Current Status of Homology Modeling Using MCSG Structures

1. 1t5b domain 1t5b domain Q92LV5 Q92LV5 template template domain model domain model Gly140/141 Gly140/141 Gly140/141 Gly140/141 Gly154/155 Gly154/155 Gly154/155 Gly154/155 Tyr95 Tyr95 Tyr95 Tyr95 Phe97 Phe97 Phe97 Phe97 Phe103 Phe103 Phe103 Phe103 Asp96 Asp96 Asp96 Asp96 Trp102 Trp102 Trp102 Trp102 Current Status of Homology Modeling Using MCSG Structures • 319 MCSG structures in PDB have over 400,000 sequence homologues. • These structures represent ~350 domains. • Models are built by MODELLER (Sali) and quality is assessed using PROSA (Sippl). • High-quality models can be generated for ~80,000 proteins. • Web site has been established that allows automated modeling of sequence homologues and evaluate the quality of the models. www.biochem.ucl.ac.uk/~dlee/GeMMA

2. Protein Structure Initiative - the Need for Large-Scale Homology Modeling • In the next five years PSI can determine approximately 3,000-4,000 protein structures, mainly at course granularity. • Reality check: novel structures in PDB will represent very small fraction of sequences in GenBank – reliable homology modeling is critical for obtaining 3D models and extending experimental work. • In PSI2 targets for structure determination are selected from large families, therefore determined structures have a large number of sequence homologues at wide range of sequence similarity. Protein often display different function. • Homology modeling must provide tools and 3D proteins models that can be used for high-confidence, reliable interpretation of specific structural features in distant (15-25%) sequence homologues, protein function assignment and evolution. • Models should provide guide for increasing number of more sophisticated experiments including: (i) aid mutagenesis and biochemical studies, (ii) predicting ligand binding, (iii) predicting oligomerization state, (iv) predicting cellular interactions (protein/protein/DNA/RNA). • We need to consider how PSI target selection of protein sequences and subsequent structure determination can improve homology modeling and the quality of the models.

3. Major Issues with Large-Scale Homology Modeling for Structural Genomics • 3D proteins models for distant (15-25%) sequence homologues are often not suitable. • Because of sequence divergence for very large families only small fraction of sequences can be reliably modeled (10-20%). • Homology modeling must provide input to target selection in fine coverage of protein families. • Domain parsing needs improvement. • We should be able to model multi-domain proteins from structures of individual domains. • We should be able to model neighbouring side chains and important structural and functional features that currently are difficult to assigned and predict correctly. • We need methods to predict unusual features and departures from the structure that is used for modelling. • Modelling loop and high B factor regions needs improvement.

4. Structure of P5CR Exemplifies Challenges for Homology Modeling • Two structures of P5CR were determined. • The proteins share 22% sequence identity and 47% sequence similarity. • Structures of monomer are very similar but show individual features. • Problems: • Protein has two domains and forms oligomers, one domain shows major swapping and protein forms different oligomeric forms in different species

5. Human Aldose Reductase – SeMet MAD at 0.9 Å Comparison – Experimental vs. Refined Map Refined map @ 0.9 Å, sigmaA (2mFo-DFc), contour level: 1 sigma Experimental map @ 0.9 Å, Fo, contour level: 1 sigma

6. MAD Map at 3.2 Å, 1.8 Å, 1.6 Å and 1.1 Å

7. Inhibitor Head Existing in Double Conformation Hard to Interpret at RT (1.45 Å), Clear at 100 K (0.8 Å) His 110 Tyr 48