1. Folds and motifs Using SCOP, CATH, CE to find structural homologs.
What class/architecture, etc does your molecule fall in?
Use CE to identify structural homologs for your protein of interest.
Motifs: classic turn types. Extended turn types.
Use Rasmol to find the glycines in your structure. That are the phi angles? What type of turn is it?

2. The SCOP database
Contains information about classification of protein structures and within that classification, their sequences

3. SCOP classification heirarchy
global characteristics (no evolutionary relation)
(1) class
(2) fold
(3) superfamily
(4) family
(5) protein
(6) species
Similar “topology” . Distant evolutionary cousins?
Clear structural homology
Clear sequence homology
functionally identical
unique sequences

4. protein classes number of sub-categories
1. all 
2. all 


5. multidomain (21)
6. membrane (21)
7. small (10)
8. coiled coil (4)
9. low-resolution (4)
10. peptides (61)
11. designed proteins (17)
number of sub-categories
possibly not complete, or erroneous

5. Mainly parallel beta sheets (beta-alpha-beta units)
class: proteins
Mainly parallel beta sheets (beta-alpha-beta units)
Folds:
TIM-barrel (22)
swivelling beta/beta/alpha domain (5)
spoIIaa-like (2)
flavodoxin-like (10)
restriction endonuclease-like (2)
ribokinase-like (2)
chelatase-like (2)
Many folds have historical names. “TIM” barrel was first seen in TIM. These classifications are done by eye, mostly.

6. fold: flavodoxin-like
3 layers, ; parallel beta-sheet of 5 strand, order 21345
Superfamilies:
1.Catalase, C-terminal domain (1)
2.CheY-like (1)
3.Succinyl-CoA synthetase domains (1)
4.Flavoproteins (3)
5.Cobalamin (vitamin B12)-binding domain (1)
6.Ornithine decarboxylase N-terminal "wing" domain (1)
7.Cutinase-like (1)
8.Esterase/acetylhydrolase (2)
9.Formate/glycerate dehydrogenase catalytic domain-like (3)
10.Type II 3-dehydroquinate dehydratase (1)
Note the term: “layers”
These are not domains. No implication of structural independence.
Note how beta sheets are described: number of strands, order (N->C)

7. fold-level similarity
common topological features
catalase
flavodoxin
At the fold level, a common core of secondary structure is conserved. Outer secondary structure units may not be conserved.

8. Superfamily:Flavoproteins
Flavodoxin-related (7)
NADPH-cytochrome p450 reductase, N-terminal domain
Quinone reductase
These molecules do not superimpose well, but side-by-side you can easily see the similar topology. Sec struct’s align 1-to-1, mostly.

9. Family: quinone reductases
binds FAD
Proteins:
NADPH quinone reductase
quinone reductase type 2
Different members of the same family superimpose well. At this level, a structure may be used as a molecular replacement model.

10. CATH Class Architecture Topology Homology

11. Remote homologs are more likely than close ones
likelihood
percent identity for structural homologs

12. Structural conservation is stronger than sequence conservation
The existence of large numbers of remote homologs shows us that true structural similarity is hard to see in the amino acid sequence.

13. Structural homology is sometimes difficult to see
beta-catenin versus clathrin (from FSSP database)

14. Rasmol practice Using your protein of interest: Find all glycines.
select gly
Label each alpha-carbon by number
label %r
Measure the phi-angle by eye (align N and CA. R-handed is positive) Write down the residue number and phi angle.

15. Rasmol exercises
(1) Color the ribbon drawing of 1qajA.pdb (it’s in your home directory) from the N-terminus (blue) to C-terminus (red)
(2) Divide 1qajA.pdb into domains of different color.
(3) In 1qajA.pdb, display only residues What are the first and last residues with helical backbone angles?
(4) Find all of the atoms that H-bond to the sidechain of Ser92.

16. Secondary structure Alpha helix
Right-handed Overall dipole N+->C residues/turn i->i+4 H-bonds

17. 3 types of Alpha helix
non-polar
amphipathic
polar a
Two ways to display position of sidechain on a helix. d e
For amphipathic and non-polar, sidechains line up in a favorable way. b g f c

18. Helix caps Capping motifs stabilize helices.
Please go to
Click on:
Glycine alpha (Schellman) C-cap Type 1
Glycine alpha (Schellman) C-cap Type 2
Proline alpha C-cap
Frayed alpha helix
Serine alpha N-cap (capping box)
Exercise: Find a helix cap in 1QAJ. What type is it?

19. Proline helix C-cap structural variability
note: hydrophobic cluster
structural variability
Sequence pattern= ...nppnnpp[HNYF]P[DE]n
Pro blocks helix
D or E stabilizes tight turn
Locations of non-polar (magenta) and polar (green) sidechains
“n”=non-polar “p”=polar [...]=alternative aa’s

20. Two Helix motifs
a-a corner (helix-turn-helix)
EF-hand
(binds Ca2+)

21. motif pattern (summary): nnpp[nH]Gn[PDS][Px]pn
a-a corner motif
backbone angles: green=psi red=phi
red=favorable
blue = unfavorable green=neutral
I-sites motif
(Also described by A. Efimov)
“n”=non-polar “p”=polar [...]=alternative aa’s
motif pattern (summary): nnpp[nH]Gn[PDS][Px]pn
Exercise: Find a a-a corner motif in myoglobin.

22. Find a a-a corner in 1DWT
Download 1dwt from Open using Rasmol
Rasmol commands:
Display->Cartoons
Color->structure
select alpha
label %m
color labels yellow
Find the a-a corner pattern (sequence and structure).

23. Can you see the a-a motif in the sequence?
1 GLSDGEWQQV LNVWGKVEAD IAGHGQEVLI RLFTGHPETL EKFDKFKHLK
HHHHHHH HHHHHHHHHT HHHHHHHHHH HHHHHTHHHH HTTTTTTT
51 TEAEMKASED LKKHGTVVLT ALGGILKKKG HHEAELKPLA QSHATKHKIP
SHHHHHTTHH HHHHHHHHHH HHHHHHHTTT HHHHHHHH HHHHHTS
101 IKYLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFRN DIAAKYKELG
HHHHHHHHHH HHHHHHHHST TSS HHHHHH HHHHHHHHHH HHHHHHHHHT
151 FQG

24. EF-hand
Exercise: Rasmol 1CLL. Follow instructions on next page

25. 1CLL exercise. How does the EF-hand bind calcium?
restrict hetero and CA (comments in blue...) spacefill (this displays only calciums) click to get residue # (x = residue number) restrict within (10., x) center selected (so you can rotate it easily) Display->ball-and-stick (from menu item. you see atoms and bonds) select protein and within (10., x) Display->sticks (now protein part has think bonds) select hoh and within (10., x) ( selects nearby waters) spacefill color cyan (or your favorite water color...) select within (3., x) ( atoms within bonding distance) label ( these are the atoms bonded to calcium x) color labels yellow (..so you can see them)

26. Calcium causes a conformational change in the EF hand.
red=1CLL
green=1cfd

27. Create a Rasmol script
Write a file using jot or vi (for example: vi script.ras) Put keyboard commands in the script. Start Rasmol Invoke the script using the ‘script’ command (for example: script script.ras)
Useful keyboard commands for scripts:
load xxx.pdb (loads a new PDB file) zap (closes PDB file, blanks the display) pause (stops the script until you hit a key)

28. Create a Rasmol script to survey many structures
List the pdb files in the xtal directory (ls xtal/*.pdb)
Create a script file (myscript.ras) as follows:
load xtal/file1.pdb (use one of the PDB files listed) backbone 0.6 color blue select hoh spacefill 1.0 color red set unitcell on pause zap load xtal/file2.pdb
(repeat as before for each file listed)
Run the script: script myscript.ras
Next, change the script and run it again (i.e. change the colors to green and magenta)

29. Nested scripts
Tired of having to edit the script 10 times over?? Write a “nested” script.
Create a file called “mynestedscript.ras” Put in all the commands except the ‘load’ line. Just one copy. The last line is ‘zap’.
Create a file called “mymainscript.ras” containing:
load file1.pdb script mynestedscript.ras load file2.pdb script mynestedscript.ras etc. etc. etc

30. Further exercises: just a few examples!
Select atoms by temperature factor: select temperature > (Rasmol uses B*100, so this selects all atoms with B > 50.00)
Find crystal contacts: select :B & within (8., :A) (This gets all atoms of chain B that are close to chain A. Your chain IDs may vary.)
Can you determine the crystal form (triclinic, orthorhombic, etc) just by looking at the unit cell? (set unitcell on)
Survey the B-factors of waters in several structures using a nested script. (Use: color temperature)
Use logic operators! Find alpha carbons within 10.A or residue x but not within 6.A. (select within (10.,x) and not within (6., x) )
Practice stereo viewing. (stereo -7 for walleyed viewing)

31. Proline helix C-cap structural variability
note: hydrophobic cluster
structural variability
Sequence pattern= ...nppnnpp[HNYF]P[DE]n
Pro blocks helix
D or E stabilizes tight turn
Locations of non-polar (magenta) and polar (green) sidechains
“n”=non-polar “p”=polar [...]=alternative aa’s

32. Two Helix motifs
a-a corner (helix-turn-helix)
EF-hand
(binds Ca2+)

33. beta-strand
middle strand
edge strand
Antiparallel:
Parallel:

34. Sequence motifs for beta strands
Hydrophobic
Amphipathic
Note preference for beta-branched aa’s: I,V,T
Found at the edges of a sheet, or when one side of the sheet is exposed to solvent (i.e. 2-layer proteins).
Found in the buried middle strands of sheets in 3-layer proteins.

35. beta hairpins Two adjacent antiparallel beta strands = a beta hairpin
Shown are “tight turns”, 2 residues in the loop region (shaded). Hairpins can have as many as 20 residues in the loop region.

36. hairpin motifs
“Serine beta-hairpin” (my naming). A specific pattern (DPESG) forms an incomplete alpha-helical turn 4-residues long.
“Extended Type-1 hairpin”. A type-1 “tight turn” has only 2 residues in the turn. This motif, more common than the tight turn, has an additional Pro or polar sidechain. Pattern: PDG.

37. diverging turn Exercise: Find the diverging turn in 1CSK.
“Diverging turns” have a Type-2 beta turn and two strands that do not pair. The consensus sequence pattern is PDG. The residue before G can be anything polar, but not a D or an N.

38. Greek key motif One of the most common arrangements of four strands.
Two permutations and 3214
beta meander
Exercise: Download 2PLT. Find the Greek key motifs.

39. bab motif
When a helix occurs between two strands, they are often paired in a parallel sheet.
The cross-over from one strand to the next is almost always right-handed. It is not known why this is true.
(NOTE: the cross-over is always right-handed even when the two strands are not paired!!)

40. TOPS diagrams beta strand pointing up beta strand pointing down
alpha helix
bab motif C N
Note R-handed crossover!

41. babab motif 1 2 3 Only 3 are possible. (with R-handed crossovers) 2 1

42. Efimov’s theory
A. Efimov showed that almost all protein structures can be classified as being one of 7 trees, each starting with a motif and “growing” by one secondary structure unit at time.
Does this recapitulate evolution? the folding pathway?

43. Domains Proteins fold heirarchically.
secondary structure --> motifs --> subdomains-->domains --> multidomain proteins --> complexes
Most domains fold independently of the rest of the chain.

44. All alpha folds 3-helix bundle 1CUK 156-203 4-helix bundle 1NFN
Globin 1DWT
...many many other

45. alpha/beta folds “TIM barrel” 8TIM “Rossman fold” 1HET 176-318
many others...
Exercise: in 1HET, find the “crevice” between two babab motifs where NAD binding commonly occurs.
Exercise: Draw TOPS diagram for 1HET

46. all beta folds “Jelly roll” 1JMU 371-500:D, 2PLT barrels 1EMC
propellers
beta-helix
Exercise: Draw TOPS diagrams for the two domains above.

47. Term projects Look at the description online.
Choose a molecule to study. Check with me before you start.
Look at “Molecule of the Month” at RCSB.org for inspiration. But don’t pick one of these molecules.