1. EMBL-EBI MSDpisa a web service for studying Protein Interfaces, Surfaces and Assemblies Eugene Krissinel http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html

2. EMBL-EBI What PISA is about Crystal = translated Unit Cell More than 80% of protein structures are solved by means of X-ray diffraction on crystals. An X-ray diffraction experiment produces atomic coordinates of the crystal’s Asymmetric Unit (ASU). In general, neither ASU nor Unit Cell has any relation to Biological Units, or stable protein complexes which act as units in physiological processes. PISA attempts to recover Biological Units from the protein crystallography data. Unit Cell = all space symmetry group mates of ASU PDB file

3. EMBL-EBI Crystal interfaces Stability of protein complexes depends on properties of protein-protein interfaces, such as free energy of formation  G int solvation energy gain  G S interface area hydrogen bonds and salt bridges across the interface hydrophobic specificity PISA calculates crystal contacts and their physical-chemical properties

4. EMBL-EBI Interface assessment A crystal may be viewed as a packing of assemblies with biologically insignificant contacts between them. Protein assembly is a packing of monomeric units with biologically relevant interfaces between them. PISA is a tool to assess crystal interfaces for biological significance.

5. EMBL-EBI Properties of assemblies Protein assemblies may exist in certain conditions and dissociate in other. Sometimes aggregation properties are the key to biological function. PISA calculates properties of protein assemblies, such as free energy of dissociation  G diss, and predicts a probable dissociation pattern. Dissociation into stable subunits with minimum

6. EMBL-EBI PISA workflow summary 1.Calculate properties of all structures 2.Calculate all crystal contacts and their properties 3.Find all assemblies which are possible in given crystal 4.Evaluate all assemblies for chemical stability and leave only potentially stable ones 5.Range assemblies by chances to be a biological unit

7. EMBL-EBI Enumerating assemblies in crystal Enumerating assemblies in crystal  crystal is represented as a periodic graph with monomeric chains as vertices and interfaces as edges  each set of assemblies is identified by engaged interface types  all assemblies may be enumerated by a backtracking scheme engaging all possible combinations of different interface types Example: crystal with 3 interface types Assembly set Engaged interface types 1000- only monomers 2001- dimer N1 3010- dimer N2 4011 Assembly set Engaged interface types 5100- dimer N3 6101 7110 8111- all crystal

8. EMBL-EBI  It is not properties of individual interfaces but rather chemical stability of protein complex in general that really matters  Protein chains will most likely associate into largest complexes that are still stable  A protein complex is stable if its free energy of dissociation is positive: Chemical stability of protein complexes

9. EMBL-EBI Protein affinity Solvation energy of protein complex Solvation energies of dissociated subunits Free energy of H-bond formation Number of H- bonds between dissociated subunits Free energy of salt bridge formation Number of salt bridges between dissociated subunits Dissociation into stable subunits with minimum Choice of dissociation subunits:

10. EMBL-EBI Entropy of dissociation Fitted parameter Mass of i-th subunit k-th principal moment of inertia of i-th subunit Murray C.W. and Verdonik M.L. (2002) J. Comput.-Aided Mol. Design 16, 741-753.

11. EMBL-EBI Benchmark results Assembly classification on the benchmark set of 218 structures published in Ponstingl, H., Kabir, T. and Thornton, J. (2003) Automatic inference of protein quaternary structures from crystals. J. Appl. Cryst. 36, 1116-1122. 198+20 198 homomers and 20 heteromers Fitted parameters: 1.Free energy of a H-bond : 2.Free energy of a salt bridge : 3.Constant entropy term : 4.Surface entropy factor : = 0.51 kcal/mol = 0.21 kcal/mol = 11.7 kcal/mol = 0.57·10 -3 kcal/(mol*Å 2 ) Classification error in  G diss : ± 5 kcal/mol

12. EMBL-EBI What is beyond the benchmark set? Classification results obtained for 366 recent depositions into PDB in reference to manual classification in MSD-EBI : 321+45 321 homomers and 45 heteromers Classification error in  G diss : ± 5 kcal/mol

13. EMBL-EBI Is it ever going to be 100%?  theoretical models for protein affinity and entropy change upon protein complexation are primitive  coordinate (experimental) data is of a limited accuracy  there is no feasible way to take conformations in crystal into account  experimental data on multimeric states is very limited and not always reliable - calibration of parameters is difficult  protein assemblies may exist in some environments and dissociate in other - a definite answer is simply not there Nobody should be that naive, because :

14. EMBL-EBI Web-server PISA http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html

15. EMBL-EBI