [1383/1389] 0.0000 bin 10: 4.4538 - 4.3001 [1390/1393] 0.0000 unused: 4.3001 - [ 0/0 ]

The full resolution range seems to contain a useful ammount of anomalous signal. Depending on your specific substructure, you could use all the data available for the location of the heavy atoms, or cut the resolution to speed up the search.

If values larger than 0.05 are encouraging why is it telling me that the entire resolution contains a useful amount of anomalous signal? Did I process this incorrectly?

Thanks

FR

--------------------------------------------- Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder

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The presence of a peak in the fluorescence doesn't guarantee low enough standard deviations in intensities to ensure 'measurable' anomalous differences. How well did your crystal diffract? The problem reported earlier is most likely a bug (which I hope can be reproduced). Cheers Peter 2009/8/11, [email protected] :

Hi, I had similar problems recently, I did a wavelength scan of my crystal at the synchrotron (selenium edge) and collected one dataset at the peak wavelength.The scan gives me a distinct peak. However when I processed the data and evaluated in xtriage it says that there is no anomolous signal. I am unable to explain this? any advice? Shya

...

Uber Odd

I reprocessed it and it seems to have resolved itself.

Unfortunately though there seems to be no real significant anomalous differences in this dataset. Back to the drawing board.

FR

On Aug 11, 2009, at 8:54 AM, Francis E Reyes wrote:

...

1.4-87

FR

On Aug 11, 2009, at 8:53 AM, Peter Zwart wrote:

...

which version are you using?

P

2009/8/11 Francis E Reyes :

...

Hi all

I ran xtriage on my dataset to 4.5.here's the relevant section pertaining to anomalous signal

Analyses of anomalous differences

Table of measurability as a function of resolution

The measurability is defined as the fraction of Bijvoet related intensity differences for which |delta_I|/sigma_delta_I > 3.0 min[I(+)/sigma_I(+), I(-)/sigma_I(-)] > 3.0 holds. The measurability provides an intuitive feeling of the quality of the data, as it is related to the number of reliable Bijvoet differences. When the data are processed properly and the standard deviations have been estimated accurately, values larger than 0.05 are encouraging. Note that this analyses relies on the correctness of the estimated standard deviations of the intensities.

unused: - 90.6596 [ 0/1 ] bin 1: 90.6596 - 9.2614 [1371/1382] 0.0375 bin 2: 9.2614 - 7.3520 [1369/1376] 0.0033 bin 3: 7.3520 - 6.4230 [1380/1388] 0.0000 bin 4: 6.4230 - 5.8358 [1353/1359] 0.0016 bin 5: 5.8358 - 5.4176 [1367/1371] 0.0000 bin 6: 5.4176 - 5.0982 [1361/1369] 0.0000 bin 7: 5.0982 - 4.8429 [1378/1382] 0.0000 bin 8: 4.8429 - 4.6321 [1351/1359] 0.0000 bin 9: 4.6321 - 4.4538 [1383/1389] 0.0000 bin 10: 4.4538 - 4.3001 [1390/1393] 0.0000 unused: 4.3001 - [ 0/0 ]

The full resolution range seems to contain a useful ammount of anomalous signal. Depending on your specific substructure, you could use all the data available for the location of the heavy atoms, or cut the resolution to speed up the search.

If values larger than 0.05 are encouraging why is it telling me that the entire resolution contains a useful amount of anomalous signal? Did I process this incorrectly?

Thanks

FR

--------------------------------------------- Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder

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Did you "backsoak" the crystal? Wouldn't heavy atom in the mother liquor give a fluorescence peak even if none of it bound? Ed [email protected] wrote:

Hi, I had similar problems recently, I did a wavelength scan of my crystal at the synchrotron (selenium edge) and collected one dataset at the peak wavelength.The scan gives me a distinct peak. However when I processed the data and evaluated in xtriage it says that there is no anomolous signal. I am unable to explain this? any advice? Shya

On Aug 11, 2009, at 11:37 AM, [email protected] wrote:

Is there a chance of backsoaking even when the protein has been expressed as a selenomethionine derivative? I just overexpressed the protein by shutting down the methionine biosynthesis in E.coli and incorporated SeMet instead of methionine. The protein has three methionine and is 26kDa

You could mass-spec the SeMet protein to make sure the expression worked, but these protocols rarely fail. Assuming the incorporation of SeMet is nearly complete, you'd see a huge fluorescence peak even if the methionine sidechains were disordered. Especially if the crystals diffract weakly and your overall signal-to-noise ratio is relatively low, it's not terribly surprising that you don't have much anomalous signal. I've seen lots of data like this (sometimes with many more Met residues); unfortunately, I've never found any solution other than growing better crystals. It might help to collect more frames to improve the signal, but this is also going to increase the radiation damage. -------------------- Nathaniel Echols Lawrence Berkeley Lab 510-486-5136 [email protected]

Sounds like you killed the signal during collection. Radiation damage is the most common cause of (probable) failure of SAD experiments (see e.g. James Holton's analyses). Try using fewer images of your dataset, that might help. And look at B-factor decay in scaling, if it's more than 5A**2, you're almost certainly screwed (but no guarantees if it's less.) phx. [email protected] wrote:

Hi, I had similar problems recently, I did a wavelength scan of my crystal at the synchrotron (selenium edge) and collected one dataset at the peak wavelength.The scan gives me a distinct peak. However when I processed the data and evaluated in xtriage it says that there is no anomolous signal. I am unable to explain this? any advice? Shya

...

Uber Odd

I reprocessed it and it seems to have resolved itself.

Unfortunately though there seems to be no real significant anomalous differences in this dataset. Back to the drawing board.

FR

On Aug 11, 2009, at 8:54 AM, Francis E Reyes wrote:

...

1.4-87

FR

On Aug 11, 2009, at 8:53 AM, Peter Zwart wrote:

...

which version are you using?

P

2009/8/11 Francis E Reyes :

...

Hi all

I ran xtriage on my dataset to 4.5.here's the relevant section pertaining to anomalous signal

Analyses of anomalous differences

Table of measurability as a function of resolution

The measurability is defined as the fraction of Bijvoet related intensity differences for which |delta_I|/sigma_delta_I > 3.0 min[I(+)/sigma_I(+), I(-)/sigma_I(-)] > 3.0 holds. The measurability provides an intuitive feeling of the quality of the data, as it is related to the number of reliable Bijvoet differences. When the data are processed properly and the standard deviations have been estimated accurately, values larger than 0.05 are encouraging. Note that this analyses relies on the correctness of the estimated standard deviations of the intensities.

unused: - 90.6596 [ 0/1 ] bin 1: 90.6596 - 9.2614 [1371/1382] 0.0375 bin 2: 9.2614 - 7.3520 [1369/1376] 0.0033 bin 3: 7.3520 - 6.4230 [1380/1388] 0.0000 bin 4: 6.4230 - 5.8358 [1353/1359] 0.0016 bin 5: 5.8358 - 5.4176 [1367/1371] 0.0000 bin 6: 5.4176 - 5.0982 [1361/1369] 0.0000 bin 7: 5.0982 - 4.8429 [1378/1382] 0.0000 bin 8: 4.8429 - 4.6321 [1351/1359] 0.0000 bin 9: 4.6321 - 4.4538 [1383/1389] 0.0000 bin 10: 4.4538 - 4.3001 [1390/1393] 0.0000 unused: 4.3001 - [ 0/0 ]

The full resolution range seems to contain a useful ammount of anomalous signal. Depending on your specific substructure, you could use all the data available for the location of the heavy atoms, or cut the resolution to speed up the search.

If values larger than 0.05 are encouraging why is it telling me that the entire resolution contains a useful amount of anomalous signal? Did I process this incorrectly?

Thanks

FR

--------------------------------------------- Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder

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-- ----------------------------------------------------------------- P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net -----------------------------------------------------------------

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--------------------------------------------- Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder

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