1. Identifying structural templates using alignments of designed sequences Stefan M. Larson Pande Group Biophysics Program December, 2002 smlarson@stanford.edu

2. Structure prediction & sequence space ASDJFHLKASDLFH ASDFLHUHOUIQWE QWEONBLQWEROKJ ASDFPOIQWERUHO QWEORSADFLKJIJ ASDJFHLKASDLFHTJYH ASDFLHUHOUIQWEDFGH QWEONBLQWEROKJDGHJ ASDFPOIQWERUHODHGR QWEORSADFLKJIJGHFG QWOIEGTXKNBVALHERT ASDLFHIUWERHSDDFGH KBJDDURMWOFBMFERTJ FGJDKEGORTMVIRGHRT ASDJFHLKASDLFHTJYH ASDFLHUHOUIQWEDFGH QWEONBLQWEROKJDGHJ ASDFPOIQWERUHODHGR QWEORSADFLKJIJGHFG ASDJFHLKASD ASDFLHUHOUI QWEONBLQWER ASDFPOIQWER QWEORSADFLK

3. Multiple sequence alignments aid comparative protein modeling 1 in 3 sequences are recognizably related to at least one protein structure. A significant fraction of the remaining 2/3 have solved structural homologues, but they are not recognized through sequence similarity searching techniques. Marti-Renom et al. (2000) Multiple sequence alignments greatly improve the efficacy and accuracy of almost all phase of comparative modeling. Venclovas (2001)

4. Computational protein design Native structure Iterative refinement New sequence

5. Large scale sequence generation 200,000Total sequences generated 4,000Processors available 80 daysTotal time of data collection 26,400Total backbone variants 264Total structures “Reverse BLAST” study:

6. “Reverse BLAST”: finding templates for comparative modeling Larson SM, Garg A, Desjarlais JR, Pande VS. (2003) Proteins: Structure, Function, and Genetics

7. Experiment: Sequence quality ASDFASDFASDFAS FDSAFASDFASDFA FASDFASDFASDFA FHFDIDIFERIDKD ADHFYWTEFHHASD ASDFYEFHGASDFV ADHFYWTEFHHASD ASDFYEFHGASDFV DGSAHDYERCNDFK AKSLKALSDFPLAK Design BLAST E<0.01

8. Results: Sequence quality

9. Method: “Reverse BLAST” THEHYPOTHETICA LPROTEINSEQUEN CEASDFASDFASDF AASDFASDFASDFA SDFASDFASDFASD FASDFHWERHWIEN CVASDFNWEFUWEF BLAST E<0.01 THEHYPOTHETICA LPROTEINSEQUEN CEASDFASDFASDF AASDFASDFASDFA SDFASDFASDFASD FASDFHWERHWIEN CVASDFNWEFUWEF Designed SequencesHypothetical ProteinsStructural Templates

10. Do the designed sequences help? Correctly identified structural templates fold-increase in # of templates fold-increase in # of genes total hits

11. Remote homology detection

12. Optimizing structural diversity sequence entropy prediction accuracy prediction coverage mean pairwise %ID mean native %ID

13. Future work Compare “reverse BLAST” to other remote homology detection approaches (3D-PSSM, HHMER, etc). Retrodict CASP targets, especially those which were not successfully predicted by comparative modeling. Increase the coverage and accuracy of the designed sequence sets.

14. Collaborators Stanford University Amit Garg Dr. Vijay Pande Harvard University Jeremy England Xencor, Inc. Dr. John Desjarlais