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.

scholarly journals LAMBEDA PHAGE

2021 ◽  
Vol 2 (3) ◽  
Keyword(s):  
Antibacterial Agents ◽  
Spectrometric Analysis ◽  
Potential Candidate ◽  
Fragmentation Pattern ◽  
Structural Studies ◽  
Electron Microscopic ◽  
Small Proteins ◽  
Growth Test ◽  
One Step ◽  
Step Growth

Background: Recognition of bacteriophages in many aspects plays an important role such as controlling the number and variation of bacteria and participation in horizontal gene transfer, which is an important process in bacterial evolution. Bacteriophages use small proteins to take over the host molecular machinery and thus interfere with central metabolic processes in infected bacteria. In general, phages inhibit or reverse host transcription to transcribe their genome. Mechanical and structural studies of phage host transcription may lead to the development of new antibacterial agents. Result: The result shows that phage vB_Eco4M-7 must be a lytic virus. This was confirmed by monitoring faglitic development by a one-step growth test. In addition, phage of relatively small uniform plaques (1 mm in diameter) occur without lysogenesis. Electron microscopic analysis showed that vB_Eco4M-7 belongs to the family Myoviridae. Based on mass spectrometric analysis, including the fragmentation pattern of unique peptides, 33 vB_Eco4M-7 phage proteins are assigned to the annotated reading frames. The results indicate that the phage studied is a potential candidate for phage treatment and / or food protection against E. coli, which produce Shiga toxin, as most of these strains belong to the O157 serotype. Conclusion: In general, phages inhibit or reverse host transcription to transcribe their genome. Mechanical and structural studies of phage host transcription may lead to the development of new antibacterial agents. The result shows that phage vB_Eco4M-7 must be a lytic virus. This was confirmed by monitoring faglitic development by a one-step growth test.

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.

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

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

Assessment of resolution in biological electron crystallography

1991 ◽  
Vol 49 ◽  
pp. 490-491
Author(s):  
Robert M. Glaeser ◽  
Kenneth H. Downing
Keyword(s):  
Three Dimensional ◽  
Two Dimensional ◽  
Structural Studies ◽  
Electron Crystallography ◽  
Fourier Synthesis ◽  
Absolute Limit ◽  
Present Context ◽  
Practical Limit ◽  
Density Map ◽  
Definition Of

In the present context the discussion of “biological electron crystallography” will be limited to structural studies of thin, two-dimensional (2-D) crystals, although a broader definition, including even single molecules, is certainly justified. The restriction to 2-D crystals provides the useful simplification that all of the information about the specimen-structure is confined to discrete diffraction spots in the computed Fourier transform of the electron micrographs. The absolute limit of resolution in a structural study is then given by the highest resolution term in the computed Fourier series that is used to synthesize a density-map representation of the specimen, which may be either a 2-D projection or a three-dimensional (3-D) reconstruction. The apparent simplicity of the above definition of “limiting resolution” disguises the fact that the structurally useful, or practical limit of resolution must invariably be somewhat poorer than the objectively determined resolution of the “highest resolution spot” included in the Fourier synthesis of a density map.

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