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PDBsum entry 2blo
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References listed in PDB file
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Key reference
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Title
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Improving radiation-Damage substructures for rip.
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Authors
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M.H.Nanao,
G.M.Sheldrick,
R.B.Ravelli.
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Ref.
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Acta Crystallogr D Biol Crystallogr, 2005,
61,
1227-1237.
[DOI no: ]
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PubMed id
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Abstract
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Specific radiation damage can be used to solve macromolecular structures using
the radiation-damage-induced phasing (RIP) method. The method has been
investigated for six disulfide-containing test structures (elastase, insulin,
lysozyme, ribonuclease A, trypsin and thaumatin) using data sets that were
collected on a third-generation synchrotron undulator beamline with a highly
attenuated beam. Each crystal was exposed to the unattenuated X-ray beam between
the collection of a 'before' and an 'after' data set. The X-ray 'burn'-induced
intensity differences ranged from 5 to 15%, depending on the protein
investigated. X-ray-susceptible substructures were determined using the
integrated direct and Patterson methods in SHELXD. The best substructures were
found by downscaling the 'after' data set in SHELXC by a scale factor K, with
optimal values ranging from 0.96 to 0.99. The initial substructures were
improved through iteration with SHELXE by the addition of negatively occupied
sites as well as a large number of relatively weak sites. The final
substructures ranged from 40 to more than 300 sites, with strongest peaks as
high as 57sigma. All structures except one could be solved: it was not possible
to find the initial substructure for ribonuclease A, however, SHELXE iteration
starting with the known five most susceptible sites gave excellent maps.
Downscaling proved to be necessary for the solution of elastase, lysozyme and
thaumatin and reduced the number of SHELXE iterations in the other cases. The
combination of downscaling and substructure iteration provides important
benefits for the phasing of macromolecular structures using radiation damage.
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Figure 4.
Figure 4
The effect of scale factor on the structure solution of (a) insulin, (b) lysozyme, (c)
thaumatin and (d) trypsin. CC[best] is represented by open squares, wMPE is represented by
crosses and pseudo-free CC is represented as closed squares.
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Figure 6.
Figure 6
Experimental electron-density maps (a) and weighted mean phase errors (b) for lysozyme for
different subsequent iterations in SHELXE. A total of 13 substructure iterations were
made; maps were drawn after iteration Nos. 1, 6, 8, 9, 10 and 11.
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The above figures are
reprinted
by permission from the IUCr:
Acta Crystallogr D Biol Crystallogr
(2005,
61,
1227-1237)
copyright 2005.
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