1. EBI is an Outstation of the European Molecular Biology Laboratory. A web service for the analysis of macromolecular interactions and complexes PDBe Protein Interfaces, Surfaces and Assemblies

2. 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  Neutron/X-ray scattering: mainly composition and multimeric state may be found.  Dynamic light scattering: 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.

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

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. 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. A simple thing to do …

9. 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?

10.  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

11. PISA 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:

12. If you have to ask... What quaternary structure can my crystal structure have? What are the crystal contacts and interfaces in my structure? What is the size of the crystal interfaces? What are the energetics that keep my quaternary structure together? Are there any other structures in the PDB that have similar interfaces? USE PDBePisa Upload your own PDB file for analysis !!

13. Web-server PISA E. Krissinel and K. Henrick (2007). Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774--797.

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

17. Details about the interface…

18. Summary What is a protein quaternary structure (PQS)? Difficulties determining PQS Properties of protein contacts Which answers can PISA give?