Studies of symbiotically important Rhizobium meliloti exopolysaccharides EPSII and succinoglycan

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, 1999.Includes bibliographical references.by Louis LeCour, Jr.S.M

Crystal structure and computational modeling of the fab fragment from a protective anti-ricin monoclonal antibody.

Many antibody crystal structures have been solved. Structural modeling programs have been developed that utilize this information to predict 3-D structures of an antibody based upon its sequence. Because of the problem of self-reference, the accuracy and utility of these predictions can only be tested when a new structure has not yet been deposited in the Protein Data Bank.We have solved the crystal structure of the Fab fragment of RAC18, a protective anti-ricin mAb, to 1.9 Å resolution. We have also modeled the Fv structure of RAC18 using publicly available Ab modeling tools Prediction of Immunoglobulin Structures (PIGS), RosettaAntibody, and Web Antibody Modeling (WAM). The model structures underwent energy minimization. We compared results to the crystal structure on the basis of root-mean-square deviation (RMSD), template modeling score (TM-score), Z-score, and MolProbity analysis.The crystal structure showed a pocket formed mainly by AA residues in each of the heavy chain complementarity determining regions (CDRs). There were differences between the crystal structure and structures predicted by the modeling tools, particularly in the CDRs. There were also differences among the predicted models, although the differences were small and within experimental error. No one modeling program was clearly superior to the others. In some cases, choosing structures based only on sequence homology to the crystallized Ab yielded RMSDs comparable to the models.Molecular modeling programs accurately predict the structure of most regions of antibody variable domains of RAC18. The hypervariable CDRs proved most difficult to model, particularly H chain CDR3. Because CDR3 is most often involved in contact with antigen, this defect must be considered when using models to identify potential contacts between antibody and antigen. Because this study represents only a single case, the results cannot be generalized. Rather they highlight the utility and limitations of modeling programs

Features of Ab Modeling Tools.

<p>Features of Ab Modeling Tools.</p

Data Collection and Refinement Statistics.

a<p>Data in parenthesis pertain to the highest resolution shell (2.0 Å-1.9 Å).</p>b<p>Rint = ∑|I - <i>|/∑I, where I is the observed intensity of a measured reflection and <i> is the mean intensity for all observation of symmetry-related reflections.</i></i></p><i><i>c<p>R factor = Σ |F<i>_oh</i> – F<i>_ch</i>|/Σ F<i>_oh</i>, where F<i>_oh</i> and F<i>_ch</i> are the observed and calculated structure factor amplitudes for the 32,658 reflections <i>h</i> that were used in structure refinement.</p>d<p>R free = Σ |F<i>_oh</i> – F<i>_ch</i>|/Σ F<i>_oh</i>, where F<i>_oh</i> and F<i>_ch</i> are the observed and calculated structure factor amplitudes pertaining to the 2,070 reflections <i>h</i> that were <u>not</u> used in structure refinement.</p></i></i

TM-Score of Comparison of Model to Crystal Structure.

a<p>None of the comparisons among the models are significantly different from the others.</p>b<p>Before or after energy minimization of the models.</p

Z-Score of Modeling Tools.

<p>Z-Score of Modeling Tools.</p

Sequence Features of RAC18.

a<p>Assignment by IgBlast <a href="http://www.ncbi.nlm.nih.gov/igblast/" target="_blank">http://www.ncbi.nlm.nih.gov/igblast/</a>.</p>b<p>Assigned according to references <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052613#pone.0052613-AlLazikani1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052613#pone.0052613-Chothia1" target="_blank">[11]</a>, using the website <a href="http://www.bioinf.org.uk/abs/chothia.html" target="_blank">http://www.bioinf.org.uk/abs/chothia.html</a>.</p

Molprobity Analysis.

a<p>100% (100^th percentile) is the best; 0% (0^th percentile) is the worst.</p>b<p>Goal: <1%.</p>c<p>Goal: <0.2%.</p>d<p>Goal: >98%.</p>e<p>Goal: 0.</p>f<p>Goal: 0%.</p>g<p>Goal: <0.1%.</p>h<p>MolProbity score: the lower, the better; it is a composite index calculated as a combination of clash score, percentage of rotamer outliers, and percentage of Ramachandran favored regions.</p

Three-dimensional crystal structure of the RAC18 Fab.

<p>A. Ribbon structure of the Fab. B. Surface diagram of a potential Ag-binding pocket. C. Electron density map of the CDR loops contoured at 0.5 σ. Color code: light gray (light chain), dark gray (heavy chain), red (CDR L1), yellow (CDR L2), blue (CDR L3), orange (CDR H1), green (CDR H2), purple (CDR H3). Residues lining the pocket are in pink; L chain 96L; H chain 32Y, 33Y, 35N, 47W, 50L, 97A, 98R, 99R, 105L, and 106F. Pocket size: height ∼8 Å, major axis ∼12 Å, minor axis ∼5 Å. Lining the pocket are 6 hydrophobic, and 5 hydrophilic (2 charged) residues.</p

RMSD Distances Between Models and Crystal Structure.

a<p>Before or after energy minimization of the model structures.</p>b<p>None of the comparisons among the models are significantly different from the others.</p>c<p>Angstroms.</p