pdb/

1. http://www.pdb.org/

2. Experimental approaches for structural biology • X-ray crystallography • NMR • cryoEM

3. Nucleic acids • four letters (DNA, RNA) • sequence - AACTAACG (5’ → 3’) • DNA – double helix • RNA – “single stranded” helix, folding (double helical regions, C2’ -OH → secondary and tertiary motifs)

4. nucleotide nucleoside

5. Proteins • 20 letters • primary structure - sequence AMNTSSTVG (N-end → C-end) Alberts, Molecular Biology of the Cell, 5th Ed.

6. Where to get structural data? • biological molecules • PDB – Protein Data Bank http://www.pdb.org free • NDB – Nucleic Data Bank http://ndbserver.rutgers.edu/ • organic molecules • CSD – Cambridge Structural Database paid

7. PDB History 1957 Myoglobin structure determined 1970’s Discussions how to establish an archive of protein structures PDB established at Brookhaven Oct 1971, 7 structures 1980’s Technology takes off molecular biology, instrumentation, computer hardware and software Number of structures increases Structural biology is able to focus on medical problems IUCr requires data deposition to the PDB 1990’s Complexity of structures increases Structural genomics begins

8. Current state of the PDB ~61,000 structures in the PDB archive Over 7,000 new structures deposited in 2008 Depositions by macromolecule type 92% Protein 3.4 % Nucleic acid 4% Protein-nucleic acid complexes Depositions by experimental technique: 86% x-ray diffraction 13.2% solution NMR 0.4% cryo-EM (256 structures) data as of 9. 11. 2009 http://www.pdb.org/pdb/static.do?p=general_information/pdb_statistics/index.html

10. PDB ID • Each structure in the PDB is represented by a 4 character identifier of the form [0-9][a-z,0-9][a-z,0-9][a-z,0-9] • 1B3T

11. PDB Format legacy format http://www.wwpdb.org/docs.html fortran-like 80 column-wide not structured enough to describe complicated 3D objects its limits have been broken several times 99,999 atoms, 34 (or 58) chains readable by most programs

12. model – chain – residue – atom

13. Introduction to Pymolhttp://www.pymol.orghttp://pymolwiki.org

15. how do I load DPB file? • Plugin -> PDB Loader Service (2bhg) • File -> Open (locally stored) • command: load • fetch 2bhg, load 2bhg • command is a keyword, followed optionally by one or more comma-separated arguments • color red, hetatm • color red • help show

16. Using the mouse • Unmodified controls • Left - rotate molecule (x, y and, at edges, z) • Middle - translate molecule (x, y) • Right - zoom (=MovZ) • Wheel - slab/clip

17. Object menus (ASHLC)

18. Show Navigation Quick draw Manipulation Action

19. Color Hide • same content as Show • Use Show and Hide to toggle things on and off Label

20. Rule: Once a selection is shown (S) it must be selectively hidden (H) as it is not removed when another selection (S) is made. Selections are therefore additive, which allows for the creation of images with mixed graphical representations. • S -> Cartoon, H -> Lines • S -> as – you do not have to click S,H • Most options can be set within the “Setting” menu within the top menu bar. • Settings -> Cartoon

21. Changing the background to white: • Display -> Background -> White • The “Display” menu within the top menu bar contains options for most options pertinent to displaying the image witin the PyMol viewer. • “fog” within the back of the molecule – Display -> Depth Cue

22. Contextual menus • Left double click or right single click to activate • click on object or part of object you want to manipulate • more or less the same menus as ASHLC

23. Ligand rescue squad • load 1BIW into Pymol • show as cartoon • recolor by secondary element • ligand is there, but it has disappeared! • rescue ligand: • S > organic > spheres

24. Mouse selection • Click on one of the spheres of the carotenoid ligand • pink dots (click anywhere to unselect) • a new name called (sele) • The name of the atom that was clicked appears within the top text window of the “external GUI.” You clicked /2BIW.pdb2//B/TYR`322/OH Selector: selection "sele" defined with 12 atoms. • This could be read as “you clicked atom OH which is on the 322nd atom in the file, and belongs to Tyrosine of chain B in the object created when opening file 2BIW.pdb2. The complete selection contains 12 atoms • (sele) -> C -> by element -> CHNOS

25. Making images • File -> Save Image As -> PNG • Raytracing (Ray button, top right), then Save Image • even better quality, use command • ray x, y (e.g. ray 2000, 2000 - dimension)

26. Action presets • A -> Preset -> Default is similar to hide everything, show lines • preset options will set some variables that are specific to these views and may change further drawings. To remove the effect of these presets affecting an object representation, use the A -> preset -> default. • Let’s explore other presets.

27. simple • b factor putty • The segments with the highest temperature factor are shown as thicker cylinders. Regions of better resolution have thinner diameter and are usually found at the core of the protein. Mostly loops in the outside of the protein wobble: the core portions of the proteins usually appear more stable than the external loops. This is mostly useful for crystallographers but is a cool representation. • technical • Color domains in separate rainbow colors and shows backbone and side chains. • pretty and publication

28. ligands • zoom in on the ligand site and show the protein as backbone except in the near vicinity of the ligand where side chains are shown. The ligand is depicted as a thicker cylinder • to zoom out, simply click on the A > Zoom • ligand sites • pertinent to looking closely at the ligand in it’s binding pocket • solid surface, 2biw > C > Yellows > Sand, select ligand stick, sele > C > Blues > slate • Note: If you are preparing a figure for a black and white print publication, it might be advantageous to use the various gray scales, black, and white within the “grays” option. • explore others by yourself

29. Measuring distances • from menu: Wizard -> Masurement • click on the first atom in ligand, click on the second atom in ligand • change the color to white (ASHLC) • When you are done using the “Measurement” panel on the bottom right click Done. • If you no longer need to display the distance object, click Delete All Measurements. Alternatively you can use the corresponding “A” menu and select the delete option.

30. Clipping • Clipping planes are imaginary planes in the front and back of the molecule. Parts of the molecule that are outside the planes are “clipped” and therefore invisible. This is very useful for complex or large structures. • To move clipping planes press shift and the right mouse button simultaneously while dragging up and down. As an exercise, try to remove some of the molecular surface

31. Save your work • File -> Save session • *.pse, binary file • save your objects, settings • File -> Open

32. Pymol command language

33. most interaction with PyMOL is via a scripting language, not all functions are available from menus • keyword followed optionally by one or more comma-separated arguments, e.g. color red, hetatm – colors all non-protein atoms red, color red – all atoms are red • in most cases 1st argument is specific to command used, while the 2nd argument is the name of the object/selection

34. commonly used commands more in PyMol docs (paid, obsolete at http://pymol.sourceforge.net/html ) or at wiki http://www.pymolwiki.org/index.php/Category:Commands PyMOL Reference card: http://pymolwiki.org/images/7/77/PymolRef.pdf

35. download 1GBV in PDB format • load it into Pymol as 1gbv object help show load 1gbv.pdb, 1gbv hide show cartoon hide cartoon, chain A show ribbon, chain A set ribbon_width, 5 hide cartoon, chain B show spheres, chain B hide cartoon, chain C show sticks, chain C color red, chain A color blue, chain B color orange, chain C

36. Save your work • PyMOL script – text file with commands, each at one line (see example.pml at the website) • *.pml • comments begin with # end_of_line • run script from PyMOL: @example.pml • or from command line as pymol example.pml • save the image • ray • png

37. Selections • chain A is an example of a selection • two selection syntaxes: hierarchical and algebraic • hierarchical /model/segment/chain/residue/atom • if patterns start with slash, the selection is worked out left-to-right, if the first slash is missing, the patterns are worked out right-to-left

38. select ///A/10 #residue 10 in chain A color red, sele color atomic • select ////10-20/CA # atoms CA in residues 10-20 # (any chain) • select 42/C+N (C,N?) #atoms C and N in residue 42 show spheres, sele • algebraic • combine terms (e.g. chain A) using logical operators (AND, OR, NOT) • select wc, chain A# wc is the name of selection • select chain A and resi 10:20 # residue number • select resn ALA and name N #backbone nitrogens # from alaresidues • select elem O and not name OH # oxygen atoms # except hydroxyls

39. The items in a list of identifiers are separated by plus signs (+) only. Do not add spaces within a list of identifiers. The selector resi takes (+)-separated lists of identifiers: • select nt, resi 1+2+3 • select nt, resi 1-3 • don’t combine these two (1-3+6 is wrong)

41. Selection algebra • and, or, not etc. • how would you select resid 27 and resid 28?

43. # selects atoms that are part of chain a, but not # residue number 125. select chain a and (not resi 125) # The following two selections are equivalent select (name cb or name cg1 or name cg2) and chain A select name cb+cg1+cg2 and chain A

44. select residue 10 in chain B (name it cpk10) • show it as cpk, zoom at it • hide cpk10 selection • make a selection called bb containing C, O and N atoms • count atoms in selection (count_atoms) • remove residue no. 5 (remove) • count atoms again

45. objects vs. named-selections • PyMOL creates an object-name to locate data when you load a data file. • Making selections is a way of pointing to a subset of that data. • Selections are in parentheses in control panel. • When you delete a selection-name, the data are still found under the object-name, but the data are no longer organized as the selection. In contrast, after you delete an object, you must reload the data to have access to it again. delete • PyMOL can hold several objects at the same time (load 1gbv and 1bna) disable, enable TIP: get rid of pink dots select bb, name c+o+n+ca, disable bb, color red, bb

46. Named selections are static. Only atoms that exist at the time the selection is defined are included in the selection, even if atoms which are loaded subsequently fall within the selection criterion • clear your workplace, load 1gbv • make selection called static containing all 1gbv atoms • count atoms in static • add hydrogens (h_add) • count atoms in static, compare with count atoms in the whole structure

47. Single word selectors

48. Point of view • zoom • orient • aligns the object or selection so its largest dimension is shown horizontally, and its second largest dimension is shown vertically • view • store orientations and recall them later • view v1, store; view v1, recall; view v1