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We have determined the three-dimensional structure of bacteriophage SPP1 portal protein (gp6) using electron microscopy at liquid-helium temperatures and angular reconstitution. The 13-fold symmetric gp6 oligomer is a turbine-shaped structure with three distinct regions: a conical stem with a central channel; the turbine wings region; and a fringe of small 'tentacles' at the end of the channel exposed to the viral head interior. The tentacle region appears flexible and may be associated with a particular function — sensing when the correct amount of DNA has been packaged. The three-dimensional structure of the gp6 SizA mutant, which packages a smaller chromosome, reveals significant differences in that region.

This tutorial paper is the mannscript of a lecture given in the Harnack-Haus in Berlin during the antumn school 1998 on Metal Clusters. The aim of this lecture was to give some hints for quantitative electron microscopy. The interpretation of electron microscopic images depends on the mode of contrast. In particular the phase contrast is described and how it affects the images.

Lattice images of dislocations, growth boundaries and surface structure, of the n-paraffin series have been obtained by high resolution electron microscopy. The crystals were grown so that the chai...

A series of diffraction patterns from two-dimensional protein crystals of bacteriorhodopsin (purple membrane) at different temperatures (294 K, 98 K and 4 K) were recorded as the diffraction spots faded due to radiation damage. The patterns were then computationally evaluated in order to obtain a quantitative measurement of the structural preservation while irradiating the specimen. To provide statistically significant results, diffraction spots corresponding to spacings of 3A(1200 spots) and 7A(600 spots) were measured. A substantial increase of the lifetime of high resolution spots was found using liquid nitrogen as a coolant, whereas further structural preservation at liquid helium temperature was significant but smaller. It appears likely therefore that high resolution images are accessible even at liquid nitrogen temperature. Mechanical stability and the absence of thermal specimen drift are certainly of equivalent importance for successful high resolution imaging.

A high resolution projection at 2.6 A of deoxycholate-treated purple membrane using only images has been obtained with a 200 keV FEG microscope operated at liquid helium temperature. Examination of this high quality map has allowed the following conclusions to be made: Comparison between the internal structure of the trimers of the native and the deoxycholate-treated crystal forms shows that almost every detail of the structure at high resolution is identical. The cell dimension change from 62.4 A to 57.9 A is accompanied by a loss of about half the normal lipids and a 2 degrees anticlockwise rotation of the trimer as a rigid body. Three of the lipids per bacteriorhodopsin molecule remain in identical positions relative to the trimer. In addition, from the projection map together with a packing analysis using the atomic model for bacteriorhodopsin, space for three further lipids has been identified making a total of six lipids per bacteriorhodopsin molecule in this crystal form. Finally, the surprisingly small rotation of the trimer between the two crystal forms with completely different Van der Waals contacts suggest that the crystals are held together by strong, long-range electrostatic interactions.

This work examines the effect of temperature on the rate of damage caused by the electron beam in the microscope. Oriented single-crystal films of aromatic organic compounds have been used as specimens between 4 and 293 K. It is shown that the rate of damage is a function of the thickness and specimen temperature for any one compound, and that the rates of damage to the same specimen are exponentially related to reciprocal temperature. The Arrhenius plots reveal two activation energies for the degradation reactions with the higher activation energy ceasing at approximately 80 K. It would be expected that significant advantages in specimen lifetime would be obtained in specimens examined below this temperature.

In recent years, electron crystallography has been a very successful means of determining the high-resolution structure of many proteins, such as bacteriorhodopsin, the light-harvesting complex, porins (PhoE and OmpF), protein extracted from the ootheca of the praying mantis, archaebacterial surface layers, acetylcholine receptor, Ca 2+ - ATPase and gp32 ∗ l crystals. This success was based on advanced low-dose cryoelectron microscopy and a highly sophisticated digital image-processing methodology which takes into account the radiation damage caused by electron exposure. Particularly successful has been the structure determination of the purple membrane where, by combining biochemical information with the electron-microscopical density maps, the amino acid chains of bacteriorhodopshin have been localized with high precision.

Crystal structure analyses of polymethylene compounds via direct phasing methods are notoriously difficult because of the dominance of the diffraction intensity by the scattering from polymethylene lattice. However, by combination of high resolution low-dose images with conventional direct phasing via structure-invariant relationships, one can assign phase values to the most important regions of the diffraction pattern (i.e. “lamellar” and “polyethylene” reflections, respectively). As demonstrated here with experimental data from the epitaxially crystallized paraffin, n -hexatriacontane, the phased observed structure factor magnitudes produce an electrostatic-potential map with atomic positions identical to the previously published X-ray crystal structure.

The mRNA codon in the ribosomal A-site is recognized by aminoacyl-tRNA (aa-tRNA) in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here we report the 13 A resolution three-dimensional reconstruction determined by cryo-electron microscopy of the kirromycin-stalled codon-recognition complex. The structure of the ternary complex is distorted by binding of the tRNA anticodon arm in the decoding center. The aa-tRNA interacts with 16S rRNA, helix 69 of 23S rRNA and proteins S12 and L11, while the sarcin-ricin loop of 23S rRNA contacts domain 1 of EF-Tu near the nucleotide-binding pocket. These results provide a detailed snapshot view of an important functional state of the ribosome and suggest mechanisms of decoding and GTPase activation.

Ordering of a system of particles into its thermodynamically stable state usually proceeds by thermally activated mass transport of its constituents. Particularly at low temperature, the activation barrier often hinders equilibration--this is what prevents a glass from crystallizing and a pile of sand from flattening under gravity. But if the driving force for mass transport (that is, the excess energy of the system) is increased, the activation barrier can be overcome and structural changes are initiated. Here we report the reordering of radiation-damaged protein crystals under conditions where transport is initiated by stress rather than by thermal activation. After accumulating a certain density of radiation-induced defects during observation by transmission electron microscopy, the distorted crystal recrystallizes. The reordering is induced by stress caused by the defects at temperatures that are low enough to suppress diffusive mass transport. We propose that this defect-induced reordering might be a general phenomenon.

Low-dose, high-resolution electron microscopy combined with conventional direct-phasing methods based on the estimates of triplet-structure invariants are used to determine phase values for all observed electron-diffraction-structure factor magnitudes from epitaxially oriented multilamellar paracrystals of the phosphospholipid 1,2-dihexadecyl-sn-glycerophosphoethanolamine. The reverse Fourier transform of these phase-structure factors is a one-dimensional electrostatic potential map that strongly resembles the electron-density maps calculated from similar x-ray-diffraction data. Determination of the phase values for the electron-diffraction data with structure invariants alone is nearly as successful as the combined use of two separate methods, assigning values to 13 of the 16 reflections--i.e., the electrostatic potential map closely resembles the one calculated with all data.

The light-driven proton pump bacteriorhodopsin occurs naturally as two-dimensional crystals. A three-dimensional density map of the structure, at near-atomic resolution, has been obtained by studying the crystals using electron cryo-microscopy to obtain electron diffraction patterns and high-resolution micrographs. New methods were developed for analysing micrographs from tilted specimens, incorporating methods previously developed for untilted specimens that enable large areas to be analysed and corrected for distortions. Data from 72 images, from both tilted and untilted specimens, were analysed to produce the phases of 2700 independent Fourier components of the structure. The amplitudes of these components were accurately measured from 150 diffraction patterns. Together, these data represent about half of the full three-dimensional transform to 3.5 A. The map of the structure has a resolution of 3.5 A in a direction parallel to the membrane plane but lower than this in the perpendicular direction. It shows many features in the density that are resolved from the main density of the seven alpha-helices. We interpret these features as the bulky aromatic side-chains of phenylalanine, tyrosine and tryptophan residues. There is also a very dense feature, which is the beta-ionone ring of the retinal chromophore. Using these bulky side-chains as guide points and taking account of bulges in the helices that indicate smaller side-chains such as leucine, a complete atomic model for bacteriorhodopsin between amino acid residues 8 and 225 has been built. There are 21 amino acid residues, contributed by all seven helices, surrounding the retinal and 26 residues, contributed by five helices, forming the proton pathway or channel. Ten of the amino acid residues in the middle of the proton channel are also part of the retinal binding site. The model also provides a useful basis for consideration of the mechanism of proton pumping and allows a consistent interpretation of a great deal of other experimental data. In particular, the structure suggests that pK changes in the Schiff base must act as the means by which light energy is converted into proton pumping pressure in the channel. Asp96 is on the pathway from the cytoplasm to the Schiff base and Asp85 is on the pathway from the Schiff base to the extracellular surface.

High-resolution cryo-electron microscopy yielded remarkable results in biological molecular structure research. These successes encouraged to proceed in developing even better instruments and procedures. In our laboratory we built the cryo-electron microscope "SOPHIE", a modified Philips CM 20 FEG. SOPHIE stands as an acronym for Superconducting Objective in a PHIlips Electronmicroscope. The main features of this microscope are the following: 1.200kV acceleration voltage2.Schottky field emitter3.Helium cooled superconducting objective The Helium-cooled cryo-objective from the former microscope SULEIKA was adapted into the column of the original Philips CM20 FEG [Fig. 1], This new SOPHIE microscope, thus, combines two important features: a modern computer controlled electron microscope with a high coherent beam source and the proven advantages of the superconducting cryo-objective. The advantage of the field emission gun at 200 kV over a thermionic cathode at 100 kV can be best visualized while comparing the respective phase-contrast-transfer functions at Scherzer focus [Fig.2]. Moreover, SOPHIE is equipped with the small spot scanning procedure.

The process of electron beam irradiation of orthorhombic monolamellar microcrystals of the n-paraffin n-tetratetracontane cooled < 15K is monitored by changes in the crystal structure to give a much clearer view of the beam damage process than is afforded by plots of diffraction intensity variation. The observed structural change in the chains, observed in the projection down the chain axes as an increasingly circular cross-section, is consistent with previous structural analyses of structural damage in an orthogonal view and also other observations of conformational flexibility of long chains induced by trans-vinylene groups. Nevertheless, within the 2.5 A resolution available for direct lattice images (reported elsewhere), there is virtually no detectable structural change at the 10 e A-2 electron dose used for these exposures.

Improvements in technique have produced electron micrographs of purple membrane that provide, after computer analysis, reproducibly measurable diffraction peaks extending to 2.8 A (1 A = 0.1 nm). The improvements include better specimen preparation, a more stable cryo-electron microscope with better alignment and the addition of an image-processing step, which gives weights to local areas of the image according to the local strength of the periodic component of the image. These improvements have enabled the calculation of a directly phased projection map at 2.8 A resolution.

Porin is an integral membrane protein that forms channels across the outer membrane of Escherichia coli. Electron microscopic studies of negatively stained two-dimensional porin crystals have shown three stain accumulations per porin trimer, revealing the locations of pores spanning the membrane. In this study, reconstituted porin lattices embedded in glucose were investigated using the low-dose technique on a cryo-electron microscope equipped with a helium-cooled superconducting objective lens. The specimen temperature was maintained at 5 K to yield an improved microscopic and specimen stability. Under these conditions, we obtained for the first time electron diffraction patterns from porin lattices to a resolution of 3.2 A and images showing optical diffraction up to a resolution of 4.9 A. Applying correlation averaging techniques to the digitized micrographs, we were able to reconstruct projected images of the porin trimer to a resolution of up to 3.5 A. In the final projection maps, amplitudes from electron diffraction and phases from these images were combined. The predominant feature is a high-density narrow band (about 6 A in thickness) that delineates the outer perimeter of the trimer. Since the molecule consists of almost exclusively β-sheet structure, as revealed by spectroscopic data, we conclude that this band is a cylindrical β-pleated sheet crossing the membrane nearly perpendicularly to its plane. Another intriguing finding is a low-density area (about 70 A2) situated in the centre of the trimer.

Electron micrographs of the purple membrane have been recorded using liquid nitrogen and liquid helium cooling on three cryoelectron microscopes. The best micrographs show optical diffraction spots, arising from the two-dimensional crystal, out to resolutions of around 6 A. Large areas of several of these micrographs have been analysed using a procedure which determines the strength of the very weak high resolution Fourier components of the image of the crystal. The procedure consists of reciprocal space filtering followed by real space correlation analysis to characterise image distortions, removal of the distortions by interpolation, and finally extraction of the amplitudes and phases of the Fourier components from the distortion-corrected image of the crystal. These raw image amplitudes and phases are then used, together with previously measured amplitude and phase information, to refine the beam tilt and crystal tilt, phase origin and amount of defocus and astigmatism of the image. The phases can then be corrected for the effects of the contrast transfer function, beam tilt and phase origin. The amplitudes of all the spots which are expected to be strong from their known electron diffraction intensity are observed to be significantly above the background noise level, and the independent phases from different images, and from symmetry-related directions in the same image, show excellent agreement out to a resolution of 3.5 A. Although only images from untilted or slightly tilted (

Crotoxin complex forms thin crystals which are suitable for electron crystallographic analysis. We have used a 100 kV electron microscope equipped with a superconducting lens to image this crystal embedded in glucose. Optical diffraction analysis of the micrographs show unambiguously a structural resolution of 3.9 A which has not been obtained with the conventional microscope at room temperature. A density map with a nominal resolution of 3.5 A has been synthesized from these images by computer processing techniques.

Phase-contrast noise inherent in high resolution bright field images can be reduced considerably by using hollow-cone illumination instead of the usual axial illumination. It is shown by experiment that there are definite values of hollow-cone angle and defocus for imaging heavy-atom structures on an amorphous carbon film with minimum disturbing noise. The imaging is distinguished by a point-spread function whose side lobes extend over only a short range. Thus the present results may be considered as apodization in phase-contrast electron microscopy. The method is advantageous in investigating atomic structures where phase-contrast noise is a limiting factor for the visualization of high resolution details.

The effect of radiation exposure on visual recognition of individual macromolecules of the protein protamine was examined using high resolution electron microscopy in scanning transmission, fixed beam transmission, dark field and bright field, and recording of images on plates or via video imaging systems including image intensifiers and digital image storage. Loss of recognition of protamine, including its 5 A substructure, followed approximately three-hit kinetics with a D 0 on the exponential portion of the curve of about 470 e/A 2 . In spite of the inevitable chemical damage at high doses, virtually all molecules in orientations that exhibited the characteristic protamine structure could still be recognized in dark field scanning transmission or fixed beam bright field at 100–200 e/A 2 . Recognition fell from 30 to 6% when doses were increased from 1000 to 2000 e/A 2 in fixed beam dark field. Exposure rates, varied over seven orders of magnitude from 0.15 e/A 2 -s to 3 × 10 e/A 2 -s, had no effect at all on the recognition of structures.

A liquid helium-cooled cryoelectron microscope, operated to expose the specimen to only a very low electron dose, was used to obtain structural images of monolamellar n-tetratetracontane (n-C44H90) crystals at 0.25-nanometer resolution. These results are in contrast to earlier predictions that such extremely beam-sensitive materials could not be studied directly at this level of detail. Analysis of the resultant lattice images gives direct evidence for crystal bending as well as direct visualization of edge dislocations in this material.

Porin (ompF gene product), an integral channel-forming protein in the outer membrane of Escherichia coli, is regarded as a molecular sieve allowing non-specific passive diffusion for low-molecular-weight (Mr < 700d) hydrophilic solutes.Electron microscopic studies of the negatively stained membrane vesicles reconstituted with porin and phospholipids have shown the presence of the stain-filled triplet indentation. Subsequent 3D image reconstruction demonstrated that three channels on the outer surface of the cell merge into a single channel at the periplasmatic face. Both 2D and 3D image reconstructions, based on electron micrographs from negatively stained preparations, are very informative about the size and distribution of the transmembrane channels but they contain very little or no information on the protein trimers which form the channels. This lack of information about the trimer structure and location is not surprising since the diffraction resolution limit, in our previous studies, is around 22Å.

Porin (OmpF) is one of the major outer membrane proteins of Escherichia coli /1/. This integral membrane protein functions as a molecular sieve for hydrophilic molecules with an exclusion limit of around 600 dalton /2/.For our investigation, purified porin was reconstituted in di-myristoyllecithin vesicles, where it formed 2D-lattices. The crystallinity was considerably improved by subsequent treatment with phospholipase. Electron microscopy was done on the “Suleika”, a cryo-electron microscope which permits low-dose imaging under “cryo-protection” conditions (4K) /3/. The embedding medium was glucose, which allows the visualisation of integral membrane proteins at high resolution /4/. Under these conditions, we obtained several electron micrographs showing optical diffraction of up to 0.8nm resolution. Image prossessing was performed by using the correlation averaging method to overcome the imperfections of the porin lattice. Only about 80% of the lattice shows well defined cross-correlation peaks which are marked in Figure 1. Dislocations along the lattice lines are clearly discernable. The averaged image of the porin trimer (Fig. 2 ) was built up from unit cells corresponding to positions with correlation peaks above the noise level.

The secondary structure of porin, which form voltage-dependent channels across outer membrane of Escherichia coli, consists mostly (two-thirds of polypeptides) of anti-parallel (β-pleated strands. However, information on the tertiary and quaternary structure of porin is still far from complete. The β-pleated strands, which have a mean length of 10-12 residues, span the outer membrane in an orientation approximately perpendicular to the membrane surface. Porins are so far the only class of membrane proteins for which the predominant β-structure has been established unequivocally. However, the exact arrangement of β-strands (β- barrels or stacked sheets) is still unknown. Depending on the method of investigation, one being the turn identification, and the second the Raman spectroscopy, 16 or 18 β-strands respectively were calculated as a building block required for one protein monomer. Both models predict the β-strands delineating the pore, as amphipatic molecules with hydrophobic residues forming protein-lipid interface and hydrophilic residues facing the lumen of the pore.