On Sat, Mar 26, 2011 at 11:29 AM, Nathaniel Echols wrote:

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1) Synchrotron Fluorescence scan indicates the heavy atom definitely presents in my protein (my control is a second protein with the same

On Fri, Mar 25, 2011 at 9:20 PM, Jason wrote: ligand

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showed no absorbance spectrum)

Keep in mind that a positive fluorescence scan does not necessarily mean that the element of interest is bound and well-ordered in the crystal - it is easy to get an excellent scan without seeing anything in the maps later.

Indeed, but hopefully it's not the case.

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When I load the mtz file to phenix.maps GUI, the mtz label pulldown menu indicates 4 possible choices for the column to use:(1) IMEAN, SIGIMEAN (2) I(+), sigI(+), I(-), sigI(-) merged (3) F(+), sigF(+), F(-), sigF(-) merged (4) F, sigF, Dano SigDano. I have tried the choices of (2) (3) and (4). However, there is barely any anomalous signal at 4sigma, which makes me wondering if something is not right. The first thing coming to my mind is the mtz labels: I(+), sigI(+), I(-), sigI(-) merged. What does merged mean? Could this be the reason? Other issues that could causing the trouble?

The "merged" means that the input data were only partially merged - this usually happens when processing in HKL2000 using the "no merge original index" setting, where the reflections are not merged to the asymmetric unit (anomalous or not). I've never used XDS, but I guess it must do something similar. Phenix doesn't really deal with data like that; it always merges equivalents (while leaving Friedel pairs alone by default). This usually doesn't have any impact on the anomalous signal. If XSCALE has an option to merge the data more completely, this should make the "merged" tag go away.

I went through the xds menu again and found that the "merged" seems ok in terms of anomalous signal.

It sounds like your element of interest isn't very well ordered; do you see the rest of the ligand in the normal maps?

I was hoping to identify the ligand binding position by resolving the anomalous map first. Shouldn't this be the procedure to locate ligand? If you really want

to be sure that it's not a data-handling issue, you could try reprocessing in other programs and confirming the result.

I know CCP4 can also generate anomalous difference map. But I myself have never done it (I googled online and found it not that straight forward). Can anybody go through it for me please, or there are other handy programs that can make anomalous difference map?

I don't know if radiation damage could be at fault, but it's always a possibility.

-Nat

====================== Jason Structural Biology Department University of Pittsburgh ======================

On Sat, Mar 26, 2011 at 11:57 AM, Nathaniel Echols wrote:

On Sat, Mar 26, 2011 at 9:45 AM, Jason wrote:

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I was hoping to identify the ligand binding position by resolving the anomalous map first. Shouldn't this be the procedure to locate ligand?

It depends on the ligand, I guess. If it's large enough and actually bound, it should be detectable in the Fo-Fc map - this would be a good sanity check to make sure that the anomalous map generation is working properly.

Unfortunately my ligand is fairly small, about 12 atoms.The Fo-Fc map won't be that helpful.

...

I know CCP4 can also generate anomalous difference map. But I myself have never done it (I googled online and found it not that straight forward). Can anybody go through it for me please, or there are other handy programs that can make anomalous difference map?

You don't need to use CCP4 to make the map, just to process the data - i.e. use MOSFLM/SCALA (xia2 will automate this), or you could also try HKL2000, then use that as input for Phenix. But I doubt this is going to give you a different answer.

XDS is the only program that is able to process my data (small oscillation angle and huge data file). On the other hand I am pretty sure xds is doing what it's supposed to do. I was more thinking of another program to generate anomalous map. Since it's phenixbb, probably it's not the right place :)

Also: run Xtriage and look at the plot of anomalous signal vs. resolution.

-Nat

====================== Jason Structural Biology Department University of Pittsburgh ======================

Since you have a molecular replacement model, the other option you should try is to ask Phaser to look for the anomalous scatterers with log-likelihood-gradient maps in the SAD likelihood target. In our experience, this gives significantly better signal-to-noise on average than simple model-phased anomalous difference Fouriers. By default, Phaser will put in anomalous scatterers where there are peaks above 6 times the rms of the log-likelihood-gradient map, and it does this iteratively, i.e. putting the anomalous scatterers into the model makes the model and makes the log-likelihood-gradient map more sensitive, so it carries on doing rounds of this until there are no more peaks to interpret as anomalous scatterers. At the end, you'll have a flat log-likelihood-gradient map but you'll also have a list of sites that were all 6sigma in at least one of the maps. As a bonus, you'll also have phases and a map that combine the information from your molecular replacement model and the anomalous scattering. You can do this either through AutoSol (if you did the molecular replacement with AutoMR you will have been given the option to press a button to run AutoSol, which is convenient) or directly through Phaser-EP. In each case you have to give a PDB file identified as a partial molecular replacement solution. Let me know if you have any difficulty finding the right options. Best wishes, Randy Read On Mar 26 2011, Jason wrote:

On Sat, Mar 26, 2011 at 11:29 AM, Nathaniel Echols wrote:

...

...

1) Synchrotron Fluorescence scan indicates the heavy atom definitely presents in my protein (my control is a second protein with the same

On Fri, Mar 25, 2011 at 9:20 PM, Jason wrote: ligand

...

showed no absorbance spectrum)

Keep in mind that a positive fluorescence scan does not necessarily mean that the element of interest is bound and well-ordered in the crystal - it is easy to get an excellent scan without seeing anything in the maps later.

Indeed, but hopefully it's not the case.

...

...

When I load the mtz file to phenix.maps GUI, the mtz label pulldown menu indicates 4 possible choices for the column to use:(1) IMEAN, SIGIMEAN (2) I(+), sigI(+), I(-), sigI(-) merged (3) F(+), sigF(+), F(-), sigF(-) merged (4) F, sigF, Dano SigDano. I have tried the choices of (2) (3) and (4). However, there is barely any anomalous signal at 4sigma, which makes me wondering if something is not right. The first thing coming to my mind is the mtz labels: I(+), sigI(+), I(-), sigI(-) merged. What does merged mean? Could this be the reason? Other issues that could causing the trouble?

The "merged" means that the input data were only partially merged - this usually happens when processing in HKL2000 using the "no merge original index" setting, where the reflections are not merged to the asymmetric unit (anomalous or not). I've never used XDS, but I guess it must do something similar. Phenix doesn't really deal with data like that; it always merges equivalents (while leaving Friedel pairs alone by default). This usually doesn't have any impact on the anomalous signal. If XSCALE has an option to merge the data more completely, this should make the "merged" tag go away.

I went through the xds menu again and found that the "merged" seems ok in terms of anomalous signal.

...

It sounds like your element of interest isn't very well ordered; do you see the rest of the ligand in the normal maps?

I was hoping to identify the ligand binding position by resolving the anomalous map first. Shouldn't this be the procedure to locate ligand?

If you really want

...

to be sure that it's not a data-handling issue, you could try reprocessing in other programs and confirming the result.

I know CCP4 can also generate anomalous difference map. But I myself have never done it (I googled online and found it not that straight forward). Can anybody go through it for me please, or there are other handy programs that can make anomalous difference map?

...

I don't know if radiation damage could be at fault, but it's always a possibility.

-Nat

====================== Jason Structural Biology Department University of Pittsburgh ======================