Template Assignment: FastA search on nonredundant set of PDB variable domain sequences - the best 5 templates are used.

3D-Alignment of Template Structures: 3D Structures of the templates are aligned by least square fit of the core C-alpha positions (as defined in Slide 8)

Comparison of Template Sequences and Structures to Target Sequences: Evaluation of potental effects of mutations on the model structure, possibly additon of auxilliary templates for loop structures

Coordinate Assignment: Automatic where a suitable template is available, based on template structure and side chain rotamer library. Loop searches or conformational searches are used to generate loop structures for which no suitable template could be found.

Identification and Correction of Steric Problems: Bump check, checking mutated side chains for potental Hbonds, cavity formation etc. If necessary, optimize side chain conformation by switching through the rotamer library. It is important to do this check before any energy minimization is performed, since energy minimization often will only serve to spread out the problem and hide it. Once all major conflicts have been resolved, energy minimization is used to refine the model: first: splice sites only, then mutated side chains, the all side chains, loop main chain segments, finally all atoms of the molecule mobile

Relative Orientation of VH and VL Domain: If the FV fragment containing the primary template for the VL domain and that containing the primary template for the VH domain a have very similar domain orientation, the relative orientation for the model can be taken from the least square fit od the two parental FV fragments. If there is a large discrepancy, the dimer interface has to be scrutinized for interactions which might explain the discrepancy. Particularely VL lambdas seem to lead to increased variability in the domain orientation. The dimer interface has to be again screen for possible conflicts before the domain models are merged to an FV model and further minimized. Molecular dynamics may be used to explore the conformational freedom loops and side chains.