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PDBsum entry 2pxt
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Signaling protein/RNA
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PDB id
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2pxt
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References listed in PDB file
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Key reference
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Title
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A general strategy to solve the phase problem in RNA crystallography.
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Authors
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A.Y.Keel,
R.P.Rambo,
R.T.Batey,
J.S.Kieft.
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Ref.
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Structure, 2007,
15,
761-772.
[DOI no: ]
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PubMed id
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Abstract
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X-ray crystallography of biologically important RNA molecules has been hampered
by technical challenges, including finding heavy-atom derivatives to obtain
high-quality experimental phase information. Existing techniques have drawbacks,
limiting the rate at which important new structures are solved. To address this,
we have developed a reliable means to localize heavy atoms specifically to
virtually any RNA. By solving the crystal structures of thirteen variants of the
G*U wobble pair cation binding motif, we have identified a version that when
inserted into an RNA helix introduces a high-occupancy cation binding site
suitable for phasing. This "directed soaking" strategy can be
integrated fully into existing RNA crystallography methods, potentially
increasing the rate at which important structures are solved and facilitating
routine solving of structures using Cu-Kalpha radiation. This method already has
been used to solve several crystal structures.
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Figure 2.
Figure 2. Engineered Cation Binding
(A) Electron density
and structure of the wild-type SRP RNA-M domain complex, showing
the portion of the helix that was varied. The view is into the
major groove. Two cobalt (III) hexammine ions (magenta) are
located near the phosphate backbone (upper right site) and two
adjacent G-C pairs (lower left).
(B) Electron density and
structure of the variant PM04, with the tandem wobble pairs
shown in cyan and the resultant major groove-bound cobalt (III)
hexammine in magenta.
(C) Anomalous difference Fourier map
(contoured at 7 σ in red) of PM04 superimposed on the
structure. The tandem wobble pairs are shown in cyan. The
endogenous reference site is at the top, and the new engineered
site at the bottom.
(D) Comparison of the very well-ordered
reference site to the new site in PM04 in a 2F[o] − F[c] map,
at 2 Å resolution, contoured at 2 σ.
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Figure 4.
Figure 4. General Rules for Cation Binding in the Major
Groove of G•U Motifs
(A) Schematic of the arrangement of
major groove functional groups for PM01, PM05, and PM09. These
three variants vary only in the orientation of flanking G-C
pairs relative to the tandem G•U pairs. Blue circles are
amine, red are carbonyls, and orange are purine N7 groups. In
PM01, the two major groove amines (from cytosine) are placed
away from the binding site due to the turn of the helix. In PM05
and PM09, the amines are placed in position to limit the
mobility of the ion (shown in green). In addition, the location
of carbonyls and N7 groups in the flanking sequences and close
to the binding site make the ion more mobile as it attempts to
satisfy multiple potential hydrogen binding partners.
(B)
Schematic of the arrangement of major groove functional groups
in the four single G•U pair containing variants. In single
G•U pairs, the amines are best placed in the 3′ positions to
withdraw them from the pocket.
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The above figures are
reprinted
from an Open Access publication published by Cell Press:
Structure
(2007,
15,
761-772)
copyright 2007.
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