1. Workshop in Computational Structural Biology
2015
81855 & 81813, 4 points
Ora Schueler-Furman
TA: Orly Marcu

2. Introduction – When, Where, How?
Thursdays, Givat Ram
Lecture: 14:00-15:45, Sprinzak 25
Exercise: 16:00-18:45, Sprinzak computer class #2
Lectures & exercises available in moodle
How:
Make sure you have an account in CS ✓
Exercises
Submit 7/10 exercises
Due within 2 weeks
Submit by to
1/3 of grade
Contact:
Ora or
Orly
Acknowledgements: Sources of figures and slides include slides from Branden & Tooze; some slides have been adapted from members of the Rosetta Community, especially from Jens Meiler
Exercises in Pyrosetta have been adapted from teaching material by Jeff Gray

3. What will we learn: Part I: Protein structure in the eye of the computational biologist
1. Introduction to computational structural biology
The basics of protein structure
Challenges in computational biology and bioinformatics
Protein structure prediction and design

4. Part I: Protein structure in the eye of the computational biologist
2. Introduction to Rosetta and structural modeling
Approaches for structural modeling of proteins
The Rosetta framework and its prediction modes
Cartesian and polar coordinates
Sampling (find the structure) and
Scoring (select the structure)
3. Optimization techniques
Energy minimization
Monte Carlo (MC) Sampling
MC with minimization (MCM)

5. Part II: Protein modeling and design
4. Ab initio modeling: Principles and approaches
5. Full-atom refinement
Local optimization
Side chain modeling
The representation of side chains as rotamers
Rotamer and off-rotamer sampling
Finding minimum energy rotamer combinations

6. Part II: Protein modeling and design
6. Homology modeling
Selection of template and alignment of query sequence to template
Loop modeling approaches (modeling of unaligned regions)
7. Protein design
The theoretical basis of protein design; how different design goals are achieved
Success and challenge in computational design

7. Part III: Protein interactions
8. Protein-protein docking
Challenges and approaches in protein docking
The theoretical basis of low-resolution and high-resolution docking
9. Interface analysis and design
Determinants of binding affinity and specificity
Identification of interface residue hotspots: Computational alanine scanning
Success and challenge in interface design
10. Summary

8. What will we learn: Exercises
Exercises will span a variety of subjects and involve both Rosetta and other widely-used protocols
Basic introduction: how to look at proteins
Protein structure evaluation and classification: What does my protein do, how good is its structure?
Structure comparison
Running Rosetta
Pyrosetta and Rosettascripts: running and programming
ab initio modeling
Homology modeling
Structure refinement
Modeling side chains
Loop modeling
Protein docking
Interface analysis – Computational alanine scanning
Protein design and protein interface design

9. 1. Introduction to Computational Structural Biology
The Basics of Protein Structure

10. The central dogma

11. The code: 4 bases, 64 triplets, 20 amino acids

12. 4 Hierarchies of protein structure
Anfinsen: sequence determines structure

13. The building blocks: 20 amino acids
Differ in size, polarity, charge, secondary structure propensity …

14. Special amino acids N CO C H The simplest aa No sc Very flexible bb N
H2C
Cyclic aa
sc Connects bb N
Very constrained bb

15. Aliphatic amino acids sc contains only carbon and hydrogen atoms
hydrophobic

16. Amino acids with hydroxyl group

17. Negatively charged amino acids
Asp does not like alpha and beta, glu does not like beta
Alpha: Ala, Leu, Arg, Met, Lys, Gln, Glu, Ile, Trp, Ser, Tyr, Phe, Val, His, Asn, Thr, Cys, Asp, Gly
Beta: Tyr, Thr, Ile, Phe, Trp, Val, Ser, Met, Cys, Leu, Arg, Asn, His, Gln, Lys, Glu, Ala, Asp, Gly, Pro
Different size → different tendency for 2. structure

18. Amide amino acids

19. Positively charged amino acids
pKa 11.1
pKa 12
large sc

20. Aromatic amino acids
pKa 7
benzene ring
sc contains aromatic ring

21. Amino acids with sulfur

22. Cystine
Oxidation of Sulfur atoms creates covalent disulfide bond (S-S bond)
between two cysteines

23. S-S bonds stabilize the protein
A chain
G I V E Q C C A S V C S L Y Q L E N E N Y C N s s s s
B chain
F V N Q H L C G S H L V E A L Y L V C G E R G F.. s s N
Insulin
A chain C
B chain

24. Post-translational modifications
Processing (pro-insulin/insulin)
control of protein activity
Glycosylation
protein trafficking
Phosphorylation (Tyr, Ser, Thr)
regulation of signaling
Methylation, Acetylation
histone tagging ….

25. Metal binding proteins
aa: HCDE
Fe, Zn, Mg, Ca Fe
blood: red hemoglobin
electro-transfer: cytochrome c Zn
in DNA-binding “Zn-finger” proteins
Alcohol dehydrogenase: oxidation of alcohol

26. Important bonds for protein folding and stability
Dipole moments attract each other by van der Waals force (transient and very weak: kcal.mol)
Hydrophobic interaction –hydrophobic groups/ molecules tend to cluster together and shield themselves from the hydrophilic solvent

27. Hydrogen bonding potential of amino acids

28. Primary sequence: concatenated amino acids

29. Primary sequence: concatenated amino acids

30. Formation of a peptide bondH O ||
+H3N Ca C O- R
CPK = Corey, Pauling, Kultin.
Condensation or dehydration synthesis reaction
cpk colors
O - oxygen
H - hydrogen
N - nitrogen
C - carbon

31. The geometry of the peptide backbone
The peptide bond is planar & polar:
W=180o (trans) or 0o (cis) W W W
Peptide bond length and angles do not change
Peptide dihedral angles define structure

32. Dihedral angles c1-c4 define side chain
Dihedral angle: defines geometry of 4 consecutive atoms (given bond lengths and angles)
From wikipedia

33. Ramachandran plot  F
All except Glycine
Glycine: flexible backbone

34. Ramachandran plot  F

35. Secondary structure: local interactions

36. Secondary structure – built from backbone hydrogen bonds

37. a helix discovered 1951 by Pauling 5-40 aa long average: 10aa
right handed
Oi-NHi+4 : bb
atoms satisfied
p helix: i - i+5
310 helix: i - i+3
1.5Å/res
Favored: Ala, Leu, Arg, Met, Lys Disfavored: Asn, Thr, Cys, Asp, Gly

38. a helix: dipole binds negative charges at N-terminus
Specific binding through bb
binds negative charges at N-terminus

39. a helix: side chains point out
View down one helical turn

40. Frequent amino acids at the N-terminus of a helices
Ncap, N1, N2, N3 …….Ccap
Pro
Blocks the continuation of the helix by its side chain
Asn, Ser
Block the continuation of the helix by
hydrogen bonding with the donor (NH) of N3

41. Helices of different character
buried
partially exposed
exposed

42. Representation: helical wheel
buried
partially exposed: amphipathic helix
exposed

43. b-sheet Involves several regions in sequence Oi-NHj Parallel and
anti-parallel
sheets
Favored: Tyr, Thr, Ile, Phe, Trp Disfavored: Glu, Ala, Asp, Gly, Pro

44. Antiparallel b-sheet Parallel Hbonds
Residue side chains point up/down/up ..
Pleated

45. Parallel b-sheet
less stable than antiparallel sheet
angled
hbonds

46. Connecting elements of secondary structure define tertiary structure

47. Loops
connect helices and strands
at surface of molecule
more flexible
contain functional sites

48. Hairpin Loops (b turns)
Connect strands in antiparallel sheet
G,N,D G G
S,T
70% of beta hairpins <7aa long; mostly 2res long;
Type I'.The first residue in this turn adopts the left-handed a-helical conformation and therefore shows preference for glycine, asparagine or aspartate. These residues can adopt conformations with positive F angles due to the absence of a side chain with glycine and because of hydrogen bonds between the side chain and main chain in the case of asparagine or aspartate. The second residue of a type I' turn is nearly always glycine as the required F and Y angles are well outside the allowed regions of the Ramachandran plot for amino acids with side chains. Were another type of amino acid to occur here there would be steric hindrance between its side chain and the carbonyl oxygen of the preceding residue.
Type II'. The first residue of these turns has a conformation which can only be adopted by glycine. The second residue shows a preference for polar amino acids such as serine and threonine.

49. Super secondary structures –
Greek Key Motif
Most common topology for 2 hairpins
Staphylococcus nuclease

50. Super Secondary Structures-
b-a-b Motif
connects strands in parallel sheet
always right-handed
In certain proteins the loop linking the carboxy terminal end of the first b-strand to the amino terminal end of the helix is involved in binding of ligands or substrates.

51. Repeated b-a-b motif creates
b-meander: TIM barrel
TIM – triose phosphate isomerase

52. Tertiary structure defines protein function

53. The quaternary structure of a protein defines its biological functional unit

54. Quaternary structure: Hemoglobin consists of 4 distinct chains

55. Quaternary structure: assembly of protein domains
(from two distinct protein chains, or two domains in one protein sequence)
Glyceraldehyde phosphate dehydrogenase:
domain 1 binds the substance for being metabolized,
domain 2 binds a cofactor

56. 1. Introduction to Computational Structural Biology
Experimental determination of protein structure: X-ray diffraction and NMR

57. Experimental determination of structure
X-ray crystallography
Determines electron density – positions of atoms in structure
Highly accurate
Static: depends on crystal
NMR
Determines constraints between labeled spins
Allows measure of structure in solution
Resolution not defined: more constraints – better defined structure

58. X-ray diffraction

59. X-ray diffraction
If direction is such that
-> Constructive addition
-> Reflection spot in the diffraction pattern
Wavelength of x-ray ~ crystal plane separations
Rotation of crystal relative to beam allows recording of different diffractions
Diffraction maps are translated to electron density maps using Fourier Transform
Path length difference: 2d sin theta
Resolution measures diffraction angles (high angle peaks – high resolution data)

60. X-ray diffraction Iterative refinement allows improvement of structure
R-factor measures quality
Fo – observed
Fc - calculated

61. X-ray diffraction
1950’s first protein structure solved by Kendrew & Perutz: sperm whale myoglobin
Today: ~107’000 structures solved, most by x-ray crystallography
Challenges
Grow crystal
Determine phase

62. NMR (Nuclear Magnetic Resonance)
NMR-active nuclei (w spins) 1H, 13C Application of magnetic field reorients spins – measure resonance between close nuclei Extract constraints & determine structure
Spin: odd number of protons and/or neutrons

63. 1. Introduction to Computational Structural Biology
Challenges in Computational Structural Biology

64. Protein structure prediction and design
Protein sequence
Protein structure
FASTA
>2180 hSERT
METTPLNSQKQ……
PDB
ATOM N GLN A N
ATOM CA GLN A C
ATOM C GLN A C
ATOM O GLN A O
….. ….
Protein Design

65. Additional topics in computational structural biology
Nucleic acids - Prediction of binding and structure
RNA stem & loops, pseudoknots; protein-RNA binding
DNA curvature; protein-DNA binding
Prediction of macromolecular structures
Reconstruction of protein assemblies from low-resolution cryo-EM maps
Protein-ligand interactions
Docking of small ligands
Design of inhibitors
… and many many more!