This tutorial originally comes from the Phaser
The files for this tutorial can be found at:
MR using BETA/BLIP
This tutorial demonstrates a difficult molecular replacement
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.
- 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
- 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
Run Phaser for solving BETA/BLIP
- Think about handedness (enantiomorphs)
Has Phaser solved the structure?
- Bring up the GUI for Phaser
- All the yellow boxes need to be filled in.
- Search for BETA and BLIP in the one job.
What search order was used?
- Look at the Z-scores for the rotation and translation
Which space group was the solution in?
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.
Why doesn't Phaser perform the rotation function in the two
enantiomorphic space groups?
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
How big is the anisotropic correction for the data? How does
this compare to TOXD?
Run Phaser again with the anisotropy correction turned off.
What effect does this have on the structure solution?
- If you wanted, you could force the other search order and
see what difference this makes.