Ginalski (P0453) - 71 predictions: 71 3D
structural and sequential constraints identified above. In some cases close
homologues were also submitted to the Meta Server as the query sequences.
For regions that displayed low stability (i.e. highly dependent on the server),
possible alignment variants were derived manually, guided mainly by
secondary structure predictions.
Modeling of CASP5 Target Proteins with 3D-CAM
K. Ginalski1,2
1
- Interdisciplinary Centre for Mathematical and Computational Modelling,
Warsaw University, Warsaw, Poland, 2 - BioInfoBank Institute, Poznań, Poland
kginal@bioinfo.pl
All plausible alternative sequence-to-structure alignments were tested by
building 3D molecular models for the target sequence with the Homology
module of InsightII (Accelrys Inc., San Diego, CA). Backbone conformation
was taken from the template structure, and only non-conserved side chains
were substituted. Modeling of loops that contained insertion and deletion
regions was skipped in this procedure. Models were then subjected to detailed
evaluation, mainly by visual inspection of structural consistency and using
Verify3D [5] and ProsaII [6] energy profiles. Such a 3D evaluation procedure
enabled selection of final sequence-to-structure alignments.
For the fifth round of Critical Assessment of Techniques for Protein Structure
Prediction (CASP5), 67 target proteins were modeled using the 3D-Consensus
Alignment Method (3D-CAM). The issue of sequence-to-structure alignment of
target sequences with their respective parent structures was the main emphasis,
and as shown in previous rounds of CASP, this part of the modeling procedure
is the major source of errors. The critical steps in modeling: selection of
template(s) and generation of sequence-to-structure alignment, were based on
the results of secondary structure prediction and tertiary fold recognition
carried out using the Meta Server [1].
Final models of target proteins were built using the MODELLER program [7].
Where possible, more than one template protein was used, after
superimposition of their molecular structures. The overall quality of each
modeled structure was checked in detail with the WHAT_CHECK program [8].
No energy minimization procedures were employed.
Initially, related proteins with known structures were identified from the
consensus of the Meta Server results. For difficult targets, template (fold)
identification was based on the results of the 3D-Jury method (Rychlewski L.,
unpublished). Structural determinants of the fold were then analyzed: all the
structures representing a given fold, and corresponding structural alignment
extracted from the FSSP database [2], were inspected for both conservation and
variability of the structural elements. Conservation of specific residues and
contacts responsible for maintaining tertiary structure, and critical for substrate
binding and/or catalysis, were also established. Additionally, homologous
sequences that matched the targets were collected with PSI-BLAST searches
[3] performed against the non-redundant protein sequence database and
unfinished genomes until profile convergence. The CLUSTAL W program [4]
was used to generate multiple sequence alignments for sets of sequences
containing target, and other closely-related proteins, to identify conserved
residues within the family.
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All alignments produced by different servers interacting with the Meta Server
were inspected for both variability and violation of structural integrity. Initial
alignment was obtained by taking, in most cases, the common alignment for
each region (mainly for each secondary structure element), taking into account
the structural alignment of templates where possible, within the context of the
7.
8.
A-1
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