Aberration-corrected microscopy for structural biology applications.

This study explores the potential of a C(s)-corrected transmission electron microscope for structural studies of biological samples, in particular isolated macromolecular complexes. A 300-kV transmission electron microscope, equipped with a C(s) corrector was employed to record sets of images at different defocus and C(s) settings. The experiments were designed to determine whether imaging with large defocus benefits from C(s) correction. Defocus contrast in biological imaging has a stronger influence on image resolution than any other parameter. We find the results are in good agreement with theoretical framework, verifying that the typical imaging conditions required for biological investigations are not affected by C(s) correction.

Structural model of phospholipid-reconstituted human transferrin receptor derived by electron microscopy.

BACKGROUND: The transferrin receptor (TfR) regulates the cellular uptake of serum iron. Although the TfR serves as a model system for endocytosis receptors, neither crystal structure analysis nor electron microscopy has yet revealed the molecular dimensions of the TfR. To derive the first molecular model, we analyzed purified, lipid-reconstituted human TfR by high-resolution electron microscopy. RESULTS: A structural model of phospholipid-reconstituted TfR was derived from 72 cryo-electron microscopic images. The TfR dimer consists of a large extracellular globular domain (6.4 x 7.5 x 10.5 nm) separated from the membrane by a thin molecular stalk (2.9 nm). A comparative protein sequence analysis suggests that the stalk corresponds to amino acid residues 89-126. Under phospholipid-reconstitution conditions, the human TfR not only integrates into vesicles, but also forms rosette-like structures called proteoparticles. Scanning transmission electron microscopy revealed an overall diameter of 31.5 nm and a molecular mass of 1669 +/- 26 kDa for the proteoparticles, corresponding to nine TfR dimers. The average mass of a single receptor dimer was determined as being 186 +/- 4 kDa. CONCLUSIONS: Proteoparticles resemble TfR exosomes that are expelled by sheep reticulocytes upon maturation. The structure of proteoparticles in vitro is thus interpreted as being the result of the TfR's strong self-association potential, which might facilitate the endosomal sequestration of the TfR away from other membrane proteins and its subsequent return to the cell surface within tubular structures. The stalk is assumed to facilitate the tight packing of receptor molecules in coated pits and recycling tubuli.

Electron cryomicroscopy of two-dimensional crystals of the H(+)-ATPase from chloroplasts.

The H(+)-ATPase from spinach chloroplasts was isolated and purified. Two-dimensional crystals were obtained from the protein/lipid/detergent micelles by treatment with phospholipase and simultaneous removal of detergent and fatty acids by Biobeads. The resulting two-dimensionally ordered arrays were investigated by electron cryomicroscopy. The ordered arrays showed top view projections of CF0F1. The images were analysed by correlation averaging. In this view CF0F1 has dimensions of 11.4 x 9 nm. The average view shows a strongly asymmetric molecule, in contrast to the rather hexagonal features of CF1, previously analyzed from two-dimensional arrays. It is concluded that this is due either to an asymmetric structure and positioning of CF0 relative to CF1 or to a rearrangement of CF1 subunits induced by binding of CF0 to CF1.

Structure of the ATP synthase complex (ECF1F0) of Escherichia coli from cryoelectron microscopy.

The structural relationship of the catalytic portion (ECF1) of the Escherichia coli F1F0 ATP synthase (ECF1F0) to the intact, membrane-bound complex has been determined by cryoelectron microscopy and image analysis of single, unordered particles. ECF1F0, reconstituted into membrane structures, has been preserved and examined in its native state in a layer of amorphous ice. Side views of the ECF1F0 show the same elongated bilobed and trilobed projection of the ECF1 views shown previously to be normal to the hexagonal projection. The elongated aqueous cavity of the ECF1 is perpendicular to the membrane bilayer profile in the bilobed view. ECF1 is separated from the membrane-embedded F0 by a narrow stalk approximately 40 A long and approximately 25-30 A thick. The F0 part extends from the lipid bilayer by approximately 10 A on the side facing the ECF1. There is no clear extension of the protein on the opposite side of the membrane.

Molecular architecture of Escherichia coli F1 adenosinetriphosphatase.

The structure of the E. coli F1 ATPase (ECF1) has been studied by a novel combination of two specimen preparation and image analysis techniques. The molecular outline of the ECF1 was determined by three-dimensional reconstruction of images of negatively stained two-dimensional crystals of ECF1. Internal features were revealed by analysis of single particles of ECF1, preserved in their native state in a thin layer of amorphous ice, and examined by cryoelectron microscopy. Various projections of the unstained ECF1 were interpreted consistently with the three-dimensional structure in negative stain, yielding a more informative description of the enzyme than otherwise possible. Results show that the ECF1 is a roughly spherical complex approximately 90-100 A in diameter. Six elongated protein densities (the alpha and beta subunits, each approximately 90 A X approximately 30 A in size) comprise its hexagonally modulated periphery. At the center of the ECF1 is an aqueous cavity which extends nearly or entirely through the length of the complex. A compact protein density, located at one end of the hexagonal barrel and closely associated with one of the peripheral subunits, partially obstructs the central cavity.

The stalk connecting the F1 and F0 domains of ATP synthase visualized by electron microscopy of unstained specimens.

E. coli F1F0 ATP synthase has been reconstituted into membranes and visualized by electron microscopy of unstained samples preserved in thin layers of amorphous ice. Unlike previous observations in negative stain, these specimens are not exposed to potentially denaturing or perturbing conditions, having been rapidly frozen from well-defined conditions in which the enzyme is fully active. The structures visualized in views normal to the lipid bilayer clearly show the presence of a narrow stalk approx. 45 A long, connecting the F1 to the membrane-embedded F0.

Total number and differentiation of nucleotide binding sites on mitochondrial F1-ATPase. An approach by photolabeling and equilibrium binding studies.

In this study 3'-O-[3-(4-azido-2-nitrophenyl)propionyl]-ADP was used as a photoaffinity analog for nucleotide binding sites on nucleotide-depleted F1-ATPase. Catalytic and binding properties of the labeled enzyme were investigated. The analog behaves as a competitive inhibitor in the dark (Ki = 50 microM). Photoirradiation of F1 in the presence of the analog leads to inactivation depending linearly on the incorporation of label. Complete inactivation is achieved at a stoichiometry of 3 mol/mol F1. The label is distributed between alpha and beta subunits in a ratio of 30%:70%. Although three sites were blocked covalently by photolabeling, three reversible sites of much higher affinity than the labeled sites were preserved. Mild alkaline treatment of photoinactivated enzyme leads to almost complete reactivation which is due to hydrolysis of the 3'-ester bond and release of the ADP moiety from the covalently bound analog. The conclusions drawn are as follows. The total number of sites which can be simultaneously occupied by nucleotides on F1 is six. Adopting the finding [Grubmeyer, C. & Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727] that the high-affinity sites are the catalytic ones which can be covalently labeled by 3'-O-[5-azidonaphthoyl(1)]-ADP [Lübben, M., Lücken, U., Weber, J. & Schäfer, G. (1984) Eur. J. Biochem. 143, 483-490], it appears likely that azidonitrophenylpropionyl-ADP is a specific photolabel for the lower-affinity sites on nucleotide-depleted F1. This means that both types of sites can be differentiated by specific photoaffinity analogs. The labeled low-affinity sites interact with the catalytic sites, abolishing enzyme turnover, when steadily occupied by ADP kept in place by the covalently linking residue, which by itself has no inhibitory effect on the enzyme.

Azidonaphthoyl-ADP: a specific photolabel for the high-affinity nucleotide-binding sites of F1-ATPase.

3'-O-[5-azidonaphthoyl]-ADP has been synthesized as a photoreactive analog to 3'-O-naphthoyl(1)-ADP which is known to bind to the high-affinity nucleotide sites of mitochondrial F1-ATPase, considered to be the catalytic sites. The photolabel in the dark acts as a ligand to F1-ATPase and as a competitive inhibitor with Ki = 11 microM. Binding to the enzyme is accompanied by a quench of endogenous protein fluorescence leveling off at an occupancy of 1 mol/mol F1, whereas the total number of reversible sites accessible to the analog is 3 mol/mol F1 as measured by isotope studies. Covalent insertion by near ultraviolet activation of the probe yields labeling of both alpha and beta polypeptides of F1; it is accompanied by corresponding removal of reversible high-affinity sites for ADP or naphthoyl-ADP and by an inhibition of the enzyme; total inactivation occurs at a covalent occupancy of 2 mol/mol F1. This is the maximum number of sites accessible to covalent modification by the label; one reversible site is still available in the totally inactivated enzyme. This observation is discussed in terms of a stochastic model requiring a minimum of two interacting catalytic domains out of three in order to commence catalysis.

High-affinity binding of ADP and of ADP analogues to mitochondrial F1-ATPase.

Nucleotide-depleted F1-ATPase was prepared from beef heart mitochondria. By use of fluorescence techniques and isotope binding analyses, we investigated the occupation of the high-affinity binding sites on F1 by ADP and the ADP analogues 3'-O-(1-naphthoyl)adenosine diphosphate (N-ADP) and 3'-O-[1-(5-dimethylamino)-naphthoyl]adenosine diphosphate (DMAN-ADP). F1-ATPase was found to exhibit three binding sites for ADP (Kd = 50 nM for one site; Kd = 3 microM for the remaining two sites), two binding sites for N-ADP (Kd = 20 - 50 nM for both of the sites), and three binding sites for DMAN-ADP (Kd = 50 nM for all of the sites). Since the adenine nucleotides under consideration are bound to the same class of sites, the binding data can be explained best on the basis of the hypothesis that the binding process is anticooperative with ADP, whereas the analogues are able to overcome anticooperativity partially (N-ADP) or completely (DMAN-ADP). This binding model is consistent with the view that the exchangeable tight sites are involved directly in the catalytical process of ATP-synthesis in oxidative phosphorylation.