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

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

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. nucleoside nucleotide

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 Pymol http://www.pymol.org http://pymolwiki.org http://www.pymol.org http://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. Action Navigation Quick draw Manipulation Show

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

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 322 nd 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 1 st argument is specific to command used, while the 2 nd 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:Commandshttp://pymol.sourceforge.net/htmlhttp://www.pymolwiki.org/index.php/Category:Commands PyMOL Reference card: http://pymolwiki.org/images/7/77/PymolRef.pdfhttp://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 ala residues –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)

40. propertyexample symbole.chemical symbol, select polar, symbol (e.) o+n namen. atom names, select carbons, n. ca+cb+cg+cd resnr. residue names, select aas, r. asp+glu+asn+gln resii. residue numbers, select mults10, i. 1+10+100+1000 select one, resi 10 zoom one chainc.chain ID ss- secondary structure type, select allstrs, ss h+s+l+""

41. Selection algebra and, or, not etc. how would you select resid 27 and resid 28? propertyshortexample not s1! s1atoms that are not included in s1 and&atoms included both in s1 and s2 or|atoms included in either s1 or s2 in-s1 atoms whose name, resi, resn, chain match s2 atoms likel.s1 atoms whose name, resi match s2 atoms

42. propertyshortexample s1 around Xs1 a. Xatoms with centers within X Angstroms of the center of any atom in s1 select near10, resi 10 around 5 s1 expand Xs1 e. XExpands s1 by all atoms within X Angstroms of the center of any atom in s1 select near10x, near10 expand 3 s1 within X of s2s1 w. X of s2 Selects atoms in s1 that are within X Angstroms of the s2 byres s1br. s1 Expands selection to complete residues select cmplt, byres near10 neighbor s1nbr. s1atoms directly bonded to s1 select vic, nbr. resi 10 s1 extend Xs1 ? XExtends s1 by X bonds connected to atom in s1 select connect_x, near10 extend 3

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 SelectorShort formDescription all*All atoms currently loaded into PyMOL none-No atoms (empty selection) hydroh.All hydrogen atoms currently loaded into PyMOL hetatmhetAll atoms loaded from Protein Data Bank HETATM records visiblev.All atoms in enabled objects with at least one visible representation polymer All atoms on the polymer (not het).

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