[sharp-discuss] question about finding heavy atom position with MR phases

Sabine Schneider sabine.schneider at cup.uni-muenchen.de
Fri May 16 13:26:17 BST 2008


Hi everyone,

I have a question about how to solve the issue with alternative origins 
when combining SAD and MR phases.
I got a native dataset to 3.3A with an anomlous scatterer in the ligand. 
The molecular replacement solution is pretty good but there is different 
ligand than to the MR model. So I removed the ligand from the model and 
I am not 100% sure about its orientation at that resolution. Since the 
ligand contains a heavy atom, I thought that some anomalous data might 
help. I got a peak dataset with a good looking anomalous signal, to 4.8A 
(chooped off the data at 4.8A); unfortunately a bit crappy crystals, but 
I guess it should be suffiecient to determine the HA position.

I used ShelxD to find the sites (2 HA per asu) and the solution looks 
somewhat sensible and I also get a nice peak in the difference patterson 
(better than in the anomalous patterson).  The crystals are tetragonal 
and I therefore have four alternative origins. So my first question is, 
to run SHARP using the HL coefficients from phaser, what initial site do 
I put in or how to I make sure that the sites I got from the difference 
patterson / ShelxD are on the same origin as the MR phases? (I scaled 
the two datasets together with scaleit and than run phaser on the native 
data )

I put in 0 0 0 as initial site in SHARP and it comes up with two 
sensible looking sites in the residual maps. I fed the sites found in 
the residual maps back into SHARP, but how come that I still have to 
shift my MR model z = 0.5 that it is matching the electron density 
calculated from eden.mtz? So I shifted the sites z= -0.5 and repeated 
the SHARP run. It comes up again with the 2 more sites in the residual 
maps, but they are just the same again on a different origin? And to 
match my MR model to the ED I have to shift the origin of my MR model 
again? I thought that if I use the phases from the MR for the 
calculation of the residual maps and get some sites from there, they 
should be on the same origin than the model? Moreover the sites are not 
making sence in terms of the location in the model so I guess they are 
wrong than? I have to protein-ligand complexes in the asu with one HA 
each and the distance between the active site of the proteins in the asu 
and between the two sites found by SHARP are similar enough?

Would be very much appreciated, if someone could tell me, where I am 
going wrong!
 
Thanks a lot for your help in advance!

Sabine



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