Download 1. How many molecules are in the asymmetric unit of the NP4 crystal

Bioc585 2008 Exercise 5 – Molecular Replacement (Answers, 10 points)
1. How many molecules are in the asymmetric unit of the NP4 crystal? What is the solvent content?
For a 20,000 Da protein in space group C2 (four asymmetric units), with cell parameters of a = 70
Å, b = 43 Å, c =53 Å, α =90, β = 94°, γ = 90°, there is room for only one molecule in the
asymmetric unit. Solvent content: 38%. Thus, only one molecular replacement solution is needed.
2. Show density for an NP1 incorrect amino acid, fix the residue, refine the new model with
Refmac, and show the new density.
One possibility is Lys 16, which becomes Asp 16 in NP4.
3. Examine the distal pocket. Is NO bound?
There is density in the distal pocket (above the heme in the figure that is consistent with NO
binding.
4. Examine the loops near the heme distal pocket. Are they the same or different in NP4? Justify
your answer.
Both loop regions look poor in the map but a second position is suggested by unoccupied density.
For example, near the A-B loop (residues 31-37, see fig.), there is positive density closer to the
heme. The best justification would be to rebuild the loop into the extra density and refine the
structure to see if the fit is reasonable (not required).
5. Extra credit question: 5 points. If you would like to experience a more difficult molecular
replacement problem (but still relatively straightforward), repeat the assignment but using NP2
(1PEE.pdb) as a starting model. The lower homology makes the initial model less reliable, but by
trimming away portions of the search model that disagree (e.g. mobile loops, incompatible amino
acids, etc.) a molecular replacement solution can be found. For full credit, try one or more
strategies and describe your results.
It turns out that the top molecular replacement solution is the correct one if a search is done using
3.0 Å data, but proving this unambiguously requires several rounds of model building and
refinement. Best success is likely by slowly increasing resolution from 3.0 to high resolution,
starting with rigid body refinement and only slowly releasing constraints.