Electron Crystallography of a Prokaryotic Potassium Channel

Potassium channels are ubiquitous ion channel proteins which play a crucial role in a broad spectrum of important cell functions. KcsA is a potassium channel found in the bacterium, Streptomyces lividan, and is believed to have only two transmembrane helices.The KcsA protein has been cloned, overexpressed and purified to homogeneity, and reconstituted with phospholipids to form two-dimensional (2-D) crystals. Crystal samples were embedded in trehalose and examined in a cryo-holder (-170 °C) using a JEOL 4000EX electron microscope operated at 400kV in low dose mode. Images were recorded in spot scan-mode at a magnification of 60,000X, then digitized on a Perkin Elmer PDS flat bed microdensitometer and processed by using the MRC program suite. Processed images were brought to a common phase origin, and the amplitudes and phases from individual images were vectorially combined.

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A Lipid Bilayer Formed on a Hydrogel Bead for Single Ion Channel Recordings

Micromachines ◽

10.3390/mi11121070 ◽

2020 ◽

Vol 11 (12) ◽

pp. 1070

Author(s):

Minako Hirano ◽

Daiki Yamamoto ◽

Mami Asakura ◽

Tohru Hayakawa ◽

Shintaro Mise ◽

...

Keyword(s):

Ion Channel ◽

Potassium Channel ◽

Lipid Bilayer ◽

Drug Targets ◽

Bk Channel ◽

Voltage Dependent Anion Channel ◽

Hydrogel Bead ◽

Cell Functions ◽

Channel Proteins ◽

Measurement Efficiency

Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, α-hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion.

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3-D Structure of a Water Channel At ˜6Å Resolution as Determined by Electron Crystallography

Microscopy and Microanalysis ◽

10.1017/s1431927600012046 ◽

1997 ◽

Vol 3 (S2) ◽

pp. 1031-1032

Author(s):

B.K. Jap ◽

H.-L. Li ◽

S. Lee

Keyword(s):

Water Channel ◽

Three Dimensional ◽

Transmembrane Helices ◽

Transmembrane Segment ◽

Electron Crystallography ◽

Major Function ◽

Aquaporin 1 ◽

Channel Proteins ◽

Density Peaks ◽

Vacuolar Membranes

Aquaporin-1 (AQP1) is a class of water channels within the aquaporin superfamily. These channel proteins have been found in a wide variety of tissues such as kidney, lung, eye secretory gland and intestinal epithelium as well as in vacuolar membranes of plants. The major function of these channel proteins is to transport water exclusively into and out of cells. Based on amino acid sequence, it has been predicted that aquaporins contain six lipid bilayer-spanning a-helices. Models for the molecular folding of AQP1 containing six and four transmembrane a-helices have been proposed previously. Our earlier projection map at 3.5Å resolution revealed eight high density peaks which we interpreted as the projections of seven transmembrane α-helices and an eighth possibly transmembrane segment. The juxtaposition of structures seen in the projection map prevented us from unambiguously determining the exact number of transmembrane helices based on the projection map alone. We report here the three-dimensional (3-D) map at ˜6 Å resolution as determined by electron crystallography.

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High Resolution Microscopy of Multi-Layered Biological Crystals

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010005319x ◽

1982 ◽

Vol 40 ◽

pp. 80-83

Author(s):

H.A. Cohen ◽

T.W. Jeng ◽

W. Chiu

Keyword(s):

High Resolution ◽

Low Dose ◽

Three Dimensional ◽

Data Interpretation ◽

Two Dimensional ◽

Biological Objects ◽

Density Maps ◽

Density Map ◽

Complex Crystal ◽

High Resolution Microscopy

This tutorial will discuss the methodology of low dose electron diffraction and imaging of crystalline biological objects, the problems of data interpretation for two-dimensional projected density maps of glucose embedded protein crystals, the factors to be considered in combining tilt data from three-dimensional crystals, and finally, the prospects of achieving a high resolution three-dimensional density map of a biological crystal. This methodology will be illustrated using two proteins under investigation in our laboratory, the T4 DNA helix destabilizing protein gp32*I and the crotoxin complex crystal.

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Correlation averaging of two-dimensional crystals: basic strategy and refinements

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142323 ◽

1986 ◽

Vol 44 ◽

pp. 136-139

Author(s):

W. Baumeister ◽

R. Rachel ◽

R. Guckenberger ◽

R. Hegerl

Keyword(s):

Low Dose ◽

Signal To Noise Ratio ◽

Real Space ◽

Fourier Domain ◽

Two Dimensional ◽

Signal To Noise ◽

Unit Cells ◽

Lateral Displacements ◽

Established Technique ◽

Crystalline Array

IntroductionCorrelation averaging (CAV) is meanwhile an established technique in image processing of two-dimensional crystals /1,2/. The basic idea is to detect the real positions of unit cells in a crystalline array by means of correlation functions and to average them by real space superposition of the aligned motifs. The signal-to-noise ratio improves in proportion to the number of motifs included in the average. Unlike filtering in the Fourier domain, CAV corrects for lateral displacements of the unit cells; thus it avoids the loss of resolution entailed by these distortions in the conventional approach. Here we report on some variants of the method, aimed at retrieving a maximum of information from images with very low signal-to-noise ratios (low dose microscopy of unstained or lightly stained specimens) while keeping the procedure economical.

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Structures and crystallization of vacuum-deposited amorphous Se and Sb films

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100173832 ◽

1990 ◽

Vol 48 (4) ◽

pp. 140-141

Author(s):

Makoto Shiojiri ◽

Toshiyuki Isshiki ◽

Tetsuya Fudaba ◽

Yoshihiro Hirota

Keyword(s):

Monte Carlo ◽

Electron Microscope ◽

Monte Carlo Method ◽

Computer Program ◽

Radial Distribution ◽

Film Formation ◽

Amorphous State ◽

Two Dimensional ◽

Amorphous Films ◽

Binding Condition

In hexagonal Se crystal each atom is covalently bound to two others to form an endless spiral chain, and in Sb crystal each atom to three others to form an extended puckered sheet. Such chains and sheets may be regarded as one- and two- dimensional molecules, respectively. In this paper we investigate the structures in amorphous state of these elements and the crystallization.HRTEM and ED images of vacuum-deposited amorphous Se and Sb films were taken with a JEM-200CX electron microscope (Cs=1.2 mm). The structure models of amorphous films were constructed on a computer by Monte Carlo method. Generated atoms were subsequently deposited on a space of 2 nm×2 nm as they fulfiled the binding condition, to form a film 5 nm thick (Fig. 1a-1c). An improvement on a previous computer program has been made as to realize the actual film formation. Radial distribution fuction (RDF) curves, ED intensities and HRTEM images for the constructed structure models were calculated, and compared with the observed ones.

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Factors in Photographic Emulsions for Low Dose Electron Crystallography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s143192760000101x ◽

1980 ◽

Vol 38 ◽

pp. 230-231

Author(s):

T.W. Jeng ◽

W. Chiu

Keyword(s):

Radiation Damage ◽

Low Dose ◽

Damage Effect ◽

Photographic Emulsion ◽

Electron Crystallography ◽

Electron Microscopic ◽

X Ray ◽

Signal Contrast ◽

Diffraction Patterns ◽

Photographic Emulsions

With the advances in preparing biological materials in a thin and highly ordered form, and in maintaining them hydrated under vacuum, electron crystallography has become an important tool for biological structure investigation at high resolution (1,2). However, the electron radiation damage would limit the capability of recording reflections with low intensities in an electron diffraction pattern. It has been demonstrated that the use of a low temperature stage can reduce the radiation damage effect and that one can expose the specimen with a higher dose in order to increase the signal contrast (3). A further improvement can be made by selecting a proper photographic emulsion. The primary factors in evaluating the suitability of photographic emulsion for recording low dose diffraction patterns are speed, fog level, electron response at low electron exposure, linearity, and usable range of exposure. We have compared these factors with three photographic emulsions including Kodak electron microscopic plate (EMP), Industrex AA x-ray film (AA x-ray) and Kodak nuclear track film (NTB3).

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