scholarly journals Electron Diffraction of Frozen, Hydrated Protein Crystals

Science ◽  
1974 ◽  
Vol 186 (4168) ◽  
pp. 1036-1037 ◽  
Author(s):  
K. A. Taylor ◽  
R. M. Glaeser
Author(s):  
B. B. Chang ◽  
D. F. Parsons

The significance of dynamical scattering effects remains the major question in the structural analysis by electron diffraction of protein crystals preserved in the hydrated state. In the few cases (single layers of purple membrane and 400-600 Å thick catalase crystals examined at 100 kV acceleration voltage) where electron-diffraction patterns were used quantitatively, dynamical scattering effects were considered unimportant on the basis of a comparison with x-ray intensities. The kinematical treatment is usually justified by the thinness of the crystal. A theoretical investigation by Ho et al. using Cowley-Moodie multislice formulation of dynamical scattering theory and cytochrome b5as the test object2 suggests that kinematical analysis of electron diffraction data with 100-keV electrons would not likely be valid for specimen thickness of 300 Å or more. We have chosen to work with electron diffraction patterns obtained from actual wet protein crystals (rat hemoglobin crystals of thickness range 1000 to 2500 Å) at 200 and 1000 kV and to analyze these for dynamical effects.


Author(s):  
Robert M. Glaeser ◽  
Steven B. Hayward

Highly ordered or crystalline biological macromolecules become severely damaged and disordered after a brief electron exposure, as may be seen by observing the fading and loss of the specimen's electron diffraction pattern. Loss of the diffraction pattern intensity has, in turn, a one-to-one relationship with a loss of the possibility to see structural information in the image. The actual electron exposure that results in a significant decrease in the diffraction intensity will depend first of all upon the resolution (Bragg spacing) involved, and in some cases upon the chemical make-up and composition of the specimen material. For high resolution features (in the range 3Å to 5Å resolution) of specimens such as protein crystals and cell membranes, the structure can become damaged and disordered after an exposure of about 1 electron/Å2 or less. Roughly speaking, this exposure is about 104 times lower than that which is required to produce a statistically defined image at high resolution.


Author(s):  
Robert M. Glaeser ◽  
Bing K. Jap ◽  
Ming Hslu Ho

Single scattering approximations (the kinematic and the weak phase object approx- imation), because of their simplicity, are perhaps the most attractive formulations for structure analysis by electron diffraction. In these approximations, the diffracted wave function is linearly related to the object potential. The validities of these approximations are, however, limited to very thin crystals at low resolution. In materials science the failure of the single scattering approximations and the impor- tance of the dynamical scattering effect have been well accepted. In biological science, the large unit cell size and the low atomic number (e.g. protein crystals) have lead some to believe that the dynamical scattering effect is insignificant for crystallographic analysis. Contrary to this belief, the number of dynamically interacting beams increases with the crystal unit cell size. It is important to note here that the dynamical scattering effect depends on the values of the excitation errors and on the magnitudes of the Fourier coefficients of the crystal potential.


Author(s):  
Christoph Burmester ◽  
Kenneth C. Holmes ◽  
Rasmus R. Schröder

Electron crystallography of 2D protein crystals can yield models with atomic resolution by taking Fourier amplitudes from electron diffraction and phase information from processed images. Imaging at atomic resolution is more difficult than the recording of corresponding electron diffraction patterns. Therefore attempts have been made to recover phase information from diffraction data from 2-D and 3-D crystals by the method of isomorphous replacement using heavy atom labelled protein crystals. These experiments, however, have so far not produced usable phase information, partly because of the large experimental error in the spot intensities. Here we present electron diffraction data obtained from frozen hydrated 3-D protein crystals with an energy-filter microscope and a specially constructed Image Plate scanner which are of considerably better crystallographic quality (as evidenced in much smaller values for the crystallographic R-factors Rsym and Rmerge) than those reported before. The quality of this data shows that the method of isomorphous replacement could indeed be used for phase determination for diffraction data obtained from 3-D microcrystals by electron diffraction.


2013 ◽  
Vol 69 (7) ◽  
pp. 1223-1230 ◽  
Author(s):  
Igor Nederlof ◽  
Eric van Genderen ◽  
Yao-Wang Li ◽  
Jan Pieter Abrahams

When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e− Å−2), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electron-diffraction data from a single ∼100 nm thick protein crystal. A highly parallel 200 keV electron beam (λ = 0.025 Å) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4°. This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLMandSCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins.


2010 ◽  
Vol 16 (S2) ◽  
pp. 1080-1081
Author(s):  
P Li ◽  
M Malac ◽  
JP Glaves ◽  
H Young

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.


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