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PDBsum entry 1kmt
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Protein binding
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PDB id
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1kmt
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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The impact of glu-->Ala and glu-->Asp mutations on the crystallization properties of rhogdi: the structure of rhogdi at 1.3 a resolution.
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Authors
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A.Mateja,
Y.Devedjiev,
D.Krowarsch,
K.Longenecker,
Z.Dauter,
J.Otlewski,
Z.S.Derewenda.
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Ref.
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Acta Crystallogr D Biol Crystallogr, 2002,
58,
1983-1991.
[DOI no: ]
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PubMed id
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Abstract
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It is hypothesized that surface residues with high conformational entropy,
specifically lysines and glutamates, impede protein crystallization. In a
previous study using a model system of Rho-specific guanine nucleotide
dissociation inhibitor (RhoGDI), it was shown that mutating Lys residues to Ala
results in enhanced crystallizability, particularly when clusters of lysines are
targeted. It was also shown that one of these mutants formed crystals that
yielded diffraction to 2.0 A, a significant improvement on the wild-type protein
crystals. In the current paper, an analysis of the impact of surface mutations
replacing Glu residues with Ala or Asp on the stability and crystallization
properties of RhoGDI is presented. The Glu-->Ala (Asp) mutants are generally
more likely to produce crystals of the protein than the wild-type and in one
case the resulting crystals yielded a diffraction pattern to 1.2 A resolution.
This occurs in spite of the fact that mutating surface Glu residues almost
invariably affects the protein's stability, as illustrated by the reduced deltaG
between folded and unfolded forms measured by isothermal equilibrium
denaturation. The present study strongly supports the notion that rational
surface mutagenesis can be an effective tool in overcoming problems stemming
from the protein's recalcitrance to crystallization and may also yield dramatic
improvements in crystal quality.
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Figure 1.
Figure 1 A view of the RhoGDI  66
fragment with glutamates shown in full and colored gold. The two
sites which were mutated in the highly diffracting crystal form
are shown in pink. The sites are numbered according to the
amino-acid sequence of the full-length protein. The figure was
prepared using RIBBONS (Carson, 1991[Carson, M. (1991). J. Appl.
Cryst. 24, 958-961.]).
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Figure 3.
Figure 3 Ribbon diagram of the overall arrangement of the
molecules in the asymmetric unit, with the location of the point
mutations identified by magenta spheres. The two molecules
within the asymmetric unit are colored according to the
temperature factors. The grey sphere indicates the entrance to
the hydrophobic pocket. The figure was prepared using BOBSCRIPT
(Esnouf, 1997[Esnouf, R. M. (1997). J. Mol. Graph. 15,
132-143.]).
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The above figures are
reprinted
by permission from the IUCr:
Acta Crystallogr D Biol Crystallogr
(2002,
58,
1983-1991)
copyright 2002.
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