2D protein crystals
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Skip the boring text and go directly to the cool AFM images...
What are two-dimensional crystals?
  • Periodic arrangement of motiffs (atoms, molecules...) in two dimensions.
  • In the case of a three-dimensional crystal, the distances between the motiffs are fixed (lattice constants). The order in 3D crystals is said to be geometric.
  • In the case of a two-dimensional crystal, this is not the case: if one takes an arbitrary motiff as a reference and measures the distance between it and any other motiff in the crystal, one will find that that distance deviates from the multiple of the lattice constant by an amount that increases logarithmically with the distance. The order in 2D crystals is said to be algebraic.
  • It also follows that there is a limit on resolution that can be obtained from diffraction studies 2D crystals. It is, once again,  related to the elastic properties of the crystal. In practice, however, the resolution seems to be limited by other factors.
  • Some of these issues are discussed in the work of Lenne et al. on the grazing-angle X-ray diffraction studies from protein 2D crystals, Biophys. J. 79, 496-500 (2000), and other publications by the same author.
  • This, in turn, means that diffraction peaks observed from 2D crystals are not delta-functions, but power-laws. FWHM of the peak is a function of temperature and elastic modulus of the crystal.
Why are 2D protein crystals interesting?
  • The main driving force behind gorowing and studying protein 2D crystlas is their application in structural biology. They are used for structure determination of soluble and transmembrane proteins by electron crystallography.
  • A common procedure for growing 2D crystals of soluble proteins is the so-called lipid monolayer method due to Kornberg et al. (For review, see ) In the case of membrane-binding proteins, the structure of the biologically relevant, membrane-bound form, can be determined.
  • Convenient model systems for studying self-organisation, complex systems, and other soft condensed matter physics phenomena;
  • Some may have biological role;
  • Possbile applications in biotechnology are also being investigated.

Interesting link (web page of Jaap Brink):
  List of proteins crystallised in two dimensions on lipid monolayers
Annexin A5 (previously called Annexin V):
  • Archetype of a family of soluble proteins expressed in many cell types in eucaryotes that share structural homology (the so-called "annexin fold") and the ability to bind lipids in a Ca2+ - dependent manner.
  • Annexins participate in processes involving membrane fusion and trafficking, as well as inhibition of blood coagulation.
  • The ability of annexin A5 to bind to phospholipids has been used to grow 2D crystals of this protein on lipid monolayers with the goal of elucidating the structure of the membrane-bound form of the protein by electron crystallography (see the work by Brisson et al. on this subject). The structure of the soluble form was solved by X-ray crystallography (Huber et al., Lewit-Bentley et al.).
  • Several forms of 2D crystals of annexin A5 exist. Initially two were known. They differ with respect to their symmetry - p6 in one case and p3 in the other. See the work of Dr. Frank Oling (Oling et al. 2001, J. Struct. Biol.133 55-63) on this subject.
  • Some recent reviews on the subject: Gerke and Moss 2002 Physiological Reviews 82, 331-371 2002; Moss and Morgan 2004, Genome Biology 5: Art. No. 219.
The questions we set out to answer were:
  • How do 2D crystals of annexin A5 grow?
  • What is the relationship between the p6 and p3 crystal forms of annexin A5?
  • What is the surface structure of the protein in the crystals?
In addition, we have looked at 2D crystals of other proteins, such as streptavidin (not discussed here).

The work shown here constitutes a part of my Doctoral thesis
 Atomic Force Microscopy of Biological Macromolecules and Their Assemblies.
Advisor: Prof. A. Brisson , Department of Biophysical Chemistry, University of Groningen (the Netherlands).
The Thesis can be downloaded here.
Take a look at the more recent work by Richter and Brisson (Biophys. J. 2005, 88, 3422-3433) on this subject.
This AFM work would not be possible without both the previous and the con-current structural characterization of the annexin A5 2D crystal forms by electron crystallography and X-ray diffraction. Corresponding references can be found in the Thesis itself and in publications. Full list of Acknowledgments appears in the Thesis.
Schematic representation of the annexin V 2D crystallization process on a phosphatidylserine - containing supported phospholipid bilayer.

  1. Ca2+- dependent adsorption of the monomeric, soluble protein.
  2. Fast trimerisation step.
  3. Nucleation and growth of p6 crystalline domains.
  4. Solid-solid phase transition between the two crystal forms, p6 and p3.
p6 and p3 forms of Annexin V 2D crystals
Annexin V 2D crystals - p6 form.
B: 250x125 nm. Unit cell (blue lozenge): a=b=19.8 nm, Gamma: 1200.
C(36x36 nm), D(35x35nm): Average 2D projection (calculated from EM data) and average topography (calculated from AFM data) maps, respectively.
Here and on the right, annexin V trimer is encircled in green. Numbers (pink) refer to individual domains within the annexin V monomer. Numbering after Huber et al.		 Annexin V 2D crystals - p3 form.
B (minimal force), C(increased force) - 116 and 145 nm, respectively. Inset in B: average topography map calculated by single particle averaging.
D: Average topography map of the p3 from calculated by single particle averaging methods. 3-fold symmetry imposed. 25 x 25 nm. E: 2D projection map of the p3 from calculated from EM data. Scale bar: 5 nm.
Trimer-trimer connections encircled in turquoise.
The image on the left shows a small area of the p3 form, which appeared spontaneously within the p6 phase. A disordered region (black asterisk) is seen to accompany its appearance. A vacancy (missing annexin V trimer) is encircled in turquoise.
Defects within the 2D crystals visualized by AFM
The image on the right shows defects - stacking faults - which were found in the 2D crystals of the p6 crystal form. The two examples highlighted with different colors exhibit a somewhat different aspect. The image in A is 546 x 458 nm, and the insets are enlarged 2.2 x.
Download a pdf version of my PhD thesis....
Background image: A contact mode AFM image of the p3 from of Annexin A5 2D crystals.