scholarly journals Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

10.3791/1846 ◽  
2010 ◽  
Author(s):  
Matthew C. Johnson ◽  
Frederik Rudolph ◽  
Tina M. Dreaden ◽  
Gengxiang Zhao ◽  
Bridgette A. Barry ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Structural Biology ◽  
Two Dimensional ◽  
Electron Crystallography

scholarly journals Two-Dimensional Crystallization Procedure, from Protein Expression to Sample Preparation

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Qie Kuang ◽  
Pasi Purhonen ◽  
Hans Hebert
Keyword(s):  
Membrane Proteins ◽  
General Idea ◽  
General Description ◽  
Two Dimensional ◽  
Structural Studies ◽  
Electron Crystallography ◽  
Electron Microscopic ◽  
Small Proteins ◽  
Crystallization Procedure ◽  
Related Proteins

Membrane proteins play important roles for living cells. Structural studies of membrane proteins provide deeper understanding of their mechanisms and further aid in drug design. As compared to other methods, electron microscopy is uniquely suitable for analysis of a broad range of specimens, from small proteins to large complexes. Of various electron microscopic methods, electron crystallography is particularly well-suited to study membrane proteins which are reconstituted into two-dimensional crystals in lipid environments. In this review, we discuss the steps and parameters for obtaining large and well-ordered two-dimensional crystals. A general description of the principle in each step is provided since this information can also be applied to other biochemical and biophysical methods. The examples are taken from our own studies and published results with related proteins. Our purpose is to give readers a more general idea of electron crystallography and to share our experiences in obtaining suitable crystals for data collection.

Structure of Membrane Proteins Revealed by Electron Crystallography

2000 ◽  
Vol 6 (S2) ◽  
pp. 234-235
Author(s):  
K. Mitsuoka
Keyword(s):  
Membrane Proteins ◽  
Structure Determination ◽  
Two Dimensional ◽  
Asymmetric Unit ◽  
Electron Crystallography ◽  
Aquaporin 1 ◽  
Diffraction Patterns ◽  
2D Crystals

Because membrane proteins are localized in a continuous lipid bilayer in the native environment, the situation of membrane proteins in the two-dimensional (2D) crystals is quite similar to the environment in vivo. Thus, electron crystallography using 2D crystals is one of the suitable techniques for structure determination of membrane proteins at atomic or near-atomic resolution. Here we describe the structures of the two membrane proteins, bacteriorhodopsin and aquaporin-1, which were solved by electron crystallography at 2.5 and 4.0 Å resolution, respectively.Bacteriorhodopsin (bR) is a light-driven proton pump found in Halobacterium salinarium. The atomic model of the protein was first proposed by electron crystallography and we improved the resolution of the structure determination up to 3.0 Å by collecting 366 electron diffraction patterns and 129 images. The resulted map showed not only a bR molecule but also eight surrounding lipids in the asymmetric unit.

Testing Parameters for Two-Dimensional Crystallization and Electron Crystallography on Two Eukaryotic Membrane Proteins

2008 ◽  
Vol 14 (S2) ◽  
pp. 1292-1293
Author(s):  
G Zhao ◽  
D Müller ◽  
D Stafford ◽  
Y Kanaoka ◽  
KF Austen ◽  
...  
Keyword(s):  
New Mexico ◽  
Membrane Proteins ◽  
Two Dimensional ◽  
Electron Crystallography ◽  
Eukaryotic Membrane Proteins

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

scholarly journals Preparation of single-layered enzyme 2D crystals for electron crystallography

2014 ◽  
Vol 70 (a1) ◽  
pp. C1065-C1065
Author(s):  
Matthew Johnson ◽  
Yusuf Uddin ◽  
Maureen Metcalfe ◽  
Ingeborg Schmidt-Krey
Keyword(s):  
Image Processing ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Electron Diffraction Data ◽  
Two Dimensional ◽  
Electron Crystallography ◽  
Negative Stain ◽  
Wide Range ◽  
Tubular Crystals ◽  
2D Crystals

Electron crystallography allows for a wide range of membrane proteins to be studied once conditions for two-dimensional (2D) crystallization have been identified. Two-dimensional crystallization is most frequently achieved via the dialysis approach, where the detergent-solubilized membrane protein is reconstituted into a lipid bilayer [1]. Vesicles, planar-tubular crystals, and sheets are the three most common 2D crystal morphologies. Vesicle and planar-tubular morphologies are observed for the largest percentage of 2D crystals of membrane proteins. Upon negative stain as well as electron cryo-microscopy (cryo-EM) grid preparation, each planar-tubular and vesicle 2D crystal will result in two ordered bilayers that can be analyzed separately by image processing. If any of these morphologies, however, contains a larger number of stacked crystals, data of tilted crystal stacks in particular can currently not be analyzed. Sheets constitute the most desirable morphology, allowing for the preparation of very flat samples for cryo-EM [2]. This is at present the only type of morphology that may be amenable to collection and analysis of electron diffraction data of highly ordered samples [3]. We could reproducibly induce single-layered sheet formation in the large majority of 2D crystals of two different enzyme samples and are working towards a general protocol applicable to other membrane protein 2D crystals.

A Novel Reconstitution Method for Inducing the Formation of Regular 2-Dimensional Arrays of Membrane Proteins and Lipids

1988 ◽  
Vol 46 ◽  
pp. 152-153 ◽  
Author(s):  
A. Engel ◽  
A. Holzenburg ◽  
K. Stauffer ◽  
J. Rosenbusch ◽  
U. Aebi
Keyword(s):  
Membrane Proteins ◽  
Flow Rate ◽  
Lipid Membranes ◽  
Functional Characterization ◽  
Dimensional Structure ◽  
Electron Crystallography ◽  
Peltier Element ◽  
Protein Ratio ◽  
Precise Control ◽  
3 Dimensional

Reconstitution of solubilized and purified membrane proteins in the presence of phospholipids into vesicles allows their functions to be studied by simple bulk measurements (e.g. diffusion of differently sized solutes) or by conductance measurements after transformation into planar membranes. On the other hand, reconstitution into regular protein-lipid arrays, usually forming at a specific lipid-to-protein ratio, provides the basis for determining the 3-dimensional structure of membrane proteins employing the tools of electron crystallography.To refine reconstitution conditions for reproducibly inducing formation of large and highly ordered protein-lipid membranes that are suitable for both electron crystallography and patch clamping experiments aimed at their functional characterization, we built a flow-dialysis device that allows precise control of temperature and flow-rate (Fig. 1). The flow rate is generated by a peristaltic pump and can be adjusted from 1 to 500 ml/h. The dialysis buffer is brought to a preselected temperature during its travel through a meandering path before it enters the dialysis reservoir. A Z-80 based computer controls a Peltier element allowing the temperature profile to be programmed as function of time.

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