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., © 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 (b) The plant aquaporin SoPIP2;1 assembles into 2D crystal that diffract to 2.5‐Å resolution (unpublished, courtesy of Wanda Kukulski).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 (unpublished, courtesy of Mohamed Chami) (d) The bacterial toxin aerolysine assembles into highly ordered 2D crystals (Iacovache et al., ).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).


Abeyrathne PD, Chami M, Pantelic RS, Goldie KN and Stahlberg H (2010) Preparation of 2D crystals of membrane proteins for high‐resolution electron crystallography data collection. Methods in Enzymology 481: 25–43.

Baumeister W (2004) Mapping molecular landscapes inside cells. Biological Chemistry 385: 865–872.

Chae PS, Rasmussen SG, Rana RR, et al. (2010) Maltose‐neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nature Methods 7: 1003–1008.

Cheng A, Leung A, Fellmann D, et al. (2007) Towards automated screening of two‐dimensional crystals. Journal of Structural Biology 160: 324–331.

Coudray N, Hermann G, Caujolle‐Bert D, et al. (2011) Automated screening of 2D crystallization trials using transmission electron microscopy: a high‐throughput tool‐chain for sample preparation and microscopic analysis. Journal of Structural Biology 173: 365–374.

Degrip WJ, Vanoostrum J and Bovee‐Geurts PH (1998) Selective detergent‐extraction from mixed detergent/lipid/protein micelles, using cyclodextrin inclusion compounds: a novel generic approach for the preparation of proteoliposomes. The Biochemical Journal 330 (Pt 2): 667–674.

Del Valle E (2004) Cyclodextrins and their uses: a review. Process Biochemistry 39: 1033–1046.

Dolder M, Engel A and Zulauf M (1996) The micelle to vesicle transition of lipids and detergents in the presence of a membrane protein: towards a rationale for 2D crystallization. FEBS Letters 382: 203–208.

Engel A, Hoenger A, Hefti A, et al. (1992) Assembly of 2‐D membrane protein crystals ‐ dynamics, crystal order, and fidelity of structure analysis by electron microscopy. Journal of Structural Biology 109: 219–234.

Engel A and Müller DJ (2000) Observing single biomolecules at work with the atomic force microscope. Nature Structural Biology 7: 715–718.

Faruqi AR and McMullan G (2011) Electronic detectors for electron microscopy. Quarterly Reviews of Biophysics 44: 357–390.

Faruqi AR and Subramaniam S (2000) CCD detectors in high‐resolution biological electron microscopy. Quarterly Reviews of Biophysics 33: 1–27.

Fernandez JJ, Li S and Crowther RA (2006) CTF determination and correction in electron cryotomography. Ultramicroscopy 106: 587–596.

Fotiadis D (2012) Atomic force microscopy for the study of membrane proteins. Current Opinion in Biotechnology 23: 510–515.

Gipson B, Zeng X, Zhang ZY and Stahlberg H (2007) 2dx‐‐user‐friendly image processing for 2D crystals. Journal of Structural Biology 157: 64–72.

Grigorieff N (2013) Direct detection pays off for electron cryo‐microscopy. eLife 2: e00573.

Gyobu N, Tani K, Hiroaki Y, et al. (2004) Improved specimen preparation for cryo‐electron microscopy using a symmetric carbon sandwich technique. Journal of Structural Biology 146: 325–333.

Hattori M, Hibbs RE and Gouaux E (2012) A fluorescence‐detection size‐exclusion chromatography‐based thermostability assay for membrane protein precrystallization screening. Structure 20: 1293–1299.

Henderson R, Baldwin JM, Ceska TA, et al. (1990) Model for the structure of bacteriorhodopsin based on high‐resolution electron cryo‐microscopy. Journal of Molecular Biology 213: 899–929.

Henderson R and Unwin PN (1975) Three‐dimensional model of purple membrane obtained by electron microscopy. Nature 257: 28–32.

Hirai T, Murata K, Mitsuoka K, Kimura Y and Fujiyoshi Y (1999) Trehalose embedding technique for high‐resolution electron crystallography: application to structural study on bacteriorhodopsin. Journal of Electron Microscopy 48: 653–658.

Hite RK, Schenk AD, Li Z, Cheng Y and Walz T (2010) Collecting electron crystallographic data of two‐dimensional protein crystals. Methods in Enzymology 481: 251–282.

Hu M, Vink M, Kim C, et al. (2010) Automated electron microscopy for evaluating two‐dimensional crystallization of membrane proteins. Journal of Structural Biology 171: 102–110.

Iacovache I, Biasini M, Kowal J, et al. (2009) The 2DX robot: a membrane protein 2D crystallization Swiss Army knife. Journal of Structural Biology 169: 370–378.

Ilgu H, Jeckelmann JM, Gachet MS, et al. (2014) Variation of the detergent‐binding capacity and phospholipid content of membrane proteins when purified in different detergents. Biophysical Journal 106: 1660–1670.

Judge PJ and Watts A (2011) Recent contributions from solid‐state NMR to the understanding of membrane protein structure and function. Current Opinion in Chemical Biology 15: 690–695.

Kaufmann TC, Engel A and Remigy HW (2006) A novel method for detergent concentration determination. Biophysical Journal 90: 310–317.

Kevany BM, Tsybovsky Y, Campuzano IDG, et al. (2013) Structural and functional analysis of the native peripherin‐ROM1 complex isolated from photoreceptor cells. Journal of Biological Chemistry 288: 36272–36284.

Kimura Y, Vassylyev DG, Miyazawa A, et al. (1997) Surface of bacteriorhodopsin revealed by high‐resolution electron crystallography. Nature 389: 206–211.

Kuhlbrandt W (1992) Two‐dimensional crystallization of membrane proteins. Quarterly Reviews of Biophysics 25: 1–49.

Lasala R, Coudray N, Abdine A, et al. (2015) Sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Journal of Structural Biology 189: 123–134.

Levy D, Chami M and Rigaud JL (2001) Two‐dimensional crystallization of membrane proteins: the lipid layer strategy. FEBS Letters 504: 187–193.

Li X, Mooney P, Zheng S, et al. (2013) Electron counting and beam‐induced motion correction enable near‐atomic‐resolution single‐particle cryo‐EM. Nature Methods 10: 584–590.

Mariani V, Schenk AD, Philippsen A and Engel A (2011) Simulation and correction of electron images of tilted planar weak‐phase samples. Journal of Structural Biology 174: 259–268.

Moraes I, Evans G, Sanchez‐Weatherby J, Newstead S and Stewart PD (2014) Membrane protein structure determination – the next generation. Biochimica et Biophysica Acta 1838: 78–87.

Murata K, Mitsuoka K, Hirai T, et al. (2000) Structural determinants of water permeation through aquaporin‐1. Nature 407: 599–605.

Philippsen A, Engel HA and Engel A (2007) The contrast‐imaging function for tilted specimens. Ultramicroscopy 107: 202–212.

Racker E, Violand B, O'Neal S, Alfonzo M and Telford J (1979) Reconstitution, a way of biochemical research; some new approaches to membrane‐bound enzymes. Archives of Biochemistry and Biophysics 198: 470–477.

Rask‐Andersen M, Almen MS and Schioth HB (2011) Trends in the exploitation of novel drug targets. Nature Reviews Drug Discovery 10: 579–590.

Remigy HW, Caujolle‐Bert D, Suda K, et al. (2003) Membrane protein reconstitution and crystallization by controlled dilution. FEBS Letters 555: 160–169.

Rigaud JL, Mosser G, Lacapere JJ, et al. (1997) Bio‐beads: an efficient strategy for two‐dimensional crystallization of membrane proteins. Journal of Structural Biology 118: 226–235.

Ringler P, Heymann B and Engel A (2000) Two‐dimensional crystallization of membrane proteins. In: Membrane Transport, pp. 229–268. Oxford: Oxford University Press.

Schenk AD, Philippsen A, Engel A and Walz T (2013) A pipeline for comprehensive and automated processing of electron diffraction data in IPLT. Journal of Structural Biology 182: 173–185.

Scherer S, Kowal J, Chami M, et al. (2014) 2dx_automator: implementation of a semiautomatic high‐throughput high‐resolution cryo‐electron crystallography pipeline. Journal of Structural Biology 186: 302–307.

Scheuring S, Müller DJ, Stahlberg H, Engel HA and Engel A (2002) Sampling the conformational space of membrane protein surfaces with the AFM. European Biophysics Journal 31: 172–178.

Signorell GA, Kaufmann TC, Kukulski W, Engel A and Remigy HW (2007) Controlled 2D crystallization of membrane proteins using methyl‐beta‐cyclodextrin. Journal of Structural Biology 157: 321–328.

Sonoda Y, Newstead S, Hu NJ, et al. (2011) Benchmarking membrane protein detergent stability for improving throughput of high‐resolution X‐ray structures. Structure 19: 17–25.

Vink M, Derr K, Love J, Stokes DL and Ubarretxena‐Belandia I (2007) A high‐throughput strategy to screen 2D crystallization trials of membrane proteins. Journal of Structural Biology 160: 295–304.

Further Reading

Jensen GJ (ed) (2010) Methods in enzymology. In: Cryo‐EM, Part A: Sample Preparation and Data Collection, vol. 481. London: Academic Press.

Jensen GJ (ed) (2010) Methods in enzymology. In: Cryo‐EM, Part B: 3‐D Reconstruction, vol. 482. London: Academic Press.

Schmidt‐Krey I and Cheng Y (eds) (2013) Electron Crystallography of Soluble and Membrane Proteins: Methods and Protocols. New York, Heidelberg, Dordrecht, London: Springer.

Hite RK, Raunser S and Walz T (2007) Revival of electron crystallography. Current Opinion in Structural Biology 17 (4): 389–395.

Fujiyoshi Y (2011) Structural physiology based on electron crystallography. Protein Science 20 (5): 806–817.

Pope CR and Unger VM (2012) Electron crystallography – the waking beauty of structural biology. Current Opinion in Structural Biology 22 (4): 514–519.

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]