1. Workshop in Computational Structural Biology
2017
81855 & 81813, 6 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. 4/13/2018
What will we learn?
Structure prediction (mainly Rosetta; also I-TASSER, MODELLER):
from sequence alone to high resolution models (Ab-initio modeling)
From homologous structures to high resolution models
MSKAVGIDLGTTYSC……
The course will revolve around the properties of the proteins we have enough structural data about – soluble globular proteins. ||
MSKAVGIDLGTTYSC…… 3

4. 4/13/2018
What will we learn?
Protein design – Engineering novel proteins not found in nature to fit a desired fold/function
Gordon et. Al. JACS (2012). Computational Design of an α‑Gliadin Peptidase

5. 4/13/2018
What will we learn?
Protein-protein docking – achieve models of protein complexes given two monomers (Rosetta, PatchDock, PIPER, HADDOCK)
Interface analysis and design – identify interface “hotspots” (via computational alanine scanning); change protein specificity

6. What will we learn? Optimization techniques: Side chain modeling
4/13/2018
What will we learn?
Optimization techniques:
Energy Minimization; concepts and implementation in Rosetta
Monte-Carlo methods
Side chain modeling
Deterministic and heuristic methods for finding preferred side chain combinations given a certain backbone
START
energy
conformations

7. What we will not learn Existing protocols, out of this course’s scope:
4/13/2018
What we will not learn
Existing protocols, out of this course’s scope:
Protein-ligand docking
Membrane proteins modeling and design
Peptide-protein docking
DNA & RNA modeling
Antibody modeling

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

9. 4 Hierarchies of protein structure
Anfinsen: sequence determines structure

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

11. The building blocks: amino acids
4/13/2018
The building blocks: amino acids

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

13. 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

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

15. Amino acids with hydroxyl group

16. 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

17. Amide amino acids

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

19. Aromatic amino acids sc contains aromatic ring 4/13/2018
Figure from Wikipedia
Tyr is common in interfaces because its aromatic ring can form stacking interactions (pi-pi). The aromatics can form pi-pi and cation-pi interactions
Figure from Proteopedia 19

20. Amino acids with sulfur

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

22. 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

23. 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 ….

24. 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

25. Hydrogen bonding potential of amino acids

26. Primary sequence: concatenated amino acids

27. Primary sequence: concatenated amino acids

28. 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

29. 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

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

31. The geometry of the peptide backbone
4/13/2018
The geometry of the peptide backbone
The peptide bond is planar & polar:
=180o (trans) or 0o (cis)   
Phi – formed by C-N-Ca-C, a rotation around N-Ca; Psi – formed by N-Ca-C-N, a rotation around Ca-C; Omega – defines plane of Ca-C-N-Ca, a rotation around C-N, the fact that it’s either 0 or 180 reduces complexity (allowed only by a cis trans isomerase, because this C-N bond is partially double, so it’s not rotatable).
Peptide bond length and angles do not change
Peptide dihedral angles define structure 31

32. The search for the native fold
4/13/2018
The Levinthal paradox: a 100 residue protein would require 1016 seconds to explore all possible conformations and choose the native one.
10^16 is more time than the universe has existed, so the protein must have another way of finding it native conformation..
Quick collapse to intermediate state, followed by accurate contacts formation
Quick collapses followed by unfolding until near native state achieved 32

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. 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

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

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

53. Quaternary structure: Hemoglobin consists of 4 distinct chains

54. 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

55. Tertiary structure defines protein function

56. 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

57. Interplay of enthalpy and entropy in protein folding
4/13/2018
Interplay of enthalpy and entropy in protein folding
Formation of the aformentioned bonds contributes to the enthalpy of the system, decreasing protein entropy
energy contribution for a system created in environment temperature T from a negligible initial volume.
At constant pressure, the enthalpy change equals the energy transferred from the environment to the system.
Entropy is the measure of molecular disorder of the system. 
change in Gibbs free energy
change in enthaply
change in the entropic term 57

58. The hydrophobic effect
4/13/2018
The hydrophobic effect
A central effect in protein folding
Driven by entropy – gain of entropy of water molecules
The release of these water molecules to the bulk solvent increases the system’s entropy.
Water molecules near hydrophobic elements have less freedom to form and break hydrogen bonds with neighboring waters
More water molecules not in direct contact with hydrophobic elements
Figures from post by Dr. Steve Mack on 58

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

60. X-ray diffraction
4/13/2018
Hard to find a way to crystalize a structure. An X-ray beam is shot towards a crystal, passing through a detector to yield a diffraction pattern. Can be decomposed by Fourier transform to an electron density map. Taking the created model and checking its diffraction pattern allows refinement of the information. 60

61. 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

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

63. X-ray diffraction
1950’s first protein structure solved by Kendrew & Perutz: sperm whale myoglobin
(Nobel Prize 1962)
Today: ~127’000 structures solved, most by x-ray crystallography
Challenges
Grow crystal
Determine phase

64. NMR (Nuclear Magnetic Resonance)
NMR-active nuclei (w spins)
1H, 13C, 15N
Application of magnetic field reorients spins – measure resonance between close nuclei
Extract constraints & determine structure
More constraints – better defined structure
Spin: odd number of protons and/or neutrons
Nobel Prize 2002 Kurt Wuthrich

65. Experimental determination of structure
4/13/2018
Experimental determination of structure
X-ray crystallography
Determines electron density – positions of atoms in structure
Highly accurate
Technically challenging
Depends on crystal (static; artifacts?)
NMR
Determines constraints between labeled spins
Allows measure of structure in solution
You need to find the conditions that allow it to organize symmetrically in a crystal – therefore it does not move as it does in biological conditions. High concentrations of the solved structure might also stabilize the wrong conformation. 65

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

67. 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

68. 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!