1. EBI is an Outstation of the European Molecular Biology Laboratory. A web service for the analysis of macromolecular interactions and complexes MSD Protein Interfaces, Surfaces and Assemblies Glen van Ginkel PDB Depositions http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html

2. Protein Data Bank in Europe http://www.ebi.ac.uk/pdbe1.11.092 Protein Quaternary Structures (PQS)‏  PQS is often a Biological Unit, performing a certain physiological function  PQS is a difficult subject for experimental studies Assembly of protein chains, stable in native environment  Light/Neutron/X-ray scattering: mainly composition and multimeric state may be found. 3D shape may be guessed from mobility measurements.  Electron microscopy: not a fantastic resolution and not applicable to all objects  NMR is not good for big chains, even less so for protein assemblies. In PDB, very few quaternary structures have been identified experimentally.

3. 3 PQS are difficult to calculate… 50 - 90% Secondary Structure (CASP 5), depending on method 2 10 - 90% Tertiary Structure (CASP 5), depending on method and target 3 Probably 0% Quaternary Structure. Docking of given number of given structures: 5 - 20% success (CAPRI 5)‏ 4 VNKERTFLAVKPDGVARGLVGEIIARYEKKGFVLVGLKQLVPTKDLAESHYAEHKERPFF then we can calculate... If we know the sequence... 1

4. But PQS are assigned to many PDB entries! Most of those are PROBABLE Quaternary Structures. 4 1.Depositor’s say prevails. 2.Accept everything which passes formal validation checks. 3.No experimental evidence for PQS is required. 4.If a depositor does not know or does not care (60-80% of instances for PQS), the curator is to decide. 5.The curator may use computing/modeling tools to assist the PQS annotation. The wwPDB “rules” are: www.wwpdb.org

5. 5 Crystallography is special in that … A) crystal is made of assemblies

6. 6 Crystallography is special in that … B) there is no need to dock subunits – the docking is given by crystal structure Macromolecular interfaces should be viewed as an additional and important artifact of protein crystallography

7. 7 Wealth of experimental data on PQS in PDB Crystal = translated Unit Cells More than 80% of macromolecular structures are solved by means of X-ray diffraction on crystals. It is reasonable to expect that PQS make building blocks for the crystal. An X-ray diffraction experiment produces atomic coordinates of the Asymmetric Unit (ASU), which are stored as a PDB file. In general, neither ASU nor Unit Cell has any direct relation to PQS. The PQS may be made of Unit Cell = all space symmetry group mates of ASU PDB file (ASU)‏ a single ASU part of ASU several ASU several parts of ASU

8. 8 ? no image or bad image In (very) simple terms … 2 crystallisation 3 in crystal ?? good image but no associations in vivo 1

9. 9 PQS server @ EBI (Kim Henrick) Trends in Biochem. Sci. (1998) 23, 358 PITA server @ EBI (Hannes Ponstingl) J. Appl. Cryst. (2003) 36, 1116 A simple thing to do …

10. 10  PQS server @ MSD-EBI (Kim Henrick) Trends in Biochem. Sci. (1998) 23, 358 http://pqs.ebi.ac.uk Method: progressive build-up by addition of monomeric chains that suit the selection criteria. The results are partly curated. http://www.ebi.ac.uk/thornton-srv/databases/pita/ Method: recursive splitting of the largest complexes as allowed by crystal symmetry. Termination criteria is derived from the individual statistical scores of crystal contacts. The results are not curated.  PITA software @ Thornton group EBI (Hannes Ponstingl) J. Appl. Cryst. (2003) 36, 1116 Making assemblies from significant interfaces

11. 11 Protein functionality: the interface should be engaged in any sort of interaction, including transient short-living protein-ligand and protein-protein etc. associations. Obviously important properties: Affinity (comes from area, hydrophobicity, electrostatics, H-bond density etc.)‏ Depends on the problem. Stable macromolecular complexes, PQS: the interface should make a sound binding. Important properties: Sufficient free energy of binding something else? Aminoacid composition Geometrical complementarity Overall shape, compactness Charge distribution etc. and properties that may be important for reaction pathway and dynamics: What is a significant interface?

12. 12 Jones, S. & Thornton, J.M. (1996) Principles of protein-protein interactions, Proc. Natl. Acad. Sci. USA, 93, 13-20. 10 20 30 40 50 60 204060801000 Rank ordering bins % of real interfaces PlanarNonplanar rms of least-square plane 10 20 30 40 50 60 204060801000 Rank ordering bins % of real interfaces Low protrusionHigh protrusion Protrusion index 10 20 30 40 50 60 204060801000 Rank ordering bins % of real interfaces Less hydrophobicMore hydrophobic Hydrophobicity Low ASAHigh ASA ASA 10 20 30 40 50 60 204060 801000 Rank ordering bins % of real interfaces Real and superficial interfaces

13. 13  “ No single parameter absolutely differentiates the interfaces from all other surface patches ” Jones, S. & Thornton, J.M. (1996) Principles of protein-protein interactions, Proc. Natl. Acad. Sci. USA, 93, 13-20.  Formation of N>2 -meric complexes is most probably a corporate process involving a set of interfaces. Therefore significance of an interface should not be detached from the context of protein complex  “… the type of complexes need to be taken into account when characterizing interfaces between them. ” Jones, S. & Thornton, J.M., ibid. Real and superficial interfaces

14. 14  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

15. 15  Solvation energy of protein complex  Solvation energies of dissociated subunits  Free energy of H-bond formation  Free energy of salt bridge formation  Number of H-bonds between dissociated subunits  Number of salt bridges between dissociated subunits Dissociation into stable subunits with minimum Choice of dissociation subunits: Protein affinity

16. Macromolecular Structure Database31.10.07 16  Translational entropy  Rotational entropy  Sidechain entropy  Mass  Tensor of inertia  Solvent-accessible surface area  Symmetry number Entropy of macromolecules in solutions

17. 17 crystal is represented as a periodic graph with monomeric chains as nodes 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 Enumerating assemblies in crystal

18. 18 Method Summary 1.Build periodic graph of the crystal 2.Enumerate all possibly stable assemblies 3.Evaluate assemblies for chemical stability 4.Leave only sets of stable assemblies in the list and range them by chances to be a biological unit : Larger assemblies take preference Single-assembly solutions take preference Otherwise, assemblies with higher  G diss take preference Detection of Biological Units in Crystals:

19. Macromolecular Structure Database31.10.0719 If you have to ask... What quaternary structure can my crystal structure have? What are the crystal contacts and interfaces in my structure ? What are the energetics that keep my quaternary structure together ? Are there any other structures in the PDB that have similar interfaces ? USE MSDPisa Upload your own PDB file for analysis !!

20. 20 A new MSD-EBI tool for working with Protein Interfaces, Surfaces and Assemblies http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html Web-server PISA

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23. Details about the interface…