Protein Structure

1. Protein Structure

2. Why study protein structure? Studying the structure model allows better understanding of the structure-function relationship, and is an important starting point for many kinds of research

3. F F Structure determination Crystallography: • A solution of protein molecules is assembled into a periodic lattice • The crystal is bombarded with X-ray beams • The collision of the beams with the atom electrons creates a diffraction pattern • The diffraction pattern is transformed into an electron density map of the protein from which the 3D locations of the atoms can be deduced

4. Structure determination Nuclear magnetic resonance (NMR): • A solution of the protein is placed in a magnetic field • Spins align parallel or anti-parallel to the field • RF pulses of electromagnetic energy shift spins from their alignment • Upon radiation termination spins re-align while emitting the energy they absorbed • The emission spectrum contains information about the identity of the nuclei and their immediate environment • The result is an ensemble of models rather than a single one

5. PDB: Protein Data Bankhttp://www.rcsb.org

6. Defines the 3D coordinates (x,y,z) of each of the atoms in one or more molecules (i.e., complex) There are models of proteins, protein complexes, proteins and DNA, protein segments, etc The models also include the positions of ligand molecules, solvent molecules, metal ions, etc PDB ID: integer + 3 integers/characters (e.g., 1a14) PDB model

7. The PDB file format

8. The PDB file format Residue identity Occupancy atom coordinates Atom identity chain Temperature factor Residue number Atom number X Y Z ATOM Records: Usually protein or DNA HETATM Records: Usually Ligand, ion, water

10. Background and motivation • DNA methylation at DNA CpG sites has a central role in imprinting (plants and mammals), but it is not clear how the imprinting machinery recognizes its target genes • The Dnmt3 protein family (a,b,l) are de novo methyltransferases • Dnmt3a and Dnmt3l KO mice show altered sex-specific de novo methylation in germ cells, indicating that these proteins are both required for the methylation of most imprinted loci in germ cells Goal: Conduct a structural and biochemical study of a homogeneous complex of Dnmt3L and Dnmt3a

11. Methods • Dnmt3a2, the shorter isoform of Dnmt3a that is the predominant form in embryonic stem cells was selected • For crystallographic reasons, a stable complex of the C-terminal domains from both proteins (Dnmt3a2-C and Dnmt3L-C) that retains substantial methyltransferase activity was focused on

12. Dnmt3a Dnmt3l Results • The complex is a tetramer: Dnmt3L–Dnmt3a–Dnmt3a–Dnmt3L • Mutagenesis at positions in both interfaces (a-a and a-L) indicate that these interfaces are essential for catalysis • Dnmt3a-Dnmt3a dimerization brings two active sites together • Dimeric Dnmt3a could methylate two CpGs separated by one helical turn in one binding event

13. “We observed a highly significant correlation of methylation status at distances of eight to ten base pairs between two CpG sites” Distribution of CpG sites among 12 known maternally imprinted genes, indicated to be Dnmt3a-Dnmt3l targets

14. Protein visualization Visualization tools (working on PC): RasMol / RasTop SwissPDBviewer (sPDBv) Protein Explorer (via the web) And many more…

15. Rastop / Rasmol

16. http://www.geneinfinity.org/rastop/

19. RasTop- main menu פתיחת קובץ סגירת קובץ

20. RasTop - display Wireframe קווים בין אטומים

21. Sphere VDW מנפח כל אטום לפי רדיוס ה- שלוVDW

22. Command editor

29. More on RasTop • RasMol manual • Using RasTop • Commands

30. Structure alignment • Essential for: • Protein classification • Detection of conserved protein folding cores • Detection of similarities between domains • Detection of similarities in functional binding sites • Evolutionary conservation • Construction of nonredundant databases

31. Pairwise structure alignment Outline: Given two proteins structures, find the transformation that produces the best superimposition of one protein onto the other

32. Computationally Find the rotations and translations of one of the points set (atoms of protein A) which produce “large” superimpositions on the other points set (atoms of protein B) ? Z Z X Y Y X

33. RMSD Root Mean Square Deviation Average distance between the matched superimposed atoms usually between backbonesCα atoms

34. http://bioinfo3d.cs.tau.ac.il/c_alpha_match • Matches C-alpha atoms • Rigid pairwise alignement • Sequence order independent • Input: two PDB files or PDB IDs with specific chains • Output: a set of high scoring conformations • The superimposed structures may be viewed in a PDB viewer

35. HEN EGG WHITE LYSOZYME BOBWHITE QUAIL LYSOZYME

36. Results • Ranking criteria: • Match size • RMSD

37. C-alpha correspondence

38. Aligned PDB file

39. http://bioinfo3d.cs.tau.ac.il/FlexProt • Flexible structural alignment • The first structure is assumed to be rigid, while in the second structure potential flexible regions - hinges, are automatically detected • Input: two PDB IDs (specific chain) • Output: list of alignments ranked according to the number of hinges

41. Results

42. Result with 0 hinges:

43. Result with one hinge:

44. http://bioinfo3d.cs.tau.ac.il/MultiProt • Multiple structural alignments of protein structures • Finds the common geometrical cores between the input molecules • Does not require that all the input molecules participate in the alignment • Actually, it efficiently detects high scoring partial multiple alignments for all possible number of molecules from the input • The final structural alignment can either preserve the sequence order (like sequence alignment), or be sequence order independent

46. Results

47. DALI - Distance matrix ALIgnment Concept: “Similar 3D structures have similar inter-residue distances” http://ekhidna.biocenter.helsinki.fi/dali_server/

48. DALI Algorithm • Generates an inter-residue distance matrix for each protein • The distance matrix contains all pairwise distances (symmetrical) • Dij = distance between C-alpha i and C-alpha j in the same protein • Compares the two distance matrices for a pair of proteins to be aligned

50. DALI Services DALI sever Used by crystallographers to compare a newly solved structure against structures in the PDB DALI database Contains all-vs.-all PDB 3D structure comparisons and thus enables to find structural neighbors of structures that are already in the PDB Pairwise server Pairwise comparison of two structures Dalilite A standalone version of DALI