Application of a slow-scan CCD camera in protein electron crystallography at 400 KV

1995 ◽  
Vol 53 ◽  
pp. 8-9
Author(s):  
Jaap Brink ◽  
Michael B. Sherman ◽  
Wah Chiu
Keyword(s):  
High Resolution ◽  
Fiber Optics ◽  
Electronic Device ◽  
Ccd Camera ◽  
Photographic Film ◽  
Electron Crystallography ◽  
Camera Model ◽  
Electron Dose ◽  
Voltage Electron ◽  
Diffraction Patterns

In protein electron crystallography, high resolution amplitudes and phases are required for a 3D reconstruction of a protein in which one can trace the Cα-backbone. Recording of these amplitudes and phases has so far been done on photographic film. In principle they can be obtained with higher fidelity using an electronic device, such as a slow-scan charge-coupled device (CCD) camera. We use a 1024 × 1024 Gatan CCD camera (model 679) attached to a JEOL 4000EX intermediate voltage electron cryo-microscope. The camera has a 20-30 μm thin P43 phosphor scintillator and an extra set of fiber optics, that reduces the number of x-ray pixels in acquired frames. In this abstract, we will focus on several issues related to the CCD camera in obtaining high resolution diffraction patterns and images from thin protein crystals.To obtain electron diffraction patterns with sufficient statistical definition, one needs to use an adequate electron dose. However, at exposures well above 0.25 e/Å2, the pattern will suffer from blooming due to the central beam.

Characteristic feature on structure analyzed by high-resolution electron crystallography

1995 ◽  
Vol 53 ◽  
pp. 834-835
Author(s):  
Y. Fujiyoshi ◽  
K. Mitsuoka ◽  
T. Hirai ◽  
K. Murata ◽  
A. Miyazawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Resolution ◽  
Characteristic Feature ◽  
Proton Pump ◽  
Ccd Camera ◽  
Electron Crystallography ◽  
Embedding Technique ◽  
Resolution Electron ◽  
Ph Conditions ◽  
Diffraction Patterns

The structure of bacteriorhodopsin (bR), which was already analyzed by Henderson et al., is studied by our new electron cryo-microscope equipped with Field Emission Gun (FEG) and Slow Scan CCD camera (SSCCD), because our system together with ice embedding technique enable us to solve the structure of bR at various pH conditions between pH 4.0 and 10.0. Ionization of amino acid is naturally closely related to the translocation of proton and then the function of the proton pump of bR. Therefore, observation of translocation of proton in bR is very important, if possible. Both ice embedding and high resolution techniques are essential to achieve this intention. Therefore, we intended to develop an electron cryo-microscope fit to these techniques and recently we had succeeded it.We collected whole sets of diffraction patterns for bR up to 70 degree tilt at pH 5.5 by using SSCCD, and merged these data of 300 diffraction patterns.

Merging of electron-diffraction intensities acquired with a slow-scan CCD camera of crotoxin complex crystals to 3.5Å resolution

1994 ◽  
Vol 52 ◽  
pp. 104-105
Author(s):  
Jaap Brink ◽  
Wah Chiu
Keyword(s):  
Electron Diffraction ◽  
Ccd Camera ◽  
Crystal Thickness ◽  
Local Contrast ◽  
Camera Model ◽  
Data Set ◽  
3 Dimensional ◽  
Diffraction Mode ◽  
Diffraction Patterns ◽  
Complex Crystals

Crotoxin complex is the principal neurotoxin of the South American rattlesnake, Crotalus durissus terrificus and has a molecular weight of 24 kDa. The protein is a heterodimer with subunit A assigneda chaperone function. Subunit B carries the lethal activity, which is exerted on both sides ofthe neuro-muscular junction, and which is thought to involve binding to the acetylcholine receptor. Insight in crotoxin complex’ mode of action can be gained from a 3 Å resolution structure obtained by electron crystallography. This abstract communicates our progress in merging the electron diffraction amplitudes into a 3-dimensional (3D) intensity data set close to completion. Since the thickness of crotoxin complex crystals varies from one crystal to the other, we chose to collect tilt series of electron diffraction patterns after determining their thickness. Furthermore, by making use of the symmetry present in these tilt data, intensities collected only from similar crystals will be merged.Suitable crystals of glucose-embedded crotoxin complex were searched for in the defocussed diffraction mode with the goniometer tilted to 55° of higher in a JEOL4000 electron cryo-microscopc operated at 400 kV with the crystals kept at -120°C in a Gatan 626 cryo-holder. The crystal thickness was measured using the local contrast of the crystal relative to the supporting film from search-mode images acquired using a 1024 x 1024 slow-scan CCD camera (model 679, Gatan Inc.).

Ab initio structure analysis of copper perbromophthalocyanine

1992 ◽  
Vol 50 (2) ◽  
pp. 1446-1447
Author(s):  
W.F. Tivol ◽  
J.N. Turner ◽  
D.L. Dorset
Keyword(s):  
Electron Diffraction ◽  
Ab Initio ◽  
Structure Analysis ◽  
High Energy ◽  
Electron Dose ◽  
Heavy Atoms ◽  
Voltage Electron ◽  
High Energy Electrons ◽  
Diffraction Mode ◽  
Diffraction Patterns

The use of high-energy (1200 kV) electrons has been shown to be advantageous in the ab initio structure analysis from electron diffraction of organic compounds. Dynamical scattering from compounds containing heavy atoms may make such an analysis difficult or impossible with data obtained at conventional voltages. In the case that even high-energy electrons do not produce diffraction intensities sufficiently close to the kinematic values, criteria other than the simple minimization of the R-factor must be used to seek the correct structure solution.Copper perbromophthalocyanine (Cu-BrPTCY) was grown epitaxially from the vapor phase onto a clean KCl crystal face. Electron diffraction patterns were obtained from crystals tilted at 26.5° and oriented so that the electron beam was parallel to the c-axis. The AEI EM7 high-voltage electron microscope was used at a voltage of 1200 kV in diffraction mode with a 10 μm selected area aperture. The data were obtained using a minimal electron dose and recorded on DuPont Lo-dose Mammography film (See Fig. 1). Intensities were measured on a Joyce-Loebl MkIIIC flat bed microdensitometer by integrating under the peaks.

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.

Generation, Processing, and Transferring of CCD Camera Images in Electron Crystallography

1996 ◽  
Vol 54 ◽  
pp. 426-427
Author(s):  
Z. G. Li ◽  
L. Liang ◽  
R. L. Harlow ◽  
K.E. Lehman ◽  
N. Herron
Keyword(s):  
Electron Diffraction ◽  
Diffraction Data ◽  
Dynamic Range ◽  
Structural Information ◽  
Ccd Camera ◽  
High Dynamic Range ◽  
Electron Diffraction Data ◽  
Electron Crystallography ◽  
Camera Model ◽  
University Of New Mexico

Recently, CCD camera has been more and more used in the electron microscopy particularly for electron crystallography [1]. Use of CCD camera in this field as a recording medium possesses many significant advantages over conventional photographic films. A CCD camera has a very high dynamic range (reliable) and produces images directly in digital form which can be conveniently processed and transferred. We have initiated a program to obtain crystal structural information of plate-like materials by processing electron diffraction data from a CCD detector. As part of our program, we have developed a complete and routine procedure to convert images to diffraction data (h, k, l’s and intensities).Figure 1 is a schematic representation of the procedure. Images are initially obtained using a 1024×1024 Gatan CCD camera (model 794) which was attached to JEM-2000EX electron microscope. The collection of the images is controlled by a MAC computer which also stores the data and allows the data to be viewed. Then, the digitized electron diffraction patterns are transferred to a Sun station computer where, using Khoros software, the CCD images are processed. The Khoros system is a very complete image analysis and image processing software developed by University of New Mexico [2].

Quantitative analysis of low-dose high-resolution images and diffraction patterns from zeolites using a slow-scan CCD camera

1993 ◽  
Vol 51 ◽  
pp. 670-671
Author(s):  
M. Pan ◽  
P.A. Crozier
Keyword(s):  
High Resolution ◽  
Molecular Sieves ◽  
Structural Damage ◽  
Low Dose ◽  
Signal To Noise Ratio ◽  
Ccd Camera ◽  
Real Space ◽  
High Signal ◽  
Resolution Electron ◽  
Diffraction Patterns

Zeolites are an important class of low-density aluminosilicates framework structures with applications to the field of catalysis and molecular sieves. In order to understand die origin of die unique properties of these materials and hence optimize their performance, it is essential to have a detailed description of their structures. Zeolite structure is often described in terms of the secondary building unit (SBU) which is a specific configuration of the SiO4 tetrahedrons. Structure determination of zeolites is then reduced to determine the types of SB Us and their connectivities when forming the 3-D framework structure.It has been recognized that zeolites undergo rapid structural damage under electron beam irradiation. The use of a slow-scan CCD camera to record low-dose high resolution electron microscope (HREM) images from zeolites with the combination with real-space averaging techniques has been shown to be successful in obtaining the averaged unit cell with high signal-to-noise ratio (SNR).

3-D diffraction intensities of the crotoxin complex crystal

1986 ◽  
Vol 44 ◽  
pp. 162-163
Author(s):  
B.V.V. Prasad ◽  
L.L. Degn ◽  
T.-W. Jeng ◽  
W. Chiu
Keyword(s):  
High Resolution ◽  
Intensity Data ◽  
Electron Crystallography ◽  
Data Set ◽  
Crotalus Durissus Terrificus ◽  
Cold Stage ◽  
Resolution Electron ◽  
Diffraction Patterns ◽  
Crotalus Durissus ◽  
Complex Crystal

The crotoxin complex, a neurotoxin from the venom of the Brazilian rattlesnake, Crotalus durissus terrificus, forms thin crystals suitable for high resolution electron crystallography. These crystals grow to variable completions of the unit cell, making it difficult to merge the intensity data from different crystals. This difficulty is overcome by collecting as many electron diffraction patterns (EDP) as possible from a single crystal tilted from +60° to -60°. Several series of approximately twenty EDP's were collected using a Philips 400 equipped with a cold stage at MRC, Cambridge in Dr. R. Henderson's laboratory. We report here our progress in synthesizing a 3-D intensity data set from these series.

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.

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.

Spot Scan Imaging and the Future of High Resolution

1990 ◽  
Vol 48 (1) ◽  
pp. 88-89
Author(s):  
K.H. Downing
Keyword(s):  
High Resolution ◽  
Transfer Function ◽  
Good Deal ◽  
Photographic Film ◽  
Modulation Transfer ◽  
Contrast Transfer Function ◽  
Electron Dose ◽  
High Resolution Data ◽  
Resolution Data

Electron crystallographers who have been working on determination of protein structure have set a goal of obtaining image information to a resolution of about 3.5 Å, from specimens tilted up to 60 degrees. This information would allow the construction of a three-dimensional density map within which the path of the peptide chain could be followed and locations of side chains defined. The recent determination of an atomic model of the membrane protein bacteriorhodopsin (bR) from EM data (1) which was not as complete as we would like, used a good deal of other biochemical and biophysical data to constrain the model. In cases where this type of information is not as extensive as with bR, isotropic high-resolution data would be required. Significant advances in several different areas have brought us tantalizingly close to reaching our goal, but there are still improvements to be made.The essential limitations in obtaining high resolution data from proteins arise from the radiation sensitivity of the specimen, which severely limits the electron exposure that can be used in recording an image and thus limits the signal-to-noise ratio (SNR). Increasing both the electron dose, which is possible with cold specimens, and the area processed, which required implementation of significant computer software, have each given about a factor of three improvement in SNR. Still, with conventional imaging, a study by Henderson and Glaeser (2) revealed that the best images contained only a small fraction of the signal that would be present in a perfect image. Factors such as the envelope of the contrast transfer function and the modulation transfer function of the photographic film account for some loss of contrast, but the factor causing the most loss was found to be beam-induced specimen motion. This motion results from the stress which is produced by changes in bond structure during the course of radiation damage.

Sign in / Sign up

Export Citation Format

Share Document