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We have developed two software packages for the processing of electron diffraction patterns acquired on a slow-scan CCD camera. Automated indexing and processing of these electron diffraction patterns is possible using a program called AUTO. To obtain a set of reliable reflections, AUTO applies a number of corrections, which are necessary due to the acquisition of the patterns with a slow-scan CCD camera. To index and process unknown lattices in electron diffraction patterns obtained from, for instance, a new protein or a new crystal form, an interactive program called EDP is available. It supports indexing in an interactive manner through a graphical user interface. Processing of the electron diffraction pattern is done using a script created by EDP. Both packages run on UNIX platforms, with EDP requiring the X Window System.

A Gatan 1024 × 1024 slow-scan charge-coupled device (CCD) camera has been interfaced to a JEOL4000EX electron cryomicroscope and explored for its usefulness in the electron crystallographic analysis of thin, glucose-embedded crystals of crotoxin complex kept at -125°C. We show that the camera allows for an on-line assessment of the crystals' crystallinity, flatness, and thickness. Intensities obtained from electron diffraction patterns acquired with the camera have been statistically analyzed and were found to be consistent with theoretically expected values. A quantitative analysis of the diffraction intensity as function of the accumulated electron dose suggests the possibility of recording up to 250 diffraction patterns with 3.5 A resolution from a single crotoxin complex crystal 128 A thick. Tilt series of 125 electron diffraction patterns with 3.5 A data acquired from a single crystal are shown to be practically feasible. The current study demonstrates for the first time the effectiveness of using a slow-scan CCD camera for electron diffraction data collection from thin protein crystals at near atomic resolution.

Electron energy loss spectroscopy and dark-field imaging in a scanning transmission electrton microscope were used to determine the thickness of glucose-embedded crotoxin complex crystals. The results demonstrate the feasibility of identifying protein crystals with a thickness of half a unit cell (12.8 nm) under low-dose and low-temperature conditions. The accuracy of this method is limited by the amount of surface coating of the crystal's embedding glucose used for preserving the high-resolution structure of the protein. The histogram of the crystal thickness distribution and the spread of the anticipated crystal thickness allow us to make an estimate of the uncertainty in the glucose layer thickness. This approach can be incorporated as part of the experimental procedure in the three dimensional data collection for structure determination of protein crystals with variable thicknesses. The measurement can be done on areas approximately 200 nm in diameter so that crystals of suitable thickness can be pre-selected before the high-resolution data is recorded. Accurate determination of the crystal thickness will optimize the data collection efficiency by avoiding the collection and subsequent analysis of unmatchable data for the three-dimensional reconstruction.

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.

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.).

In protein electron crystallography, both low dose electron diffraction patterns and images are needed to provide accurate amplitudes and phases respectively for a 3-dimensional reconstruction. We have demonstrated that the Gatan 1024x1024 model 679 slow-scan CCD camera is useful to record electron diffraction intensities of glucose-embedded crotoxin complex crystal to 3 Å resolution. The quality of the electron diffraction intensities is high on the basis of the measured intensity equivalence ofthe Friedel-related reflections. Moreover, the number of patterns recorded from a single crystal can be as high as 120 under the constraints of radiation damage and electron statistics for the reflections in each pattern.A limitation of the slow-scan CCD camera for recording electron images of protein crystal arises from the relatively large pixel size, i.e. 24 μm (provided by Gatan). The modulation transfer function of our camera with a P43 scintillator has been determined for 400 keV electrons and shows an amplitude fall-off to 0.25 at 1/60 μm−1.

There is an ever present need in electron microscopy of biological specimens to improve the quality of the recorded data to approach the atomic resolution capability of the modem instruments. The method adopted which has yielded the only significant advance in this direction is to directly phase theelectron diffraction amplitudes of 2-dimensional crystals using the Fourier transforms of high resolution images. Cryo methods have improved the preservation and stability of the specimen to the relatively high electron dose irradiation which is required for imaging compared to electron diffraction. In the cases, however, where the actual power of the structure factors was compared with those that were transferred by the microscope’s imaging system, a large attenuation was measured. Spot-scan imaging has been shown to improve the image quality by reducing the beam-induced movement. Charging of the specimen appears to cause similar deleterious effects on image resolution, but it requires different methods aimed at resolution improvement. Proteins are insulators and because of secondary electron loss they will become positively charged during irradiation. Defocused diffraction brightfield imagescan display the charged nature of an irradiated specimen as an apparent increase in mass. A comparison wasmade of the degree of charging and discharging between using 100 and 400 keV electrons on streptavidin crystals in glucose and catalase crystals in amorphous ice. We show that when the region of the irradiated sample includes part of the supporting carbon film, the charge build-up is prevented.

In three-dimensional structure determination of macromolccules by electron crystallography it is necessary to combine diffraction patterns and images recorded at various tilt angles from different crystals. In order to merge these data sets successfully all the crystals should have the same thickness. It has been proposed that parallel electron energy loss spectroscopy (EELS) might provide a useful means of assessing the thickness of a beam-sensitive organic crystal prior to recording the high-resolution structural data. This can be achieved by measuring the fraction of the total transmitted electrons that do not lose energy, i.e., the zero-loss intensity. If Izis the integrated zero-loss intensity and Itotis the total integrated intensity in the energy loss spectrum, then the specimen thickness, t, is given in terms of the total inelastic mean free path, λi, by, t/λi= ln(Itot/Iz). Results recently obtained fromn-paraffin crystals have shown that it is feasible to determine the number of unit cell layers under low electron dose conditions.

Protein electron crystallography can provide a reconstruction detailed enough for the polypeptide chain of a membrane protein to be traced (1). However, there remain a number of technical problems associated with this technique that must be overcome before it can become a routine tool for high resolution three-dimensional structural analysis of protein crystals. A 400 kV electron microscope has been demonstrated to be advantageous for enhancement of resolution and contrast in images of protein crystals (1,2) because of a reduction in chromatic aberration effects. This paper addresses the question of experimental strategy in data collection for thin protein crystals with a 400 kV cryo-microscope. We will use crotoxin complex and T4 DNA helix destabilizing protein as examples of thin crystals to illustrate our approach.Crotoxin complex forms highly ordered crystals with different thicknesses among different crystals. In order to simplify sorting out data from the various crystals for 3-dimensional merging, it is desirable to collect as much data as possible from a single crystal.

Two-dimensional crystals of bovine NADH: ubiquinone oxidoreductase fragments were studied by cryo-electron microscopy and computer image analysis at two focus settings. The lattice parameters of the crystals were determined as a =14.5±0.2 nm , b =14.8±0.3 nm with y =87.8°±0.2°. Translation and rotation alignment resulted in a final resolution of 1.38 (close-to-focus) or 1.9 nm (1.3 μm underfocus). When only translational alignment was employed 1.6 nm resolution resulted (close-to-focus case). The application of a further correction procedure surprisingly lowered the achieved resolution to 2.2 nm. A combination of the projected structures for the two focus settins in Fourier space resulted in a noise-free image displaying enhanced image contrast for reciprocal spacings from 1/5.0 to 1/1.4 nm -1 . The structure in amorphous ice comprises four separate quasi-identical motifs, each resembling a distorted triangle. Two symmetry-related (p2) motifs reveal a pore-like feature. In negative stain the structure has p4 mm symmetry. The absence of in-plane symmetry elements (i.e. twofold rotation and screw axes) and other differences might be accounted for (at least in part) if the crystal studied was slightly tilted.

The results of Section IV can be summarized in a simple ATP synthase model. This model implies that either the alpha or the beta subunits must be closer to the membrane. The work of Gao and Bauerlein (1987) indicates that the alpha subunits are closer to the membrane. Although the overall structure is more or less clear, important questions need to be clarified. First, the number and the arrangement of the subunits in the F0 part must be known. Second, the exact shape of F1, and particularly the shape of the large subunits needs to be elucidated. On the basis of fluorescence resonance energy transfer measurements by McCarty and Hammes (1987), a model was presented showing large oblong subunits. Such 'banana-shaped' subunits, which are also presented in the many phantasy models (e.g. Walker et al., 1982), are very unlikely in view of the electron microscopical results, although the large subunits do not need to be exactly spherical. The third and most interesting central question is on the changes in the structure that take place during the different steps in the synthesis of ATP. It can now be taken as proven that the energy transmitted to the ATP synthase is used to induce a conformational change in the latter enzyme, in such a way as to bring about the energy-requiring dissociation of already synthesized ATP (Penefsky, 1985 and reviewed in Slater, 1987). But the way in which the three parts of the ATP synthase are involved is completely unknown. It is rather puzzling that such a long distance exists between the catalytic sites, which are on the interface of the alpha and beta subunits and the F0 part where the proton movements occur, which, according to Mitchell's theory (1961), is the driving force for the synthesis of ATP. Perhaps alternative mechanisms such as the collision hypothesis formulated by Herweijer et al. (1985) are more realistic in describing the mechanism of ATP synthesis. It would bring the complexes I and V close together, not only in the artificial way treated in this paper, but in a useful way for energy conversion.

We investigated the structure of two-dimensional crystals from bovine heart mitochondrial NADH: ubiquinone oxidoreductase. A detailed description of uranyl acetate-stained crystals demonstrated that they are composed of fragments in a spatial arrangement according to space group P42 1 2 [J. Brink, S. Hovmoller, C.I. Ragan, M.W.J. Cleeter, E.J. Boekema and E.F.J. van Bruggen, European J. Biochem. 166 (1987) 287]. To gain more structural information on the crystal structure and to assess the effects of various negative stains on the structure preservation and appearance, we examined stained crystals by means of electron microscopy and image analysis. The space group P42 1 2 appeared to be present for several stains tested, i.e. ammonium molybdate, uranyl acetate, uranyl nitrate and uranyl sulphate. Use of phosphotungstic acid and silicotungstate resulted in a reduction of symmetry to pseudo-P42 1 2 or p 4 . Use of sodium tungstate led to a considerable loss of resolution to 3.8 nm at best, whereas otherwise 1.5 to 1.9 nm could be demonstrated. The lattice vectors were not affected by the stains; they were determined as a = b = 14.9 ± 0.25 nm with γ =89.8° ± 0.6°. Image analysis showed the presence of similar structures with the molybdate and uranyl compounds. Differences were observed in the case of the tungstate type of stains. Furthermore, the analysis revealed the complete absence of the four small pores of 2.0 nm diameter in the unit cell. This effect was observed irrespective of the type of stain and supporting film, and could be ascribed only to the glow-discharge treatment of the supporting film. The observed difference must be caused by changed interactions between the protein, stain and supporting film. Application of correspondence analysis and clustering algorithms to the various reconstructed images of the crystals showed that they could be separated into several clusters. Each of these clusters corresponded on the average to only one type of stain, whereas a further division according to the specific uranyl compounds was observed. This study therefore shows that under identical preparation conditions subtle differences between individual stains can be detected.

We have investigated the structure of two-dimensional crystals from preparations of NADH:ubiquinone oxidoreductase from beef-heart mitochondria. The crystal structure of these crystals was previously determined to be equivalent with two native enzyme molecules per unit cell, i.e. a p2 symmetry [Boekema, E. J., Van Heel, M. G. & Van Bruggen, E. F. J. (1984) Biochim. Biophys. Acta 787, 19-26]. However, the optical diffraction patterns of the crystals displayed a clear fourfold symmetry. A Fourier analysis carried out on the calculated diffraction pattern proved unambiguously that the crystal symmetry was p42(1)2. Following crystallographic rules the unit cell therefore contained eight identical molecules. As a consequence, only a subcomplex of the enzyme rather than the intact enzyme formed the crystal. Electron microscopy of isolated, single molecules of the iron-sulphur protein, a dissociation product of complex I, revealed the presence of square complexes with sides of approximately 15 nm. Since these complexes were indistinguishable from the building blocks (unit cells) of the two-dimensional crystals, the crystals could be composed of Fe-S protein fragments only. The nature of the fragments in the unit cell was probed by immuno-labelling with monovalent antibodies (Fab's), raised against the 75-kDa subunit from the Fe-S protein, followed by image analysis. We found at least four binding sites for the anti-(75-kDa subunit) Fab per unit cell, indicating the presence of at least four copies of the antigen. In order to account for these observations we postulate the hypothesis that the two-dimensional crystals obtained from complex I are composed of iron-sulphur protein molecules in an octameric arrangement.

The crotoxin complex is a potent neurotoxin composed of a basic subunit (Mr = 12,000) and an acidic subunit (M = 10,000). The basic subunit possesses phospholipase activity whereas the acidic subunit shows no enzymatic activity at all. The complex's toxocity is expressed both pre- and post-synaptically. The crotoxin complex forms thin crystals suitable for electron crystallography. The crystals diffract up to 0.16 nm in the microscope, whereas images show reflections out to 0.39 nm2. Ultimate goal in this study is to obtain a three-dimensional (3D-) structure map of the protein around 0.3 nm resolution. Use of 100 keV electrons in this is limited; the unit cell's height c of 25.6 nm causes problems associated with multiple scattering, radiation damage, limited depth of field and a more pronounced Ewald sphere curvature. In general, they lead to projections of the unit cell, which at the desired resolution, cannot be interpreted following the weak-phase approximation. Circumventing this problem is possible through the use of 400 keV electrons. Although the overall contrast is lowered due to a smaller scattering cross-section, the signal-to-noise ratio of especially higher order reflections will improve due to a smaller contribution of inelastic scattering. We report here our preliminary results demonstrating the feasability of the data collection procedure at 400 kV.Crystals of crotoxin complex were prepared on carbon-covered holey-carbon films, quench frozen in liquid ethane, inserted into a Gatan 626 holder, transferred into a JEOL 4000EX electron microscope equipped with a pair of anticontaminators operating at −184°C and examined under low-dose conditions. Selected area electron diffraction patterns (EDP's) and images of the crystals were recorded at 400 kV and −167°C with dose levels of 5 and 9.5 electrons/Å, respectively.