From Phaserwiki
http://www.phaser.cimr.cam.ac.uk/index.php/MR_using_CCP4i:BETA/BLIP
2. MR
using CCP4i:BETA/BLIP
Reflection data:
beta_blip_P3221.mtz
Structure files:
beta.pdb,
blip.pdb
Sequence file:
beta.seq, blip.seq
This tutorial
demonstrates a difficult molecular
replacement problem.
β-Lactamase (BETA,
29kDa) is an enzyme produced
by various bacteria, and is of interest because it is
responsible for
penicillin resistance, cleaving penicillin at the β-lactam
ring. There are
many small molecule inhibitors of BETA in clinical use, but
bacteria can become
resistant to these as well. Streptomyces clavuligerus
produces beta-lactamase inhibitory protein (BLIP, 17.5kDa),
which has been
investigated as an alternative to small molecule inhibitors,
as it appears more
difficult for bacteria to become resistant to this form of
BETA inhibition. The
structures of BETA and BLIP were originally solved separately
by experimental
phasing methods. The crystal structure of the complex between
BETA and BLIP has
been a test case for molecular replacement because of the
difficulty
encountered in the original structure solution. BETA, which
models 62% of the
unit cell, is trivial to locate, but BLIP is more difficult to
find. The BLIP
component was originally found by testing a large number of
potential
orientations with a translation function search, until one
solution stood out
from the noise.
1.
What do you think
is the best order in which to
search for BETA and BLIP? Under what circumstances could the
lower molecular
weight search model be the easiest to find by molecular
replacement?
2.
What is the
space-group recorded on the mtz
file? If you had not solved this structure, would you
know that this was the space-group? If not, what other
space-group(s) must you
consider?
á
Think about
handedness (enantiomorphs)
3.
Run Phaser for
solving BETA/BLIP
á
Bring up the GUI
for Phaser
á
All the yellow
boxes need to be filled in.
á
Search for BETA
and BLIP in the one job.
4.
Has Phaser solved
the structure?
á Look at
the Z-scores for the rotation and translation functions
5.
What search order
was used?
á If you
wanted, you could force the other search order and see what
difference this
makes.
6.
Which space group
was the solution in?
7.
Look though the job.sum
file and identify the anisotropy correction, rotation
function, translation
function, packing, and refinement modes, for the two search
molecules, and all
the space groups. Draw a flow diagram of the search strategy.
8.
Why doesn't Phaser
perform the rotation function
in the two enantiomorphic space
groups?
9.
Which reflections
in the data are particularly
important for deciding the translational symmetry of the
space-groups to
search? Under what data collection conditions might you not
have recorded these
important reflections? Are there any other space-groups that
you might want to
consider when solving BETA/BLIP?
10.
How big is the
anisotropic correction for the
data? How does this compare to TOXD?
11.
Run Phaser again
with the anisotropy correction
turned off. What effect does this have on the structure
solution?