From Phaserwiki
http://www.phaser.cimr.cam.ac.uk/index.php/Combined_MR-SAD_using_CCP4i
5. Combined
MR-SAD using CCP4i
Reflection data: lyso2001_scala1.mtz
Lactalbumin model: 1fkq_prot.pdb
Sequence file: hewl.pir
This tutorial
illustrates a common molecular
replacement/experimental phasing scenario, when refinement is
hindered by very
strong model bias, but there is some experimental phasing
signal available.
Goat
α-lactalbumin is 45%
identical to hen egg-white lysozyme. Although it is possible
to solve lysozyme
using α-lactalbumin as a model, it is very difficult to refine
the
structure, partly because of model bias. Unfortunately, low
solvent content of
this crystal form limits the ability of density modification
to remove the
bias. However, one can use anomalous scattering from intrinsic
sulfur atoms to
improve phases dramatically. It is noteworthy that the
anomalous signal from
the sulfur atoms is not sufficient for ab initio phasing (it
is not possible to
locate the anomalous scatterers from the data alone).
1.
Solve
the structure with the α-lactalbumin model. Follow the
"Molecular
replacement tutorial" if necessary.
2.
For
a fairer comparison of phase quality, we will treat the
molecular replacement
solution as a source of experimental phase information. (If
you use the
"automated model building starting from PDB file" mode, the
current
version of ARP/wARP will be able to build the structure, but
older versions
coupled with older versions of Refmac5 failed.) Do a quick
solvent flattening
with Parrot (choose "Use PHI/FOM instead of HL coefficients"
because
the MTZ file produced after MR doesn't include
Hendrickson-Lattman
coefficients; set PHI=PHIC and FOM=FOM; select "Use map
coefficients", then set F=FWT and PHI=PHWT).
3.
Start
up ARP/wARP Classic in "automated model building starting from
experimental phases" mode. Select the MTZ file from Parrot. To
start from
the Parrot map, select "Use a different (pre-weighted) Fobs
for initial
map calculation" under the ARP/wARP flow parameters folder,
then set FBEST=parrot.F_phi.F,
PHIB=parrot.F_phi.phi and FOM=Unassigned. Select the sequence
file, and note
there are 129 residues in lysozyme. To save time, do 3 cycles
of autobuilding
instead of 10.
4.
Now
add the S-SAD phase information. Bring up the GUI for Phaser
SAD pipeline in
the Automated Search & Phasing section of the Experimental
Phasing module
5.
All
the yellow boxes need to be filled in.
á Set "Mode for
experimental
phasing" to SAD with molecular replacement partial structure.
á Uncheck the
Parrot and Buccaneer
steps of the pipeline (to allow control for better comparison
with the MR model
alone).
á Set LLG-map
calculation atom type to
S.
á Under the "Define
atoms"
heading, set "Partial structure" to the molecular replacement
solution (output PDB-file) you have obtained in step 1.
6.
Run
Phaser after you entered all the information.
7.
Solvent
flatten with Parrot using a similar protocol to step 2.
However, you should
*not* choose "Use PHI/FOM instead of HL coefficients". Choose
the
HLanomA/B/C/D coefficients because these describe the phase
information
obtained only from the anomalous scattering information and
not from the
molecular replacement model; using HLA/B/C/D would include the
model phase
information, which would bias maps from subsequent cycles of
phase improvement
to look like the model.
8.
Run
ARP/wARP using a similar protocol as in step 3, except you
should open the
Refmac parameters folder and choose the option to include HL
phase restraints.
In the MTZ data section, choose the HLanomA/B/C/D
coefficients.
9.
How
many anomalous scatterers has Phaser found? Check them against
the model and
guess what they may be! Why is it not important to specify the
exact element
type in this case?
10.
If
you did not know the correct space group (from the MR step),
would you have to
run Phaser SAD-phasing twice?
11.
Compare
the two ARP/wARP runs! Which one has built more residues?