This tutorial originally comes from the
Download the data from 6_mr_sad.tgz
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, the 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).
- Solve the structure by MR with the α-lactalbumin model. Please note
that if you used Molrep for this you will need to follow by refinement with
Refmac in order to produce an MTZ file with phases.
- 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 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. You should also select
Use map coefficients, then set
F=FWT and PHI=PHWT).
This sets the map used in the first cycle of density modification, rather than
calculating it from FP and PHI/FOM.
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
- Start up ARP/wARP Classic in 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.
mode. Select the MTZ file from Parrot. To
start from the Parrot map, select
- Now add the S-SAD phase information.
- Bring up the GUI for the Phaser SAD pipeline in the Automated
Search & Phasing section of the module.
- Fill in all of the yellow boxes.
- Set SAD with
molecular replacement partial structure
- Uncheck the Parrot and Buccaneer steps of the pipeline (to allow
control for better comparison with the MR model alone).
- Under the Define atoms heading, set LLG-map completion to
Complete with atom type to S
- Also set Partial structure input as to the
molecular replacement solution (output PDB-file) you have obtained in
the first step.
- Run Phaser after you entered all the information.
- 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.
- 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 HLA/HLB/HLC/HLD
to the coefficients.
restraints. In the MTZ data section, set
- 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?
- If you did not know the correct space group (from the MR step), would
you have to run Phaser SAD-phasing twice?
- Compare the two ARP/wARP runs! Which one has built more residues?