Electron Microscopy Of Two-dimensional Crystals Research Articles

Burial of the Polymorphic Residue 129 in Amyloid Fibrils of Prion Stop Mutants

Misfolding of the natively α-helical prion protein into a β-sheet rich isoform is related to various human diseases such as Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome. In humans, the disease phenotype is modified by a methionine/valine polymorphism at codon 129 of the prion protein gene. Using a combination of hydrogen/deuterium exchange coupled to NMR spectroscopy, hydroxyl radical probing detected by mass spectrometry, and site-directed mutagenesis, we demonstrate that stop mutants of the human prion protein have a conserved amyloid core. The 129 residue is deeply buried in the amyloid core structure, and its mutation strongly impacts aggregation. Taken together the data support a critical role of the polymorphic residue 129 of the human prion protein in aggregation and disease.

Phosphorylation and Mutation Induce an Order-To-Disorder Transition in the Cytoplasmic Domain of Phospholamban

Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We have studied this interaction using electron microscopy of two-dimensional crystals of SERCA in complex with phospholamban. In previous studies, phospholamban oligomers were found interspersed between SERCA dimer ribbons and a three-dimensional model was constructed to show interactions with SERCA. In the present study, we have examined the effects of phosphorylation and mutation of phospholamban on the interaction with SERCA in the two-dimensional crystals. Based on projection maps from negatively-stained and frozen-hydrated crystals, phosphorylation of Ser16 selectively disordered the cytoplasmic domain of wild-type phospholamban. This was not the case for a pentameric gain-of-function mutant (Lys27-to-Ala), which retained inhibitory activity and remained ordered in the phosphorylated state. A partial loss-of-function mutation that altered the charge state of phospholamban (Arg14-to-Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34-to-Ala) was disordered. The functional state of phospholamban correlated with an order-to-disorder transition of phospholamban's cytoplasmic domain in the two-dimensional co-crystals. Furthermore, the residues studied (Ser16, Lys27 and Asn34) may implicate domain Ib of phospholamban in the order-to-disorder transition. In summary, the two-dimensional co-crystals with SERCA require a functional pentameric form of phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the phospholamban monomer for the inhibitory association. Phosphorylation or mutation of phospholamban alters the SERCA-pentamer interaction in a manner normally associated with inhibition by the monomer.

The Structure of a Putative Scaffolding Protein of Immature Poxvirus Particles as Determined by Electron Microscopy Suggests Similarity with Capsid Proteins of Large Icosahedral DNA Viruses

Orf virus, the prototype parapoxvirus, is responsible for contagious ecthyma in sheep and goats. The central region of the viral genome codes for proteins highly conserved among vertebrate poxviruses and which are frequently essential for viral proliferation. Analysis of the recently published genome sequence of orf virus revealed that among such essential proteins, the protein orfv075 is an orthologue of D13, the rifampin resistance gene product critical for vaccinia virus morphogenesis. Previous studies showed that D13, arranged as "spicules," is necessary for the formation of vaccinia virus immature virions, a mandatory intermediate in viral maturation. We have determined the three-dimensional structure of recombinant orfv075 at approximately 25-A resolution by electron microscopy of two-dimensional crystals. orfv075 organizes as trimers with a tripod-like main body and a propeller-like smaller domain. The molecular envelope of orfv075 shows unexpectedly good agreement to that of a distant homologue, VP54, the major capsid protein of Paramecium bursaria Chlorella virus type 1. Our structural analysis suggests that orfv075 belongs in the double-barreled capsid protein family found in many double-stranded DNA icosahedral viruses and supports the hypothesis that the nonicosahedral poxviruses and the large icosahedral DNA viruses are evolutionarily related.

Three-Dimensional Reconstruction of Protein Kinase C-delta and Its Regulatory Domain by Electron Microscopy of Two-Dimensional Crystals

Three-Dimensional Reconstruction of Protein Kinase C-delta and Its Regulatory Domain by Electron Microscopy of Two-Dimensional Crystals

Specimen Charging on Thin Films with One Conducting Layer: Discussion of Physical Principles

Although the most familiar consequences of specimen charging in transmission electron microscopy can be eliminated by evaporating a thin conducting film (such as a carbon film) onto an insulating specimen or by preparing samples directly on such a conducting film to begin with, a more subtle charging effect still remains. We argue here that specimen charging is in this case likely to produce a dipole sheet rather than a layer of positive charge at the surface of the specimen. A simple model of the factors that control the kinetics of specimen charging, and its neutralization, is discussed as a guide for experiments that attempt to minimize the amount of specimen charging. Believable estimates of the electrostatic forces and the electron optical disturbances that are likely to occur suggest that specimen bending and warping may have the biggest impact on degrading the image quality at high resolution. Electron optical effects are likely to be negligible except in the case of a specimen that is tilted to high angle. A model is proposed to explain how both the mechanical and electron-optical effects of forming a dipole layer would have much greater impact on the image resolution in a direction perpendicular to the tilt axis, a well-known effect in electron microscopy of two-dimensional crystals.

Crystal and Electron Microscopy Structures of Sticholysin II Actinoporin Reveal Insights into the Mechanism of Membrane Pore Formation

Sticholysin II (StnII) is a pore-forming protein (PFP) produced by the sea anemone Stichodactyla helianthus. We found out that StnII exists in a monomeric soluble state but forms tetramers in the presence of a lipidic interface. Both structures have been independently determined at 1.7 Å and 18 Å resolution, respectively, by using X-ray crystallography and electron microscopy of two-dimensional crystals. Besides, the structure of soluble StnII complexed with phosphocholine, determined at 2.4 Å resolution, reveals a phospholipid headgroup binding site, which is located in a region with an unusually high abundance of aromatic residues. Fitting of the atomic model into the electron microscopy density envelope suggests that while the β sandwich structure of the protein remains intact upon oligomerization, the N-terminal region and a flexible and highly basic loop undergo significant conformational changes. These results provide the structural basis for the membrane recognition step of actinoporins and unexpected insights into the oligomerization step.

Cryo-atomic Force Microscopy of Unphosphorylated and Thiophosphorylated Single Smooth Muscle Myosin Molecules

The purpose of this study was to determine whether steric blockage of one head by the second head of native two-headed myosin was responsible for the inactivity of nonphosphorylated two-headed myosin compared with the high activity of single-headed myosin, as suggested on the basis of electron microscopy of two-dimensional crystals of heavy meromyosin (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385-1390; and Wendt, T., Taylor, D., Trybus, K. M., and Taylor, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4361-4366). Our earlier cryo-atomic force microscopy (cryo-AFM) (Zhang, Y., Shao, Z., Somlyo, A. P., and Somlyo, A. V. (1997) Biophys. J. 72, 1308-1318) indicates that thiophosphorylation of the regulatory light chain increases the separation of the two heads of a single myosin molecule, but the thermodynamic probability of steric hindrance by strong binding between the two heads was not determined. We now report this probability determined by cryo-AFM of single whole myosin molecules shown to have normal low ATPase activity (0.007 s-1). We found that the thermodynamic probability of the relative head positions of nonphosphorylated myosin was approximately equal between separated heads as compared with closely apposed heads (energy difference of 0.24 kT (where k is a Boltzman constant and T is the absolute temperature)), and thiophosphorylation increased the number of molecules having separated heads (energy advantage of -1.2 kT (where k is a Boltzman constant and I is the absolute temperature)). Our results do not support the suggestion that strong binding of one head to the other stabilizes the blocked conformation against thermal fluctuations resulting in steric blockage that can account for the low activity of nonphosphorylated two-headed myosin.

Heme, chlorophyll, and bilins: Methods and protocols: Smith, A. G., Witty, M. (eds.)

Heme, Chlorophyll, and Bilins: Methods and Protocols Smith, A. G., Witty, M. (eds.); Humana Press, Totowa, New Jersey, 2001, 340 pp., ISBN 1-588-29-111-1, $112.50. This book contains strategies, detailed procedures, and practical advice for the (bio)chemical characterization of heme, chlorophyll, and bilin. Moreover, the physicochemical and biochemical properties of hemoproteins, chlorophyll proteins, phytochromes, and phycobiliproteins are reported. Tetrapyrrole (bio)chemistry includes methods for the study of tetrapyrroles, synthesis of tetrapyrroles, enzymatic preparation of tetrapyrrole intermediates, and analysis of biosynthetic intermediates. Remarkably, the (bio)chemical characterization of well known heme and hemo-protein model systems, such as tetraphenylporphyrin and octaethylporphyrin, is given, with particular emphasis on solubility, crystallization, and reconstitution of tetrapyrroles. Heme and hemoprotein (bio)chemistry deals with heme, as well as wild-type and recombinant hemoproteins characterization, with particular emphasis on combined use of spectroscopic techniques, such as UV-visible, magnetic circular dichroism, resonance Raman, and NMR. Detailed information concerning the determination of the hemoprotein structure by electron microscopy of two-dimensional crystals is reported. Chlorophyll and chlorophyll protein (bio)chemistry includes synthesis, analysis of intermediates and end products of the chlorophyll (bio)synthetic pathway, and analysis, reconstitution, and crystallization of chlorophyll proteins. Spectroscopic properties of chlorophylls and related (bio)synthetic intermediates are reported in detail. The analysis and reconstitution of phytochrome concerns spectroscopy for quantitation and assembly detection of holophytochrome. Step-by-step protocols for purification of bilins and phycobiliproteins, as well as re-constitution of holoproteins, are given. This is a specialized collection of methods and protocols that should be very useful for novices in the field. Indeed, protocols are optimized in terms of time-saving techniques, tricks, and useful tips. However, anyone who uses this book will still need access to standard works and specialized publications such as Methods in Enzymology. Finally, the editors could have made the compilation more useful from the educational viewpoint if general aspects of heme, chlorophyll, and bilin (bio)chemistry were expanded. A book like this would also benefit if protocols and methods were schematized and made available through the web.

Principles of protein X-ray crystallography (2nd edition). By Jan Drenth. Heidelberg: Springer–Verlag, 1999, pp. xv + 341. Price DM129.00. ISBN 0 387 98587 5.

Those who liked the ®rst edition of this book will be pleased with the changes in the second. It contains the same 15 chapters of the ®rst edition, with very similar section and chapter headings. Signi®cant advances in the ®eld have been added: `Cryocooling' has been added to chapter 1 (`Crystallizing a protein'), `Weighted averaging of multiplere®ned dummy atomic models' to chapter 8 (`Phase Improvement') and `Shake-andBake' as well as the `Principle of Maximum Entropy' have been added to chapter 11 (`Direct Methods'). Correspondingly, discussion of equipment or procedures less frequently used today has been reduced or omitted: `The Precession Camera' is gone from chapter 2 (`X-ray Sources and Detectors'), `The Projection of Electron Density along an Axis' is replaced by `Scattering by a Plane of Atoms' in chapter 4 (`The Theory of X-ray Diffraction by a Crystal') and `The Matthews Patterson Summation', `The Rossman Method' and `Other Common Origin Methods' have been dropped from chapter 7 (`The Solution of the Phase Problem by the Isomorphous Replacement Method'). This second edition is an appropriate text for a graduate course in macromolecular crystallography as well as being a valuable reference for the laboratory. The style continues informal and clear. Drenth often uses the second person when `addressing' his students. Terms or concepts that must be used before full de®nition or clari®cation are noted and referenced. The text is well prioritized, if not intended to treat each topic in great depth; it usually provides enough mathematical or physical background to show the plausibiliy of a procedure. Adequate references are given to original literature, though some would have bene®ted by being updated from those used in the ®rst edition. The book is well written, illustrated and indexed. Inevitably, individual instructors might choose to shift emphasis or to add a few topics. Growing crystals is now often the rate-limiting step in structure determination. The relevance of the second virial coef®cient in exploring crystallization conditions, the rationale for crystal screens, the rate of approach to saturation and the rationale for crystallization of integral membrane proteins might have been treated in greater detail. Others may want an elaboration of the interface of X-ray crystallography with neutron and electron crystallography and with electron microscopy of two-dimensional crystals. Some might argue that a course in protein crystallography should include helical diffraction theory. The rationale of and tricks for solving difference Patterson functions would be helpful. The use of charge-coupled device X-ray detectors and of bent mirrors for monochromatization and focusing might be elaborated. However, at 341 pages, Principles is a small book. It re ects a consensus of the core of basic protein crystallography courses as taught in many universities around the world.

Three-dimensional Structure of Higher Plant Photosystem I Determined by Electron Crystallography

We describe the three-dimensional structure of higher plant photosystem I (PSI) as obtained by electron microscopy of two-dimensional crystals formed at the grana margins of thylakoid membranes. The negatively stained crystalline areas displayed unit cell dimensions a = 26.6 nm, b = 27.7 nm, and gamma = 90(o), and p22121 plane group symmetry consisting of two monomers facing upward and two monomers facing downward with respect to the membrane plane. Higher plant PSI shows several structural similarities to the cyanobacterial PSI complex, with a prominent ridge on the stromal side of the complex. The stromal ridge is resolved into at least three separate domains that are interpreted as representing the three well characterized stromal subunits, the psa C, D, and E gene products. The lumenal surface is relatively flat but exhibits a distinct central depression that may be the binding site for plastocyanin. Higher plant PSI is of dimensions 15-16 x 11-12.5 nm, and thus leaves a larger footprint in the membrane than its cyanobacterial equivalent (13 x 10.5 nm). It is expected that additional membrane-bound polypeptides will be present in the higher plant PSI. Both higher plant and cyanobacterial complexes span about 8-9 nm in the direction orthogonal to the membrane. This report represents the first three-dimensional structure for the higher plant PSI complex.

Conformational Changes in the Mitochondrial Channel Protein, VDAC, and Their Functional Implications

The voltage-dependent, anion-selective channel (VDAC) is generally considered the main pathway for metabolite diffusion across the mitochondrial outer membrane. It also interacts with several mitochondrial and cytosolic proteins, including kinases and cytochrome c. Sequence analysis and circular dichroism suggest that the channel is a bacterial porin-like β-barrel. However, unlike bacterial porins, VDAC does not form tight trimeric complexes and is easily gated (reversibly closed) by membrane potential and low pH. Circular dichroism indicates that the protein undergoes a major conformational change at pH <5, involving decreased β-sheet and increased α-helical content. Electron microscopy of two-dimensional crystals of fungal VDAC provides direct information about the size and shape of its lumen and suggests that the N-terminal domain forms a mobile α-helix. It is proposed that the N-terminal domain normally resides in a groove in the lumen wall and that gating stimuli favor its displacement, destabilizing the putative β-barrel. Partial closure would result from subsequent larger-scale structural rearrangements in the protein, possibly corresponding to the conformational change observed at pH <5.

Structural study of the response regulator HupR from Rhodobacter capsulatus. electron microscopy of two-dimensional crystals on a nickel-chelating lipid

Two-dimensional crystals of the histidine-tagged-HupR protein, a transcriptional regulator from the photosynthetic bacterium Rhodobacter capsulatus, were obtained upon specific interaction with a Ni 2+-chelated lipid monolayer. HupR is a response regulator of the NtrC family; it activates the transcription of the structural genes, hupSLC, of the [NiFe]hydrogenase. The lipid (Ni-NTA-DOGA) uses the metal chelator nitrilotriacetic group as the hydrophilic headgroup and contains unsaturated oleyl tails to provide the fluidity necessary for two-dimensional protein crystallization. A projection map of the full-length protein at 18 Å resolution was generated by analysing electron microscopy micrographs of negatively stained crystals. The HupR protein appeared to be dimeric and revealed a characteristic “propeller-like” motif. Each monomer forms an L-shaped structure.

Electron microscopy of two-dimensional crystals of mitochondrial ATP synthase

Two-dimensional crystals of the mitochondrial ATP synthase up to 0.4 μm in size were obtained from the detergent-lipid-protein micelles by detergent dialysis. A projected map of the negatively stained crystal was calculated from electron microscopical images by the Fourier-filtering procedure at about 2.8 nm resolution. The unit cell (with not more than two ATP synthase molecules) has the following parameters: a = 13.0 nm, b = 25.6 nm and γ = 86°. Two alternative models for the crystal structural organization were suggested, viz. with one or two protein molecules per unit cell.

Three-dimensional model of yeast RNA polymerase I determined by electron microscopy of two-dimensional crystals.

Two-dimensional crystals of yeast RNA polymerase I dimers were obtained upon interaction with positively charged lipid layers. A three-dimensional surface model of the enzyme was determined by analyzing tilted crystalline areas and by taking advantage of the non-crystallographic internal symmetry of the dimer to correct for the missing viewing directions. The structure shows, at approximately 3 nm resolution, an irregularly shaped molecule 11 nm x 11 nm x 15 nm in size characterized by a 3 nm wide and 10 nm long groove which constitutes a putative DNA binding site. The overall structure is similar to the Escherichia coli holo enzyme and the yeast RNA polymerase II delta 4/7 structures. The most remarkable structural feature is a finger-shaped stalk which partially occludes the entrance of the groove and forms a 2.5 nm wide channel. We discuss the possible location of the catalytic centre and of the carboxy-terminal region of the beta-like subunit in the channel. The interference of different DNA fragments with RNA polymerase dimerization and crystallization indicates the orientation of the template in the putative DNA binding groove.

The structure of photosystem I from the thermophilic cyanobacterium Synechococcus sp. determined by electron microscopy of two-dimensional crystals

The structure of the Photosystem I (PS I) complex from the thermophilic cyaobacterium Synechococcus sp. has been investigated by electron microscopy and image analysis of two-dimensional crystals. Crystals were obtained from isolated PS I by removal of detergents with Bio-Beads. After negative staining, either single layers or two superimposed layers with a rotational different orientation were observed. The layers have a rectangular unit cell of 16.0 × 15.0 nm, which contains two PS I monomers. The monomers are arranged alternating up and down in each layer. For double-layer crystals, the images of the two layers could be separately processes by a combination of Fourier-peak-filtering and correlation averaging. Features in the two-dimensional plane can be seen with a resolution up to 1.5–1.8 nm. A model for the PS I structure was obtained by combining three-dimensional reconstructions from three tilt-series. The model shows an asymmetric PS I complex. On one side (presumably the stromal side) there is a 3 nm high ridge. This is most likely comprised of the psaC, psaD and psaE subunits. The other side (presumably the lumenal side) is rather flat, but in the center there is a 3 nm deep indentattion, which possibly separates partly the two large subunits psaA and psaB.

The structure of photosystem I from the thermophilic cyanobacterium Synechococcus sp. determined by electron microscopy of two-dimensional crystals

The structure of photosystem I from the thermophilic cyanobacterium Synechococcus sp. determined by electron microscopy of two-dimensional crystals

Toward the molecular structure of the mitochondrial channel, VDAC.

A summary is presented of the most recent information about the structure and mechanism of closure of the mitochondrial channel, VDAC. Considerable information has come from studies involving electron microscopy of two-dimensional crystals and from electrophysiological studies of wild-type channels and site-directed mutants. Available evidence points to a beta-barrel as the basic structural model for VDAC. Two models for voltage- or effector- induced closure have been proposed, the first involving removal of strands from the wall of the pore, the second invoking movement of protein domains into the lumen. Experimental strategies to resolve the actual mechanism are presented.

Structural analysis of mitochondrial pores

Structural information about the channel in the mitochondrial outer membrane, derived from sequence analysis and electron microscopy of two-dimensional crystals, is summarized. A model for the channel is presented, consisting of a cylindrical beta-barrel that is formed by one or two 30-kDa polypeptides, with an alpha-carbon backbone diameter of 3.8 nm. The radial distributions of basic amino acids and lipid-contact regions on the projected cylinder are mapped relative to interchannel bonding sites inferred from channel packing in the arrays. Speculation on the kinds of conformational changes that the channel might undergo is also presented.

Three-dimensional structure of (Na + + K +)-ATPase revealed by electron microscopy of two-dimensional crystals

Prolonged incubation of membrane fragments containing homogeneous (Na + + K +)-ATPase with Mg 2+, K + and VO 3 − at 4°C resulted in formation of two-dimensional crystals of this enzyme with unit cell parameters: a = 66 Å, b = 118 Å, γ = 108°. The crystals correspond to the two-sided plane group p21. By combining tilted electron microscopic views of the crystals, a three-dimensional structure of (Na + + K +)-ATPase was calculated at ~20 Å resolution. The unit cell is formed by two (αβ)-promoters which are in contact in their central parts. The structure was compared with chemical modification and immunochemical data; the arrangement of intra- and extramembrane domains was proposed.

Three-dimensional structure of the light-harvesting chlorophyll a/b–protein complex

Photosynthesis in the chloroplasts of green plants is carried out by a highly organized system of membranes. Chlorophyll–protein complexes in the photosynthetic membrane perform specialized functions which result in the biochemical fixation of solar energy. The light-harvesting chlorophyll a/b protein complex (LHC) has a key role in this process 1,2. A striking property of the LHC that distinguishes it from other chlorophyll–protein complexes and makes it suitable for structural studies by Fourier methods is its tendency to form crystalline arrays in vitro3. I report here the three-dimensional structure of pea LHC determined at 16 A resolution by electron microscopy of two-dimensional crystals and image analysis. A three-dimensional map shows that the LHC is a highly asymmetric transmembrane protein con posed of three structurally equivalent monomers. The large surface area exposed on one side of the trimeric complex suggests a functional role in membrane interaction. The ability of LHC isolated from widely different plant species to form similar crystalline arrays4–6 suggests that the structure reported here is of general significance.