Electron Crystallography of Two-Dimensional Crystals of Membrane Proteins

Walz, Thomas; Grigorieff, Nikolaus

doi:10.1006/jsbi.1998.3945

Cited by 80 publications

(39 citation statements)

References 191 publications

(263 reference statements)

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“…Once the 2-D crystals have been produced, the structure of the protein can be determined by electron crystallography and image processing to a resolution that depends on the quality of the 2-D crystal (11). The best electron micrographs are selected by optical diffraction with a laser diffractometer, digitized on a microdensitometer and computer processed.…”

Section: Structural Analysismentioning

confidence: 99%

“…Reconstitution of membrane proteins into artificial membranes to form 2-D crystals has opened a new way to solve their structures inside a native-like environment (7)(8)(9)(10). Indeed, electron crystallography of 2-D crystals has developed to the point that significant structural atomic models have been built and that many membrane protein structures have been evaluated at resolutions allowing the secondary structure to be assessed (11). These recent results have made electron crystallography an excellent alternative and a complementary discipline to X-ray crystallography in membrane protein structural biology.…”

Section: Introductionmentioning

confidence: 99%

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Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals

2002

Braz J Med Biol Res

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Reconstitution of membrane proteins into lipid bilayers is a powerful tool to analyze functional as well as structural areas of membrane protein research. First, the proper incorporation of a purified membrane protein into closed lipid vesicles, to produce proteoliposomes, allows the investigation of transport and/or catalytic properties of any membrane protein without interference by other membrane components. Second, the incorporation of a large amount of membrane proteins into lipid bilayers to grow crystals confined to two dimensions has recently opened a new way to solve their structure at high resolution using electron crystallography. However, reconstitution of membrane proteins into functional proteoliposomes or 2-D crystallization has been an empirical domain, which has been viewed for a long time more like "black magic" than science. Nevertheless, in the last ten years, important progress has been made in acquiring knowledge of lipid-protein-detergent interactions and has permitted to build upon a set of basic principles that has limited the empirical approach of reconstitution experiments. Reconstitution strategies have been improved and new strategies have been developed, facilitating the success rate of proteoliposome formation and 2-D crystallization. This review deals with the various strategies available to obtain proteoliposomes and 2-D crystals from detergent-solubilized proteins. It gives an overview of the methods that have been applied, which may be of help for reconstituting more proteins into lipid bilayers in a form suitable for functional studies at the molecular level and for high-resolution structural analysis. Correspondence

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Section: Structural Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals

2002

Braz J Med Biol Res

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“…Three different methods are being used for removing detergents, dialysis, beads, and cyclodextrin 14,31,32 . But it remains difficult to achieve a well-controlled, gradual removal of detergents from a small volume 33,34 .…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

Lee

Zheng

Shi

et al. 2013

JoVE

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To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of welldefined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels. Video LinkThe video component of this article can be found at http://www.jove.com/video/50436/ IntroductionCells exchange materials and information with their environment through the functions of specific membrane proteins 1 . Membrane proteins in cell membranes function as pumps, channels, receptors, intramembrane enzymes, linkers and structural supporters across membranes. Mutations that affect the membrane proteins have been related to many human diseases. In fact, many membrane proteins have been the primary drug targets because they are important and easily accessible in cell membranes. It is therefore very important to understand the structure and function of various membrane proteins in membranes, and make it possible to devise novel methods to alleviate the detrimental effects from the mutant proteins in human diseases.Lipids surround all membrane proteins integrated in bilayers 2,3 . In eukaryotic membranes, the various different types of lipids are known to be organized into microdomains 4, 5 . Many membrane proteins were shown to be distributed among these microdomains as well as the bulky fluid phase of membranes 3,6 . The mechanism underlying the organization of the microdomains and the delivery of membrane proteins into them and the physiological significance of such distributions are clearly important but remain poorly understood. One major technical difficulty in studying the lipid effects on membrane proteins is the reliable reconstitution of biochemically purified membrane proteins int...

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“…Commercially available thin three-dimensional microcrystals of bovine liver catalase (Serva) were recrystallized to obtain better ordered three-dimensional microcrystals (Wrigley, 1968). They have orthorhombic lattice parameters a = 69, b = 173.5 and c = 206 A Ê (Unwin, 1975).…”

Section: Determination Of Density Profile In Experimental Datamentioning

confidence: 99%

“…Electron crystallography has been extremely successful in molecular structure determination utilizing crystalline twodimensional monolayers of biological samples (for an overview, see Walz & Grigorieff, 1998). The structures of three membrane proteins (Henderson et al, 1990;Jap et al, 1991;Ku È hlbrandt et al, 1994) and of the cytoplasmic protein tubulin (Nogales et al, 1998) have been solved at the molecular level.…”

Section: Introductionmentioning

confidence: 99%

Determination of lattice-transform density profiles for multilayered three-dimensional microcrystals in electron crystallography

2000

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Electron crystallography on multilayered three-dimensional microcrystals has been limited in application by the need to de®ne precisely the three-dimensional shape of the diffraction density pro®les. A new method is presented here to obtain this pro®le from experimental spot positions which are shifted in a characteristic way from the expected Bragg positions. While the Bragg positions are de®ned by the diffraction geometry, the characteristic shift additionally depends on the density pro®le in Fourier space. In general, these two effects are intermingled. A new correlation approach is presented which uses characteristic shift patterns to separate these effects. This technique also allows the determination of all three crystallographic unit-cell dimensions from a single tilted electron diffraction pattern. It was tested on simulated diffraction patterns and applied to experimental data of frozen hydrated crystals of the protein catalase. Since multilayered catalase crystals with different numbers of crystallographic layers were studied, an inhomogeneous data set had to be evaluated. Processing of such data is now possible using the new correlation approach.

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Electron Crystallography of Two-Dimensional Crystals of Membrane Proteins

Cited by 80 publications

References 191 publications

Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals

Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

Determination of lattice-transform density profiles for multilayered three-dimensional microcrystals in electron crystallography

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