Two‐Dimensional Crystallisation of Membrane Proteins and Structural Assessment


Two‐dimensional (2D) crystallisation of membrane proteins reconstitutes them into their native environment, the lipid bilayer. Electron crystallography allows the structural analysis of these regular protein–lipid arrays up to atomic resolution. The crystal quality depends on the protein purity, its stability and on the crystallisation conditions. The basics of 2D crystallisation and different recent advances are reviewed and electron crystallography approaches summarised. Progress in 2D crystallisation, sample preparation, image detectors and automation of the data acquisition and processing pipeline makes 2D electron crystallography particularly attractive for the structural analysis of membrane proteins that are too small for single‐particle analyses and too unstable to form three‐dimensional (3D) crystals.

Key Concepts

  • Structural integrity and full biological activity require membrane proteins to be embedded in the bilayer. However, structural assessment at atomic resolution can be achieved only if milligram‐amounts of purified membrane protein are available. Therefore, their solubilisation by detergent cannot be avoided. The advantages of 2D crystallisation concern the reconstitution of the membrane protein in the bilayer and the possibility to study the structure of small and large membrane proteins by electron crystallography.

Keywords: 2D crystallisation; detergent; electron crystallography; lipid; membrane protein; protein stability; reconstitution; solubilisation; structure

Figure 1. The mean unfolding rates of the bacterial transporters correlate linearly with the micelle size of the detergents. Adapted from Sonoda et al., 2011 © Elsevier.
Figure 2. 2D crystals of different membrane proteins. (a) 2D crystals of AQP1 led to the first structure of a human channel protein (Murata et al., ). (b) The plant aquaporin SoPIP2;1 assembles into 2D crystal that diffract to 2.5‐Å resolution (unpublished, courtesy of Wanda Kukulski). (c) 2D crystals of the bacterial haemophor‐receptor complex HasA‐HasR diffract to 3‐Å resolution. HasR is the outer membrane receptor interacting with the soluble extracellular HasA found in Gram‐negative bacteria. (unpublished, courtesy of Mohamed Chami) (d) The bacterial toxin aerolysine assembles into highly ordered 2D crystals (Iacovache et al., ).
Figure 3. Light‐scattering profiles monitor the kinetics of the peripherin/ROM1 and rhodopsin reconstitution into lipid vesicles (Kevany et al., ). Peripherin/ROM1 complexes (a), peripherin/ROM1 and rhodopsin (b) and rhodopsin (c) were incorporated into protein–lipid arrays by slow addition of methyl‐beta‐cyclodextrin. The transition from low to high scattering indicates formation of larger structures and is only observed in the presence of rhodopsin (b,c). Negatively stained reconstitution products assembled upon detergent removal reveal significant differences. Peripherin/ROM1 (a) forms cylindrical rings (scale bar: 100 nm). (b) When rhodopsin is added to the peripherin/ROM1‐lipid mixture, larger discs with clear rims and rings are found (scale bar: 200 nm). (c) Rhodopsin alone reconstitutes into larger vesicles; pronounced rims are missing (scale bar: 1 µm). The assembly starts upon reaching the CMC of the DDM used to solubilise all components.
Figure 4. The approach to gather 3D information from 2D projections (Henderson and Unwin, ). Each 2D image (after being Fourier transformed) or 2D diffraction pattern (2) of the 2D crystal (1) represents a central section in the Fourier space (3). The Fourier transform of images exhibits diffraction maxima whose amplitudes and phases are extracted by image processing; electron diffraction patterns deliver the peak intensities. The z* values belonging to measured A(h,k) and phi(h,k) are calculated from the tilt geometry. Because 2D crystals are only one unit cell thick, the Fourier transform along z* is a continuous function; measurements are therefore along lattice lines (red vertical lines). Fitting experimental data and sampling allows A(h,k,l) and phi(h,k,l) to be estimated and the 3D map to be calculated (4).


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Further Reading

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Vinothkumar KR and Henderson R (2010) Structures of membrane proteins. Quarterly Reviews of Biophysics 43 (1): 65–158.

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Fotiadis, Dimitrios, and Engel, Andreas(Jun 2015) Two‐Dimensional Crystallisation of Membrane Proteins and Structural Assessment. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0003041.pub2]