Multiple isomorphous replacement for phase determination in protein crystals

1995 ◽  
Vol 53 ◽  
pp. 856-857
Author(s):  
Christoph Burmester ◽  
Kenneth C. Holmes ◽  
Rasmus R. Schröder
Keyword(s):  
Electron Diffraction ◽  
Heavy Atom ◽  
Atomic Resolution ◽  
Protein Crystals ◽  
Electron Diffraction Data ◽  
Phase Information ◽  
Electron Dose ◽  
Isomorphous Replacement ◽  
Resolution Electron ◽  
Diffraction Patterns

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 high resolution electron diffraction patterns. Therefore attempts have been made to retrieve phase information from diffraction from heavy atom labelled protein crystals. The expected differences between native and labelled crystals are small, therefore a high experimental accuracy is necessary. This is achieved by the use of energy filter TEM and image plates, as dicussed in. Here we present electron diffraction data obtained from frozen hydrated 3D protein crystals with an energy filter microspcope and a specially designed image plate scanner. Data were recorded for the native crystal as well as for two different heavy atom derivatives. Differences between the native and the derivate forms can be detected and are significant.Electron diffraction patterns from frozen hydrated catalase crystals were recorded on an EFTEM Zeiss 912 Ω (120kV, zero loss mode, energy width ΔE=10eV, electron dose 5 e-/A2) using image plates and a quasi confocal scanner readout.

Electron-diffraction patterns from frozen hydrate protein microcrystals: A new tool instructural studies

1994 ◽  
Vol 52 ◽  
pp. 102-103
Author(s):  
Christoph Burmester ◽  
Kenneth C. Holmes ◽  
Rasmus R. Schröder
Keyword(s):  
Electron Diffraction ◽  
Diffraction Data ◽  
Heavy Atom ◽  
Atomic Resolution ◽  
Protein Crystals ◽  
Electron Diffraction Data ◽  
Phase Information ◽  
Isomorphous Replacement ◽  
Diffraction Patterns

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.

Low dose electron diffraction of wet protein crystals

1978 ◽  
Vol 36 (2) ◽  
pp. 6-7
Author(s):  
B.B. Chang ◽  
D.F. Parsons
Keyword(s):  
High Resolution ◽  
Electron Diffraction ◽  
Radiation Damage ◽  
Low Dose ◽  
Protein Crystal ◽  
Protein Crystals ◽  
Electron Dose ◽  
Resolution Electron ◽  
Diffraction Patterns ◽  
Iterative Procedures

High resolution electron diffraction patterns from wet unstained protein-crystals have been successfully obtained by using very low electron dose (∽10-2 e/Å2, much smaller dose than used by Unwin and Henderson (1975) for electron diffraction of their crystals). This enabled us to follow the radiation damage of the protein crystal due to increasing dosage by recording successive diffraction patterns given by the same crystal. Changes in intensities of both the high and the low order reflections can be studied. Another important consequence is that very low dose electron diffraction can be obtained from the same crystal and iterative procedures such as Gerchberg and Saxton's technique can be applied.The electron diffraction work with very low dose was performed at 200 kV using the environmental chamber in the Jeolco 200. To achieve best electron diffraction conditions with the specimen in the hydration chamber, the objective and intermediate lens currents have been changed.

Dynamical Scattering in Electron Diffraction of wet Protein Crystals

1980 ◽  
Vol 38 ◽  
pp. 42-43
Author(s):  
B. B. Chang ◽  
D. F. Parsons
Keyword(s):  
Electron Diffraction ◽  
Scattering Theory ◽  
Protein Crystals ◽  
Electron Diffraction Data ◽  
Specimen Thickness ◽  
Major Question ◽  
X Ray ◽  
Scattering Effects ◽  
Diffraction Patterns ◽  
Acceleration Voltage

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.

Effect of diffraction crystallography on HREM

2003 ◽  
Vol 218 (4) ◽  
Author(s):  
Fang-hua Li
Keyword(s):  
Image Processing ◽  
High Resolution ◽  
Electron Diffraction ◽  
Diffraction Data ◽  
Heavy Atom ◽  
Empirical Method ◽  
Crystal Defects ◽  
Electron Diffraction Data ◽  
Crystal Thickness ◽  
Resolution Electron

AbstractA simple image contrast theory in high-resolution electron microscopy (HREM) is introduced to demonstrate that below a certain critical crystal thickness the intensity of the Scherzer focus image is linear to the projected potential of an artificial crystal that is isomorphic to the examined one. It has become the theoretical base of electron crystallographic image processing techniques relying on the weak-phase-object approximation and kinematical diffraction. Two techniques of image processing are introduced. One of them aims at determining crystal structures by combining electron diffraction data and applying diffraction analysis methods. To reduce various kinds of electron diffraction intensity distortion the diffraction data are corrected by means of an empirical method set up by referring to the heavy atom method and Wilson statistic. The other one aims at revealing crystal defects at atomic resolution from the image taken with a medium-voltage field-emission high-resolution electron microscope. The dynamical effect is corrected by forcing the integral amplitudes of reflections in the diffractogram of image equal to the amplitudes of corresponding structure factors for the perfect crystal. The principle of the two techniques is briefly introduced, and examples of applications to crystal structure and defect determination are given.

Electron diffraction of helical structures

1992 ◽  
Vol 50 (1) ◽  
pp. 518-519
Author(s):  
T. Ruiz ◽  
R. Diaz ◽  
J-L. Ranck ◽  
D.L.D. Caspar ◽  
D.J. DeRosier
Keyword(s):  
Electron Microscopy ◽  
Electron Diffraction ◽  
Helical Structure ◽  
Lipid Monolayer ◽  
Electron Diffraction Data ◽  
Helical Structures ◽  
Protein Aggregate ◽  
X Ray ◽  
Isomorphous Replacement ◽  
Diffraction Patterns

Electron microscopy has advantages over X-ray diffraction for the study of helical structures. For X-ray studies, one needs large well oriented samples which are difficult to obtain. Only one helical structure, TMV, has been solved by conventional X-ray analysis using multiple isomorphous replacement. In contrast, one requires single particles or small rafts for studies by electron microscopy. We are attempting to use a combination of imaging and electron diffraction data to analyze helical structures at 9-10 Å resolution in order to visualize α-helices. To obtain electron diffraction patterns we produced well-ordered domains (∽ 1-3 μm in diameter) for diffraction work. Several methods succeeded in aligning helical particles : the lipid monolayer technique, mica sandwiching and unidirectional blotting. The lipid monolayer technique proved to be the best for high resolution work. The three samples under study (flagellar filaments from Salmonella typhimurium, TMV and TMV stacked disk protein aggregate) gave electron diffraction patterns out to ∽10 Å resolution.

scholarly journals Three-dimensional electron crystallography of protein microcrystals

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Dan Shi ◽  
Brent L Nannenga ◽  
Matthew G Iadanza ◽  
Tamir Gonen
Keyword(s):  
Electron Diffraction ◽  
Protein Structures ◽  
Three Dimensional ◽  
Electron Diffraction Data ◽  
Electron Crystallography ◽  
Electron Dose ◽  
Tilt Series ◽  
Diffraction Patterns ◽  
A New Technique

We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1–1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. This proof of principle paves the way for the implementation of a new technique, which we name ‘MicroED’, that may have wide applicability in structural biology.

scholarly journals MicroED: Three Dimensional Electron Crystallography of Protein Micro-Crystals

2014 ◽  
Vol 70 (a1) ◽  
pp. C1063-C1063
Author(s):  
Tamir Gonen
Keyword(s):  
Electron Diffraction ◽  
Protein Structures ◽  
Three Dimensional ◽  
Electron Diffraction Data ◽  
Molecular Replacement ◽  
Electron Crystallography ◽  
Electron Dose ◽  
Tilt Series ◽  
Diffraction Patterns

We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1 - 10and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9Å resolution. In this seminar I will present our initial proof of principle study and highlight the major advances since the first publication.

Isomorphous replacement in electron diffraction of wet crystals of rat hemoglobin

1983 ◽  
Vol 41 ◽  
pp. 446-447
Author(s):  
William F. Tivol ◽  
Murray Vernon King ◽  
D. F. Parsons
Keyword(s):  
Electron Diffraction ◽  
Heavy Atom ◽  
Isomorphous Substitution ◽  
X Ray Diffraction ◽  
Salt Solutions ◽  
X Ray ◽  
Isomorphous Replacement ◽  
Structure Factors ◽  
Diffraction Structure ◽  
The Mean

Feasibility of isomorphous substitution in electron diffraction is supported by a calculation of the mean alteration of the electron-diffraction structure factors for hemoglobin crystals caused by substituting two mercury atoms per molecule, following Green, Ingram & Perutz, but with allowance for the proportionality of f to Z3/4 for electron diffraction. This yields a mean net change in F of 12.5%, as contrasted with 22.8% for x-ray diffraction.Use of the hydration chamber in electron diffraction opens prospects for examining many proteins that yield only very thin crystals not suitable for x-ray diffraction. Examination in the wet state avoids treatments that could cause translocation of the heavy-atom labels or distortion of the crystal. Combined with low-fluence techniques, it enables study of the protein in a state as close to native as possible.We have undertaken a study of crystals of rat hemoglobin by electron diffraction in the wet state. Rat hemoglobin offers a certain advantage for hydration-chamber work over other hemoglobins in that it can be crystallized from distilled water instead of salt solutions.

Electron-diffraction studies in synthetic polymer materials

1983 ◽  
Vol 41 ◽  
pp. 362-365
Author(s):  
D.T. Grubb
Keyword(s):  
Electron Diffraction ◽  
Synthetic Polymer ◽  
Polymer Materials ◽  
Crystallographic Structure ◽  
High Dose ◽  
Electron Dose ◽  
Dose Rates ◽  
First Case ◽  
Diffraction Patterns ◽  
The Many

Diffraction studies in polymeric and other beam sensitive materials may bring to mind the many experiments where diffracted intensity has been used as a measure of the electron dose required to destroy fine structure in the TEM. But this paper is concerned with a range of cases where the diffraction pattern itself contains the important information.In the first case, electron diffraction from paraffins, degraded polyethylene and polyethylene single crystals, all the samples are highly ordered, and their crystallographic structure is well known. The diffraction patterns fade on irradiation and may also change considerably in a-spacing, increasing the unit cell volume on irradiation. The effect is large and continuous far C94H190 paraffin and for PE, while for shorter chains to C 28H58 the change is less, levelling off at high dose, Fig.l. It is also found that the change in a-spacing increases at higher dose rates and at higher irradiation temperatures.

Anisotropic radiation damage of potassium tetracyano platinate (KCP)

1978 ◽  
Vol 36 (1) ◽  
pp. 392-393
Author(s):  
N. Uyeda ◽  
E. J. Kirkland ◽  
B. M. Siegel
Keyword(s):  
Electron Diffraction ◽  
Radiation Damage ◽  
Structural Changes ◽  
High Resolution Electron Microscopy ◽  
Radiation Sensitivity ◽  
Resolution Electron ◽  
Damage Process ◽  
Diffraction Patterns ◽  
Fading Rate

The direct observation of structural change by high resolution electron microscopy will be essential for the better understanding of the damage process and its mechanism. However, this approach still involves some difficulty in quantitative interpretation mostly being due to the quality of obtained images. Electron diffraction, using crystalline specimens, has been the method most frequently applied to obtain a comparison of radiation sensitivity of various materials on the quantitative base. If a series of single crystal patterns are obtained the fading rate of reflections during the damage process give good comparative measures. The electron diffraction patterns also render useful information concerning the structural changes in the crystal. In the present work, the radiation damage of potassium tetracyano-platinate was dealt with on the basis two dimensional observation of fading rates of diffraction spots. KCP is known as an ionic crystal which possesses “one dimensional” electronic properties and it would be of great interest to know if radiation damage proceeds in a strongly asymmetric manner.

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