1. IBGP/BMI 705 Lab 4: Protein structure and alignment TA: L. Cooper

2. Why Align Structures 1.For homologous proteins (similar ancestry), this provides the “gold standard” for sequence alignment – elucidates the common ancestry of the proteins. 2.For nonhomologous proteins, allows us to identify common substructures of interest. 3.Allows us to classify proteins into clusters, based on structural similarity.

3. Example of Structural Homologs 4DFR: Dihydrofolate reductase 1YAC: Octameric Hydrolase of Unknown Specificity 5.9% sequence identity (best alignment) 1YAC structure solved without knowing function. Alignment to 4DFR and others implies it is a hydrolase of some sort. DHFR:yellow & orange YAC:green & purple Sheets only Helices only

4. Example of Structural Homologs Sequence alignment SLSAAEADLAGKSWAPVFANKNANGLDFLVALFEKFPDSANFFADFK-GKSVADIKA-S VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHG PKLRDVSSRIFTRLNEFVNNAANAGKMSAMLSQFAKEHVGFGVGSAQFENVRSMFPGFVA KKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTP Structural alignment XSLSAAEADLAGKSW-APVFANKN-ANGLDFLVALFEKFPDSANFF-ADFKGKSVA—-DIK V-LSPADKTNVKAAWGK-VGAHA-GEYGAEALERMFLSFPTTKTYFPHF-------DLS-H ASPKLRDVSSRIFTRLNEFVNNAANAGKMSA-MLSQ-FAKEHV-GFGVGSAQFENVRSM-F GSAQVKGHGKKVADALTNAVAHV-D—-DMPNAL—-SALSDLHAHKLRVDPVNFKLLS-HCL PGFVA LVTLAAHLPAEFTP

5. How to Align Structures 1. Visual inspection (by eye) 2. Computational approach Point-based methods using point distances and other properties to establish correspondences Secondary structure-based methods use vectors representing secondary structures to establish correspondences. motif Global local

6. Structural Alignment Algorithms Alignment algorithms create a one-to-one mapping of subset(s) of one sequence to subset(s) of another sequence. Structure-based alignment algorithms do this by minimizing the structure difference score or root-mean-square difference (rmsd) in alpha-carbon positions. Find correspondence set Find alignment transform (protein superposition problem) Chicken-and-egg

7. Parameter Space Torsion angles (  ) are: - local by nature (error propagation) - invariant upon rotation and translation of the molecule - compact (O(n) angles for a protein of n residues) Add 1 degree To all  Problem: find the rotation matrix, R and a vector, v, that minimize the following quantity:

8. Structural Alignments Methods STRUCTAL [Levitt, Subbiah, Gerstein] Using dynamic programming with a distance metric DALI [Holm, Sander] Analysis of distance maps LOCK [Singh, Brutlag] Analysis of secondary structure vectors, followed by refinement with distances SSAP [Orengo and Taylor, 1989] VAST [Gibrat et al., 1996] CE [Shindyalov and Bourne, 1998] SSM [Krissinel and Henrik, 2004] …

9. VAST (Vector Alignment Search Tool) It places great emphasis on the definition of the threshold of significant structural similarity. By focusing on similarities that are surprising in the statistical sense, one does not waste time examining many similarities of small substructures that occur by chance in protein structure comparison. Very many of the remaining similarities are examples of remote homology, often undetectable by sequence comparison. As such they may provide a broader view of the structure, function and evolution of a protein family. At the heart of VAST's significance calculation is definition of the "unit" of tertiary structure similarity as pairs of secondary structure elements (SSE's) that have similar type, relative orientation, and connectivity. In comparing two protein domains the most surprising substructure similarity is that where the sum of superposition scores across these "units" is greatest. The likelihood that this similarity would be seen by chance is then given as a simple product: the probability that one would obtain this score in drawing so many "units" at random, times the number of alternative SSE-pair combinations possible in the domain comparison, from which one has chosen the best. http://www.ncbi.nlm.nih.gov/Structure/RESEARCH/iucrabs.html#Ref_6

10. Today’s lab Answer questions bolded on handout (There are five)

11. PDB: Protein structure viewing

12. PDB- Protein structure viewing

15. SCOP: Protein Classification

18. VAST: Alignment