Analytical ultracentrifugation for the study of biological macromolecules

2004 ◽  
pp. 73-82
Author(s):  
C. Ebel
Keyword(s):  
Analytical Ultracentrifugation ◽  
Biological Macromolecules

Observation of biological macromolecules using various electron microscopic methods

1978 ◽  
Vol 36 (2) ◽  
pp. 170-171
Author(s):  
Mitsuo Ohtsuki ◽  
Michael Sogard
Keyword(s):  
Electron Microscope ◽  
Phase Contrast ◽  
Image Interpretation ◽  
Density Lipoprotein ◽  
Biological Macromolecules ◽  
Contrast Effects ◽  
Biological Structure ◽  
Electron Microscopic ◽  
Structural Investigations ◽  
Staining Techniques

Structural investigations of biological macromolecules commonly employ CTEM with negative staining techniques. Difficulties in valid image interpretation arise, however, due to problems such as variability in thickness and degree of penetration of the staining agent, noise from the supporting film, and artifacts from defocus phase contrast effects. In order to determine the effects of these variables on biological structure, as seen by the electron microscope, negative stained macromolecules of high density lipoprotein-3 (HDL3) from human serum were analyzed with both CTEM and STEM, and results were then compared with CTEM micrographs of freeze-etched HDL3. In addition, we altered the structure of this molecule by digesting away its phospholipid component with phospholipase A2 and look for consistent changes in structure.

Measurement and Reduction of Radiation Damage in Frozen Hydrated Crystalline Specimens

1978 ◽  
Vol 36 (3) ◽  
pp. 70-77 ◽  
Author(s):  
R.M. Glaeser ◽  
S.B. Hayward
Keyword(s):  
Diffraction Pattern ◽  
Structural Information ◽  
Structural Disorder ◽  
Structural Features ◽  
Biological Macromolecules ◽  
Diffraction Intensity ◽  
Object Structure ◽  
Specimen Material ◽  
Unit Cells ◽  
Identical Unit

Highly ordered or crystalline biological macromolecules become severely damaged and structurally disordered after a brief electron exposure. Evidence that damage and structural disorder are occurring is clearly given by the fading and eventual disappearance of the specimen's electron diffraction pattern. The fading and disappearance of sharp diffraction spots implies a corresponding disappearance of periodic structural features in the specimen. By the same token, there is a oneto- one correspondence between the disappearance of the crystalline diffraction pattern and the disappearance of reproducible structural information that can be observed in the images of identical unit cells of the object structure. The electron exposures that result in a significant decrease in the diffraction intensity will depend somewhat upon the resolution (Bragg spacing) involved, and can vary considerably with the chemical makeup and composition of the specimen material.

The Nature of Rapidly Extracted Phycocyanin from the Blue-Green Alga Plectonema Boryanum

1971 ◽  
Vol 29 ◽  
pp. 366-367
Author(s):  
M. Kessel ◽  
R. MacColl
Keyword(s):  
Self Assembly ◽  
Analytical Ultracentrifugation ◽  
Uranyl Acetate ◽  
The Self ◽  
Major Protein ◽  
Subunit Structure ◽  
Blue Green Alga ◽  
Thin Sections ◽  
Blue Green Algae ◽  
Cacodylate Buffer

The major protein of the blue-green algae is the biliprotein, C-phycocyanin (Amax = 620 nm), which is presumed to exist in the cell in the form of distinct aggregates called phycobilisomes. The self-assembly of C-phycocyanin from monomer to hexamer has been extensively studied, but the proposed next step in the assembly of a phycobilisome, the formation of 19s subunits, is completely unknown. We have used electron microscopy and analytical ultracentrifugation in combination with a method for rapid and gentle extraction of phycocyanin to study its subunit structure and assembly.To establish the existence of phycobilisomes, cells of P. boryanum in the log phase of growth, growing at a light intensity of 200 foot candles, were fixed in 2% glutaraldehyde in 0.1M cacodylate buffer, pH 7.0, for 3 hours at 4°C. The cells were post-fixed in 1% OsO4 in the same buffer overnight. Material was stained for 1 hour in uranyl acetate (1%), dehydrated and embedded in araldite and examined in thin sections.

Concentration determination of chromatin in unstained resin-embedded sections by means of Z-contrast in STEM

1990 ◽  
Vol 48 (2) ◽  
pp. 186-187
Author(s):  
M. Haider ◽  
B. Bohrmann
Keyword(s):  
Energy Loss ◽  
Freeze Substitution ◽  
Biological Macromolecules ◽  
Local Concentration ◽  
E Coli ◽  
Concentration Determination ◽  
Phosphorous Content ◽  
Z Contrast ◽  
Electron Energy Loss

The technique of Z-contrast in STEM offers the possibility to determine the local concentration of macromolecules like lipids, proteins or DNA. Contrast formation depends on the atomic composition of the particular structure. In the case of DNA, its phosphorous content discriminates it from other biological macromolecules. In our studies, sections of E. coli, the dinoflagellate Amphidinium carterae and Euglena spec. cells were used which were obtained by cryofixation followed by freeze-substitution into acetone with 3% glutaraldehyde. The samples were then embedded either in Lowicryl HM20 at low temperature or in Epon at high temperature. Sections were coated on both sides with 30Å carbon.The DF- and the inelastic image have been recorded simultaneously with a Cryo-STEM. This Cryo-STEM is equipped with a highly dispersive Electron Energy Loss Spectrometer. With this instrument pure Z-contrast can be achieved either with a Filtered DF-image divided by the inelastic image or, as is used in this paper, by dividing the conventional DF-image by an inelastic image which has been recorded with an inelastic detector whose response is dependent on the total energy loss of the inelastically scattered electrons.

Resolution assessment from spectral signal-to-noise ratios

1987 ◽  
Vol 45 ◽  
pp. 746-747
Author(s):  
M. Unser ◽  
B.L. Trus ◽  
A.C. Steven
Keyword(s):  
Electron Microscopy ◽  
Limiting Factor ◽  
Biological Macromolecules ◽  
Signal To Noise ◽  
Differential Phase ◽  
Traditional Measure ◽  
Spectral Signal ◽  
Two Measures ◽  
Diffraction Patterns ◽  
Averaging Techniques

Since the resolution-limiting factor in electron microscopy of biological macromolecules is not instrumental, but is rather the preservation of structure, operational definitions of resolution have to be based on the mutual consistency of a set of like images. The traditional measure of resolution for crystalline specimens in terms of the extent of periodic reflections in their diffraction patterns is such a criterion. With the advent of correlation averaging techniques for lattice rectification and the analysis of non-crystalline specimens, a more general - and desirably, closely compatible - resolution criterion is needed. Two measures of resolution for correlation-averaged images have been described, namely the differential phase residual (DPR) and the Fourier ring correlation (FRC). However, the values that they give for resolution often differ substantially. Furthermore, neither method relates in a straightforward way to the long-standing resolution criterion for crystalline specimens.

A TEM study of electron tunneling in biological macromolecules

1987 ◽  
Vol 45 ◽  
pp. 140-141
Author(s):  
J. A. Panitz
Keyword(s):  
Stark Effect ◽  
Electron Tunneling ◽  
Imaging Modality ◽  
Tunneling Current ◽  
Biological Species ◽  
Scanning Tunneling ◽  
Biological Macromolecules ◽  
Flat Surfaces ◽  
Virus Particles ◽  
Stm Images

Tunneling is a ubiquitous phenomenon. Alpha particle disintegration, the Stark effect, superconductivity in thin films, field-emission, and field-ionization are examples of electron tunneling phenomena. In the scanning tunneling microscope (STM) electron tunneling is used as an imaging modality. STM images of flat surfaces show structure at the atomic level. However, STM images of large biological species deposited onto flat surfaces are disappointing. For example, unstained virus particles imaged in the STM do not resemble their TEM counterparts.It is not clear how an STM image of a biological species is formed. Most biological species are large compared to the nominal electrode separation of ∼ 1nm that is required for electron tunneling. To form an image of a biological species, the tunneling electrodes must be separated by a distance that would normally be too large for a tunneling current to be observed.

Depletion and Reconstitution of Active E. Coli Ribosomes

1975 ◽  
Vol 33 ◽  
pp. 640-641
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Protein Biosynthesis ◽  
Large Subunit ◽  
High Resolution Electron Microscopy ◽  
Small Subunit ◽  
Structure And Function ◽  
Biologically Active ◽  
Biological Macromolecules ◽  
E Coli ◽  
Resolution Electron ◽  
And Function

Correlations between structure and function of biological macromolecules have been studied intensively for many years, mostly by indirect methods. High resolution electron microscopy is a unique tool which can provide such information directly by comparing the conformation of biopolymers in their biologically active and inactive state. We have correlated the structure and function of ribosomes, ribonucleoprotein particles which are the site of protein biosynthesis. 70S E. coli ribosomes, used in this experiment, are composed of two subunits - large (50S) and small (30S). The large subunit consists of 34 proteins and two different ribonucleic acid molecules. The small subunit contains 21 proteins and one RNA molecule. All proteins (with the exception of L7 and L12) are present in one copy per ribosome.This study deals with the changes in the fine structure of E. coli ribosomes depleted of proteins L7 and L12. These proteins are unique in many aspects.

High-resolution scanning electron microscopy in biology

1990 ◽  
Vol 48 (3) ◽  
pp. 14-15
Author(s):  
Keiichi Tanaka
Keyword(s):  
High Resolution ◽  
Immunoglobulin M ◽  
Metal Coating ◽  
Biological Macromolecules ◽  
Ultrahigh Resolution ◽  
Preparation Methods ◽  
Low Contrast ◽  
Almost All ◽  
Charging Effect ◽  
Scanning Electron

With the development of scanning electron microscope (SEM) with ultrahigh resolution, SEM became to play an important role in not only cytology but also molecular biology. However, the preparation methods observing tiny specimens with such high resolution SEM are not yet established.Although SEM specimens are usually coated with metals for getting electrical conductivity, it is desirable to avoid the metal coating for high resolution SEM, because the coating seriously affects resolution at this level, unless special coating techniques are used. For avoiding charging effect without metal coating, we previously reported a method in which polished carbon plates were used as substrate. In the case almost all incident electrons penetrate through the specimens and do not accumulate in them, when the specimens are smaller than 10nm. By this technique some biological macromolecules including ribosomes, ferritin, immunoglobulin G were clearly observed.Unfortunately some other molecules such as apoferritin, thyroglobulin and immunoglobulin M were difficult to be observed only by the method, because they had very low contrast and were easily damaged by electron beam.

High-Resolution Cryo-Electron Microscopy of Biological Macromolecules

1990 ◽  
Vol 48 (1) ◽  
pp. 126-127
Author(s):  
Yoshinori Fujiyoshi
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Diffraction Pattern ◽  
Superfluid Helium ◽  
Copper Phthalocyanine ◽  
Optical Diffraction ◽  
Irradiation Damage ◽  
Biological Macromolecules ◽  
Cross Sectional ◽  
Instrumental Resolution

The resolution of direct images of biological macromolecules is normally restricted to far less than 0.3 nm. This is not due instrumental resolution, but irradiation damage. The damage to biological macromolecules may expect to be reduced when they are cooled to a very low temperature. We started to develop a new cryo-stage for a high resolution electron microscopy in 1983, and successfully constructed a superfluid helium stage for a 400 kV microscope by 1986, whereby chlorinated copper-phthalocyanine could be photographed to a resolution of 0.26 nm at a stage temperature of 1.5 K. We are continuing to develop the cryo-microscope and have developed a cryo-microscope equipped with a superfluid helium stage and new cryo-transfer device.The New cryo-microscope achieves not only improved resolution but also increased operational ease. The construction of the new super-fluid helium stage is shown in Fig. 1, where the cross sectional structure is shown parallel to an electron beam path. The capacities of LN2 tank, LHe tank and the pot are 1400 ml, 1200 ml and 3 ml, respectively. Their surfaces are placed with gold to minimize thermal radiation. Consumption rates of liquid nitrogen and liquid helium are 170 ml/hour and 140 ml/hour, respectively. The working time of this stage is more than 7 hours starting from full LN2 and LHe tanks. Instrumental resolution of our cryo-stage cooled to 4.2 K was confirmed to be 0.20 nm by an optical diffraction pattern from the image of a chlorinated copper-phthalocyanine crystal. The image and the optical diffraction pattern are shown in Fig. 2 a, b, respectively.

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