1. MSD database is structured around the fact that Proteins are “sticky”

2. Classes of cell adhesion molecules
cadherins
Classes of cell adhesion molecules
Ig-CAMs
selectins
integrins

3. A short biography of 1 protein whose very existence depends on being as sticky as possible

4. EMBL K02078
ttaacgcgta aattcaaaaa tctcaaattc cgacccaatc aacacacccg ataccccatg
ccaataaaaa agtaacgaaa atcggcacta aaactgacaa ttttcgacac tgccgccccc
ctacttccgc aaaccacacc cacctaaaag aaaatacaaa ataaaaacaa ttatatagag
ataaacgcat aaaatttcac ctcaaaacat aaaatcggca cgaatcttgc tttataatac
gcagttgtcg caacaaaaaa ccgatggtta aatacattgc atgatgccga tggcaagccc
tgaggctttc ccctttcaat taggagtaat tttatgaata cccttcaaaa aggctttacc
cttatcgagc tgatgattgt gatcgctatc gtcggcattt tggcggcagt cgcccttccc
gcctaccaag actacaccgc ccgcgcgcaa gtttccgaag ccatcctttt ggccgaaggt caaaaatcag ccgtcaccga gtattacctg aatcacggca aatggccgga aaacaacact tctgccggcg tggcatcccc cccctccgac atcaaaggca aatatgttaa agaggttgaa gttaaaaacg gcgtcgttac cgccacaatg ctttcaagcg gcgtaaacaa tgaaatcaaa ggcaaaaaac tctccctgtg ggccaggcgt gaaaacggtt cggtaaaatg gttctgcgga cagccggtta cgcgcaccga cgacgacacc gttgccgacg ccaaagacgg caaagaaatc
gacaccaagc acctgccgtc aacctgccgc gataaggcat ctgatgccaa atgaggcaaa ttaggcctta aattttaaat aaatcaagcg gtaagtgatt ttccacccgc ccggatcaac
ccgggcggct tgtcttttaa gggtttgcaa ggcgggcggg gtcgtccgtt ccggtggaaa taatatatcg at

5. UniProt P02974
MNTLQKGFTL IELMIVIAIV GILAAVALPA YQDYTARAQV SEAILLAEGQ KSAVTEYYLN HGKWPENNTS AGVASPPSDI KGKYVKEVEV KNGVVTATML SSGVNNEIKG KKLSLWARRE NGSVKWFCGQ PVTRTDDDTV ADAKDGKEID TKHLPSTCRD NFDAK

6. PDB 1AY2

7. MSD DATABASE
pentamer

8. MSD DATABASE

9. negatively stained TEM images

10. The pili are polar flexible filaments of about 5
The pili are polar flexible filaments of about 5.4 nm diameter and 2500 nm average length.

11. Neisseria gonorrhoeae expressing pili and interacting with epithelial cells.

12. Model for Type IV pilus biogenesis and dynamics of fiber formation and retraction

13. PDB 2PIL / 1AY2
Forest, K.T., Parge, H.E., Tainer, J.A. Nature , 32-8.
Forest, K.T., Dunham, S.A., Koomey, M., Tainer, J.A. Mol Microbiol ,
SOURCE: Neisseria gonorrhoeae
A fibre forming cell adhesion protein responsible for the virulent attachment
Pilin is a subunit of the pilus, a polar flexible filament, which consists of a single polypeptide chain arranged in a helical configuration of five subunits per turn.
PDB 1ay2 , 1dzo , 1hpw , 1kb7 , 1nil , 1pan CATH SCOP d , j

14. Type IV Pilin Structure and Assembly: X-Ray and EM Analyses of Vibrio cholerae Toxin-Coregulated Pilus and Pseudomonas aeruginosa PAK Pilin
L. Craig, R.K. Taylor, M.E. Pique, B.D. Adair, A.S. Arvai, M. Singh, S.J. Lloyd, D.S. Shin, E.D. Getzoff, M. Yeager, K.T. Forest & J.A. Tainer
Molecular Cell, 11, 1139–1150, 2003

15. EM Analysis of TCP Reveals a Three-Start Helix with a 45 Pitch
C Bundle of negatively stained TCP filaments
D computed Fourier transform of a single filament within the bundle as indicated by the box in (C).
E Left-handed representation of a three-start helix with each start shown in a different colour.

17. Pili are thin, protein tubes
The pilus has a shaft composed pilin.
At the end of the shaft is the adhesive tip structure having a shape corresponding to that of specific glycoprotein or glycolipid receptors on a host cell
Because both the bacteria and the host cells have a negative charge, pili may enable the bacteria to bind to host cells without initially having to get close enough to be pushed away by electrostatic repulsion.
Once attached to the host cell, the pili can depolymerize and enable adhesions in the bacterial cell wall to make more intimate contact.

18. Type IV pili are not merely passive sticky fibres but dynamic machines that participate in a surprising number of functions including:
Bacterial aggregation
Adhesion to host cells
Twitching motility
Pilus retraction
DNA transformation
In another bacterial species, motility.
Phage receptor in V. cholerae.

19. EMBL
UniProt
PDB
Assembly (MSD)
Microscopy
still not the full story - GENOME

20. Pilus gene organisation
Many copies of pilin gene throughout chromosome
Two are functional, pilE1 and pilE2
All other copies are silent
Antigenic variation occurs due to recombination (within mini-cassettes)

21. Antigenic variation in N. gonorrhoeae
A single cell can give rise to daughter cells expressing structurally and antigenically different pili
Gonococcus has the genetic capacity to make as many as a million different pilin variants
All able to bind to same host tissues and to cause the same disease symptoms

22. PILI are the pathogen’s answer to
Mankind's physical defence systems
One of the body's innate defences is the ability to physically remove bacteria from the body by:
constant shedding of surface epithelial cells
coughing, sneezing, vomiting, and diarrhoea
removal by bodily fluids such as saliva, blood, mucous, and urine.

23. pili enable adhere N. gonorrhoeae to receptors on target epithelial cells and thus colonize and resist flushing by the body.

24. REMEMBER – this all achieved by simple non-covalent forces

25. What has all this got to do with MSD?
PDB Entries and X-Ray results
Crystal Structure
Molecular Structure (covalent)
Oligomeric Assembly

26. MSD Relational Database
Exp. Result
Assembly
Chains
Residues
Atoms

27. KEY to MSD DataBase

28. Biological Context
PDB  MSD
Oxalate oxidase 1FI2 hexameric

29. Protein Stickiness
What does this mean?
What is the evidence?

30. PDB Xray coordinates
PDB entry the deposited coordinates usually consist of the contents of the asymmetric unit:
The contents of the ASU define a single copy of the macromolecule
The contents of the ASU consist of more than one copy of the macromolecule
The contents of the ASU require crystallographic symmetry operations to be applied to generate the complete macromolecule(s)
A combination of the above, including multiple copies and required symmetry transformations

31. A crystal is a periodic arrangement of a motif in a lattice
A crystal is a periodic arrangement of a motif in a lattice. The motif can be a single atom, a small molecule, a protein or any combination thereof. Often the motif, also referred to as to the 'asymmetric unit', is subjected to a number of symmetry operations yielding differently oriented copies.

32. Space Groups
The combination of all available symmetry operations (point groups plus glides and screws) with the Bravais translations leads to exactly 230 combinations, the 230 Space Groups.
Kathleen Yardley Lonsdale Carried out a profound and systematic study of the theory of space groups , methods for their determination, and the possibilities of molecular symmetry that are involved (1924, 1936).

33. benzene
C6H6
Covalent bonded

34. Benzene crystallised in Space Group P6/m
6-fold rotation axis
Mirror plane

35. Benzene P6/m in the PDB Entire atomic contents:
ATOM C1 x1 y1 z1 occupancy 0.5
ATOM H1 x2 y2 z2 occupancy 0.5

36. HELD TOGETHER BY WEAK FORCES
The stronger of the two is the hydrogen bond.
The weaker is the van der Waal's forces.
Both interactions depend on the same fundamental cause, the charge on electrons, and how that results in attraction and repulsion at an atomic level.

37. Johannes D. van der Waals
The equation of state for gases and liquids
Nobel Prize 1910
The origin of the London van der Waals force lies in the instantaneous dipole generated by the fluctuation of electron cloud surrounding the nucleus of electrically neutral atoms.

38. van der Waals forces
All intermolecular attractions are known collectively as van der Waals forces. The various different types were first explained by different people at different times.
Dispersion forces, for example, were described by London in 1930;
dipole-dipole interactions by Keesom in 1912.

39. van der Waals

40. Hydrogen Bonding
Linus Pauling ( )
Nobel Prize 1954

41. Hydrogen Bonds
Pauling in 1935 was the first to explain the mysterious stickiness of water molecules.
The basic principle behind hydrogen bonding is that the electron deficient hydrogen atom of one polar molecule is attracted to the electron rich side of another polar molecule.
Hydrogen bonds are somewhat stronger than van der Waal's forces, and require two components:
a donor group and
an acceptor group.

42. Hydrogen Bonds

43. Quaternary Structure
Quaternary Structure is defined as that level of form in which units of tertiary structure aggregate to form homo- or hetero-multimers.
Consideration of the presence of a quaternary state is important in the understanding of a protein's biological function.

44. Crystal Structure

45. Crystal Structure
Oligomeric Assembly

46. Proteins don’t do this –
pack by translationals

47. Symmetry There are three main types of symmetry:
symmetry with respect to a plane (mirrors)
symmetry with respect to a line (rotations)
symmetry with respect to a point (inversions)

48. Symmetry symmetry with respect to a line (rotations)
symmetry with respect to a plane (mirrors)
symmetry with respect to a point (inversions)

49. 1, 2, 3, 4, 6 -fold rotational symmetry
These are the only rotational symmetries that can exist in crystals; all others are disallowed. These five rotational axes are called the five Proper Axes
Symmetries showing 5-, 7-, 8-, 9-, 10-, 11-, & 13- fold rotations are known for biological molecules – these are observed in the Asymmetric Unit.

50. 1g8h A’ A Applying 1st 3-fold Rotation
Residues of Chain A in interface

51. A’
Residues of Chain A’ in interface A

52. A’ A” A
Applying 2nd 3-fold Rotation

53. Also has a 2-fold rotation

54. Final Assembly is a Hexamer from 23 symmetry

55. Screw Axes
If you add translations to rotation axes, you form what are call screw axes. For an nm screw axis, the rotational component is 360/n degrees, and the translations is m/n of the unit translation along the axis.
In Biological Crystallography --> Polymers
Helices are improper Screw axes – e.g. DNA

56. Screw Axes
YopM is a strongly acidic protein containing 13–20 repeats of a 19-residue leucine-rich-repeated motif (LRR). YopM has a crescent shape, formed from parallel β-sheets,with a loose amino terminus95.
Four YopM monomers form a hollow cylinder with an inner diameter of 35 Å .
YopM is an important virulence factor in Yersinia infection

57. 1jl5 YOPM

58. 4-fold screw axis

59. Also has a 2-fold rotation – infinite cylinder in crystal

60. Screw Axis

61. Screw Axes example
tubulins

62. Bacteriophage T4
Molecular Machine

63. Bacteriophage T4
Identified
Gene products

64. Bacteriophage T4 1N7Z Gp8 Baseplate Structural Protein 1QEX Gp9
1EL6 Gp11 Baseplate-tail tube complex
1CZD Gp45 Processivity Clamp
1G Gp31 Co-Chaperonin
1OCY Receptor-Binding Domain
1RFO Phagehead Fibritin - whisker antigen control
1C1K Gp59 Helicase Assembly
Bacteriophage T4

65. Connector Protein From Bacteriophage Phi29
1FOU

66. 1FOU
Molecular assembly acts as a mechanical attachment flange between the head and tail units

68. Bacteriophage T4 24 Genes give proteins in the Head+Whiskers/Neck
22 Genes give proteins for the Tail+Base Plate
7 Genes give proteins in the Tail Fibres
1 Gene gives the fin attachment protein
e.g. in the Head scaffold there are 576 copies of gp22
ALL HELD TOGETHER BY WEAK FORCES

69. Bacteriophage T4 Identify genes Identify structures Identify location
Becomes Mechanics – just balls and springs

70. Bacteriophage T4
Important
Hinge Proteins

71. Hinge-Bending, Swiveling Motions
Most large proteins are built from assemblies of domains that for the most part consist of regions of nearly rigid motions jointed by flexible regions. The activity of many proteins induces conformational transitions by hinge bending, which involves the movement of relatively rigid parts of a protein about flexible joints
The conformational switch from open to closed of the flexible loop-6 of triosephosphate isomerase (TIM)
The hinge region on the Fc fragment of human immunoglobulin G

72. Hinge-Bending, Swiveling Motions
Hinge mechanism that occurs when there is no continuously maintained interface constraining the motion. Hinge motions usually occur in proteins with two domains with one domain rotating about the hinge as a rigid body. The rotation is caused by a few large torsion angle changes within the hinge region.
shear mechanism that occurs when two interfaces slide across each other in order to maintain a well-packed interface. Shear motions are typically small so a large shear motion will be composed of a number of individual shear motions.

73. Genes  Proteins  Structure  Function

74. Genes  Proteins  Structure  Function
Some Proteins have No predicted structure or regions predicted not to fold
Genes  Proteins  Structure  DYNAMICS
Function

75.
FoldIndex© tries to answer to the question: Will this protein fold?
It's a dynamic and interactive process that estimates the local and general probability for the provided sequence, under specified conditions, to fold.

76. Bacteriophage T4

77. Bacteriophage T4 baseplate
Kostyuchenko et al., Nat. Struct. Biol., , 688
Emd Entry 1048

78. gp5/gp27 hexamer
of the tube makes up the hypodermic needle at the tip

79. gp5/gp27 hexamer Bacteriophage T4 PDB Entry 1K28 BACTERIOPHAGE T4
CELL-PUNCTURING DEVICE
KANAMARU, et.al.,

80. gp5/gp27 hexamer
PDB: ASU
3-fold Rotation
MSD: Assembly

81. PDB Example

82. Observed Asymmetric Unit
1e94 M.Bochtler
et al,
Nature, 403, (2000)

83. 1e94 - 3 separate molecules (?)
2 dodecamers
1 hexamer

84. 1e94 M.Bochtler
et al,
Nature, 403, (2000)

85. hexamer

86. hexamer
Grapple

87. 1e94 M.Bochtler
et al,
Nature, 403, (2000)

88. dodecamer

89. dodecamer

90. Heat shock proteins HslV and HslU that form a new ATP-dependent protease in Escherichia coli - ATP-dependent protease complexes rid cells of misfolded or damaged proteins and control the level of certain regulatory proteins.
M. Bochtler, C. Hartmann, H.K.Song, G.P.Bourenkov, H.D.Bartunik, and R.Huber (2000) The structures of HSLU and the ATP-dependent protease HSLU-HSLV. Nature 403, 800
Couvreur B. , Wattiez R. , Bollen A. , Falmagne P. , Le ray D. , Dujardin J.C. (2002). Eubacterial HslV and HslU subunits homologs in primordial eukaryotes.Mol. Biol. Evol. 19,

91. Easter Island Complex with topknot

92. P31059
HSLV (P31059) caps HSLU (P32168)
P32168

94. STATS on PDB Oligomers ~ 24,000 entries
Oligomer type Num Homo Hetero
Monomer
dimer (8169) 7215(6261) 1958(1908)
trimer (1628) 815( 786) 844( 842)
tetramer (2930) 1852(1828) 1111(1102)
pentamer ( 178) 91( 91) 87( 87)
hexamer ( 883) 534( 521) 364( 362)
heptamer ( 52) 29( 29) 24( 23)
octamer ( 418) 209( 208) 212( 210)
nonamer ( 62) 6( 6) 56( 56)
decamer ( 72) 43( 43) 29( 29)
undecamer ( 10) 8( 8) 2( 2)
dodecamer ( 230) 96( 96) 134( 134)

95. Windscreen bug splat examples

96. 1fmd

98. SYMMETRY Rules –BUT What about -

99. What happened to symmetry?
2:1 hetero-complex

100. Insulin/insulin receptor complex

101. The Ribosome – the champion Heterocomplex
proteins tossed around the RNA

102. protein aggregates complicate the lives of people who study proteins in vitro

103. Protein Aggregation and Amyloid Diseases
- Converting the protein from a soluble to a fibrillar structure

104. ACKNOWLEDGEMENTS
I have taken from the WWW most of the pictures used here
The list of sources is available separately.