Electron crystallography - the waking beauty of structural biology

Since its debut in the mid 1970s, electron crystallography has been a valuable alternative in the structure determination of biological macromolecules. Its reliance on single-layered or double-layered two-dimensionally ordered arrays and the ability to obtain structural information from small and disordered crystals make this approach particularly useful for the study of membrane proteins in a lipid bilayer environment. Despite its unique advantages, technological hurdles have kept electron crystallography from reaching its full potential. Addressing the issues, recent initiatives developed high-throughput pipelines for crystallization and screening. Adding progress in automating data collection, image analysis and phase extension methods, electron crystallography is poised to raise its profile and may lead the way in exploring the structural biology of macromolecular complexes.     

Two-dimensional crystallization of the ryanodine receptor Ca2+ release channel on lipid membranes

The ryanodine receptor (RyR) is the largest known membrane protein with a total molecular mass of 2.3 x 10(3) kDa. Well ordered, two-dimensional (2D) crystals are an essential prerequisite to enable RyR structure determination by electron crystallography. Conventionally, the 2D crystallization of membrane proteins is based on a 'trial-and-error' strategy, which is both time-consuming and chance-directed. By adopting a new strategy that utilizes protein sequence information and predicted transmembrane topology, we successfully crystallized the RyR on positively charged lipid membranes. Image processing of negatively stained crystals reveals that they are well ordered, with diffraction spots of IQ < or = 4 extending to approximately 20 angstroms, the resolution attainable in negative stain. The RyR crystals obtained on the charged lipid membrane have characteristics consistent with 2D arrays that have been observed in native sarcoplasmic reticulum of muscle tissues. These crystals provide ideal materials to enable structural analysis of RyR by high-resolution electron crystallography. Moreover, the reconstituted native-like 2D array provides an ideal model system to gain structural insights into the mechanism of RyR-mediated Ca2+ signaling processes, in which the intrinsic ability of RyR oligomers to organize into a 2D array plays a crucial role.     

Use of detergents in two-dimensional crystallization of membrane proteins

Structure determination at high resolution is actually a difficult challenge for membrane proteins and the number of membrane proteins that have been crystallized is still small and far behind that of soluble proteins. Because of their amphiphilic character, membrane proteins need to be isolated, purified and crystallized in detergent solutions. This makes it difficult to grow the well-ordered three-dimensional crystals that are required for high resolution structure analysis by X-ray crystallography. In this difficult context, growing crystals confined to two dimensions (2D crystals) and their structural analysis by electron crystallography has opened a new way to solve the structure of membrane proteins. However, 2D crystallization is one of the major bottlenecks in the structural studies of membrane proteins. Advances in our understanding of the interaction between proteins, lipids and detergents as well as development and improvement of new strategies will facilitate the success rate of 2D crystallization. This review deals with the various available strategies for obtaining 2D crystals from detergent-solubilized intrinsic membrane proteins. It gives an overview of the methods that have been applied and gives details and suggestions of the physical processes leading to the formation of the ordered arrays which may be of help for getting more proteins crystallized in a form suitable for high resolution structural analysis by electron crystallography.     

Osmolyte trimethylamine-N-oxide does not affect the strength of hydrophobic interactions: origin of osmolyte compatibility

Osmolytes are small organic solutes accumulated at high concentrations by cells/tissues in response to osmotic stress. Osmolytes increase thermodynamic stability of folded proteins and provide protection against denaturing stresses. The mechanism of osmolyte compatibility and osmolyte-induced stability has, therefore, attracted considerable attention in recent years. However, to our knowledge, no quantitative study of osmolyte effects on the strength of hydrophobic interactions has been reported. Here, we present a detailed molecular dynamics simulation study of the effect of the osmolyte trimethylamine-N-oxide (TMAO) on hydrophobic phenomena at molecular and nanoscopic length scales. Specifically, we investigate the effects of TMAO on the thermodynamics of hydrophobic hydration and interactions of small solutes as well as on the folding-unfolding conformational equilibrium of a hydrophobic polymer in water. The major conclusion of our study is that TMAO has almost no effect either on the thermodynamics of hydration of small nonpolar solutes or on the hydrophobic interactions at the pair and many-body level. We propose that this neutrality of TMAO toward hydrophobic interactions-one of the primary driving forces in protein folding-is at least partially responsible for making TMAO a "compatible" osmolyte. That is, TMAO can be tolerated at high concentrations in organisms without affecting nonspecific hydrophobic effects. Our study implies that protein stabilization by TMAO occurs through other mechanisms, such as unfavorable water-mediated interaction of TMAO with the protein backbone, as suggested by recent experimental studies. We complement the above calculations with analysis of TMAO hydration and changes in water structure in the presence of TMAO molecules. TMAO is an amphiphilic molecule containing both hydrophobic and hydrophilic parts. The precise balance of the effects of hydrophobic and hydrophilic segments of the molecule appears to explain the virtual noneffect of TMAO on the strength of hydrophobic interactions.     

Formation of helical protein assemblies of IgG and transducin on varied lipid tubules

Helical protein arrays on lipid tubules are valuable assemblies for studying protein structure and protein-lipid interactions through electron microscopy and crystallography. We describe conditions for forming such arrays from two proteins, IgG and transducin, the photoreceptor G protein, using a variety of lipid surfaces. Anti-dinitrophenyl (DNP) IgG arrays formed on DNP-phosphatidylethanolamine (DNP-PE) mixed with either galactosyl-ceramide lipids or phosphatidylcholine (PC) display different pH sensitivities and dimensions, yet have similar helical symmetries. DNP-PE/PC mixtures formed small crystals and large well-ordered flattened tubules. The peripheral membrane protein transducin (G(t)) formed helical arrays either on a mixture of cationic and neutral lipids or on residual photoreceptor lipids. Despite differences in lipid composition, the G(t) arrays have similar structures and show similar sensitivity to activation and variations in ionic environment. G(t) activation causes the helical assemblies to collapse to small vesicles, a process resembling the vesiculation of activated dynamin-lipid tubules. In a preliminary three-dimensional reconstruction, the hapten-bound IgG appears to make two contacts to the central lipid tubule, presumably via the F(ab) domains. The ability to generate a three-dimensional reconstruction without tilts illustrates one advantage of helical structures for two-dimensional crystallography, especially for visualizing protein-lipid interactions.     

7A projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals

Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7A resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.     

The Basic Protein of CNS Myelin: Its Structure and Ligand Binding

Consideration of the evidence presented in this review leads to the following conclusions: (a) Isolated MBP in aqueous solution has little ordered secondary or tertiary structure. (b) In this state, the protein can associate with a wide range of hydrophobic and amphiphilic compounds, these interactions involving limited sections of the protein. (c) The strength of binding to bilayers and the accompanying conformational changes in the protein are greatest for systems containing acidic lipids, presumably because of the involvement of ionic interactions. (d) When bound to bilayers of acidic lipids, MBP will have substantially more ordered secondary structure than it manifests in aqueous solution, and it is likely to be oligomeric (possibly hexameric). (e) MBP does affect the organization of lipid aggregates. It influences strongly the separation of bilayers in multilayers of purified lipids, and at present this must be viewed as its prime role within myelin. The greatest impediment to our understanding of MBP is the lack of an assayable biological activity. In contrast to the situation with enzymes, for example, we have no functional test for changes in protein structure or changes accompanying interactions with other molecules. Current evidence suggests that the protein has a structural role within myelin and that its own three-dimensional structure is strongly dependent on the molecules with which it is associated. If this picture is correct, studies of the isolated protein or of the protein in reconstituted lipid systems may yield, at best, a rough guide to the structure within its biological environment. Further clarification of the structure and function of MBP may have to await development of more powerful techniques for studying proteins bound to large molecular aggregates, such as lipid bilayers. The paucity of generally applicable methods is reflected in the fact that even low resolution structures are known for only a handful of intrinsic membrane proteins, and even more limited information exists for proteins associated with membrane surfaces. However, the increasing use of a combination of electron microscopy and diffraction on two-dimensional arrays of proteins formed on lipid bilayers (Henderson et al., 1990) offers the hope that it may not be too long before it will be possible to study at moderate resolution the three-dimensional structure of MBP bound to a lipid membrane.     

Solid-state NMR spectroscopy of 18.5 kDa myelin basic protein reconstituted with lipid vesicles: spectroscopic characterisation and spectral assignments of solvent-exposed protein fragments

Myelin basic protein (MBP, 18.5 kDa isoform) is a peripheral membrane protein that is essential for maintaining the structural integrity of the multilamellar myelin sheath of the central nervous system. Reconstitution of the most abundant 18.5 kDa MBP isoform with lipid vesicles yields an aggregated assembly mimicking the protein's natural environment, but which is not amenable to standard solution NMR spectroscopy. On the other hand, the mobility of MBP in such a system is variable, depends on the local strength of the protein-lipid interaction, and in general is of such a time scale that the dipolar interactions are averaged out. Here, we used a combination of solution and solid-state NMR (ssNMR) approaches: J-coupling-driven polarization transfers were combined with magic angle spinning and high-power decoupling to yield high-resolution spectra of the mobile fragments of 18.5 kDa murine MBP in membrane-associated form. To partially circumvent the problem of short transverse relaxation, we implemented three-dimensional constant-time correlation experiments (NCOCX, NCACX, CONCACX, and CAN(CO)CX) that were able to provide interresidue and intraresidue backbone correlations. These experiments resulted in partial spectral assignments for mobile fragments of the protein. Additional nuclear Overhauser effect spectroscopy (NOESY)-based experiments revealed that the mobile fragments were exposed to solvent and were likely located outside the lipid bilayer, or in its hydrophilic portion. Chemical shift index analysis showed that the fragments were largely disordered under these conditions. These combined approaches are applicable to ssNMR investigations of other peripheral membrane proteins reconstituted with lipids.     

7 Å projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals

Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7 Å resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.     

Solid-state NMR spectroscopy of 18.5 kDa myelin basic protein reconstituted with lipid vesicles: Spectroscopic characterisation and spectral assignments of solvent-exposed protein fragments

Myelin basic protein (MBP, 18.5 kDa isoform) is a peripheral membrane protein that is essential for maintaining the structural integrity of the multilamellar myelin sheath of the central nervous system. Reconstitution of the most abundant 18.5 kDa MBP isoform with lipid vesicles yields an aggregated assembly mimicking the protein's natural environment, but which is not amenable to standard solution NMR spectroscopy. On the other hand, the mobility of MBP in such a system is variable, depends on the local strength of the protein-lipid interaction, and in general is of such a time scale that the dipolar interactions are averaged out. Here, we used a combination of solution and solid-state NMR (ssNMR) approaches: J-coupling-driven polarization transfers were combined with magic angle spinning and high-power decoupling to yield high-resolution spectra of the mobile fragments of 18.5 kDa murine MBP in membrane-associated form. To partially circumvent the problem of short transverse relaxation, we implemented three-dimensional constant-time correlation experiments (NCOCX, NCACX, CONCACX, and CAN(CO)CX) that were able to provide interresidue and intraresidue backbone correlations. These experiments resulted in partial spectral assignments for mobile fragments of the protein. Additional nuclear Overhauser effect spectroscopy (NOESY)-based experiments revealed that the mobile fragments were exposed to solvent and were likely located outside the lipid bilayer, or in its hydrophilic portion. Chemical shift index analysis showed that the fragments were largely disordered under these conditions. These combined approaches are applicable to ssNMR investigations of other peripheral membrane proteins reconstituted with lipids.     

Temperature-mediated switching of protectant-denaturant behavior of trimethylamine-N-oxide and consequences on protein stability from a replica exchange molecular dynamics simulation study

The detailed mechanism of protein folding–unfolding processes with the aid of osmolytes has been a leading topic of discussion over many decades. We have used replica-exchange molecular dynamics simulation to propose the molecular mechanism of interaction of a 20-residue mini-protein with urea and trimethylamine N-oxide (TMAO) that act as denaturing and protecting osmolyte, respectively, in binary osmolyte solutions. Urea is found to exert its action by interacting directly with the protein residues. Temperature tolerance of TMAO’s action is particularly emphasised in this study. At lower range of temperature, TMAO acts as a successful protein protectant. Interestingly, the study discloses the tendency of TMAO molecules to prefer self-association at the protein surface at elevated temperature. A greater number of TMAO molecules in the protein hydration shell at higher temperature is also observed. Dihedral angle principal component analysis and free energy landscape plots sampled all possible conformations adopted by the protein that reveal highly folded behaviour of the protein in pure water and binary TMAO solutions and highly unfolded behaviour in presence of urea.     

Counteracting osmolyte trimethylamine N-oxide destabilizes proteins at pH below its pKa. Measurements of thermodynamic parameters of proteins in the presence and absence of trimethylamine N-oxide

Earlier studies have reported that trimethylamine N-oxide (TMAO), a naturally occurring osmolyte, is a universal stabilizer of proteins because it folds unstructured proteins and counteracts the deleterious effects of urea and salts on the structure and function of proteins. This conclusion has been reached from the studies of the effect of TMAO on proteins in the pH range 6.0-8.0. In this pH range TMAO is almost neutral (zwitterionic form), for it has a pK(a) of 4.66 +/- 0.10. We have asked the question of whether the effect of TMAO on protein stability is pH-dependent. To answer this question we have carried out thermal denaturation studies of lysozyme, ribonuclease-A, and apo-alpha-lactalbumin in the presence of various TMAO concentrations at different pH values above and below the pK(a) of TMAO. The main conclusion of this study is that near room temperature TMAO destabilizes proteins at pH values below its pK(a), whereas it stabilizes proteins at pH values above its pK(a). This conclusion was reached by determining the T(m) (midpoint of denaturation), delta H(m) (denaturational enthalpy change at T(m)), delta C(p) (constant pressure heat capacity change), and delta G(D) degrees (denaturational Gibbs energy change at 25 degrees C) of proteins in the presence of different TMAO concentrations. Other conclusions of this study are that T(m) and delta G(D) degrees depend on TMAO concentration at each pH value and that delta H(m) and the delta C(p) are not significantly changed in presence of TMAO.     

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.Download video file.^{(59M, mov)}     

Osmolyte trimethylamine N-oxide converts recombinant alpha-helical prion protein to its soluble beta-structured form at high temperature

The thermal unfolding of full-length human recombinant alpha-helical prion protein (alpha-PrP) in neutral pH is reversible, whereas, in the presence of the osmolyte N-trimethylamine oxide (TMAO), the protein acquires a beta-sheet structure at higher temperatures and the thermal unfolding of the protein is irreversible. Lysozyme, an amyloidogenic protein similar to prion protein, regains alpha-helical structure on cooling from its thermally unfolded form in buffer and in TMAO solutions. The thermal stability of alpha-PrP decreases, whereas that of lysozyme increases in TMAO solution. Light-scattering and turbidity values indicate that beta-sheet prion protein exists as soluble oligomers that increase thioflavin T fluorescence and bind to 1-anilino 8-naphthalene sulfonic acid (ANS). The oligomers are resistant to proteinase K digestion and during incubation for long periods they form linear amyloids>5 microm long. The comparable fluorescence polarization of the tryptophan groups and their accessibility to acrylamide in alpha-PrP and oligomers indicate that the unstructured N-terminal segments of the protein, which contain the tryptophan groups, do not associate among themselves during oligomerization. Partial unfolding of alpha-helical prion protein in TMAO solution leads to its structural conversion to misfolded beta-sheet form. The formation of the misfolded prion protein oligomers and their polymerization to amyloids in TMAO are unusual, since the osmolyte generally induces denatured protein to fold to a native-like state and protects proteins from thermal denaturation and aggregation.     

Alterations in the protein-synthesis, -degradation and/or -secretion rates in hepatic subcellular fractions of selenium-deficient mice.

Conformational studies of myelin basic protein (MBP) in solution generally have used protein purified in organic solvents and acid. The use of such conditions raises the possibility that the secondary structure reported for the basic protein represents a denatured state. Therefore we have purified this protein by using a procedure that avoids denaturants. Bovine myelin was extracted with 0.2 M-CaCl2 and the protein was purified from the supernatant by chromatography on Sephadex G-75. The conformation of the basic protein was characterized by using c.d. and 1H-n.m.r. spectroscopy. In solution, it appeared to be predominantly randomly coiled, with only small segments of persistent structure. However, in the presence of myristoyl lysophosphatidylcholine the secondary structure of MBP became more ordered, and sedimentation-velocity experiments showed that MBP aggregated. Comparison of our results with published data indicates that Ca2+-extracted basic protein behaves similarly to the protein purified by traditional methods with respect to its ordered conformation in solution in the absence and in the presence of lipid and with respect to its self-association. Thus its thermodynamically stable structure in aqueous solution appears to be a highly flexible coil.     

Review: electron crystallography: present excitement, a nod to the past, anticipating the future

From a modest beginning with negatively stained samples of the helical T4 bacteriophage tail, electron crystallography has emerged as a powerful tool in structural biology. High-resolution density maps, interpretable in terms of an atomic structure, can be obtained from specimens prepared as well-ordered, two-dimensional crystals, and the resolution achieved with helical specimens and icosahedral viruses is approaching the same goal. A hybrid approach to determining the molecular structure of complex biological assemblies is generating great interest, in which high-resolution structures that have been determined for individual protein components are fitted into a lower resolution envelope of the large complex. With this as background, how much more can be anticipated for the future? Considerable scope still remains to improve the quality of electron microscope images. Automation of data acquisition and data processing, together with the emergence of computational speeds of 10(12) floating point operations per second or higher, will make it possible to extend high-resolution structure determination into the realm of single-particle microscopy. As a result, computational alignment of single particles, i.e., the formation of "virtual crystals," can begin to replace biochemical crystallization. Since single-particle microscopy may remain limited to "large" structures of 200 to 300 kDa or more, however, smaller proteins will continue to be studied as helical assemblies or as two-dimensional crystals. The further development of electron crystallography is thus likely to turn increasingly to the use of single particles and small regions of ordered assemblies, emphasizing more and more the potential for faster, higher throughput.     

Two-dimensional crystallization of proteins on planar lipid films and structure determination by electron crystallography

Electron crystallography constitutes a powerful new method for determining the struture of biological macromolecules. This method is best adapted to the study of ordered assemblies of macromolecules, and principally to two-dimensional (2-D) crystals of proteins. Obtaining protein 2-D crystals ordered at high resolution constitutes the major limiting step in the application of this approach. Considerable interest has been raised by the development of a rational method of 2-D crystallization based on the specific binding of proteins to planar lipid films. The applicability of this method is quasi-general in the case of soluble proteins. Its basic principles, together with examples taken from work in our group, are presented here.     

Controlled 2D crystallization of membrane proteins using methyl-beta-cyclodextrin

High-resolution structural data of membrane proteins can be obtained by studying 2D crystals by electron crystallography. Finding the right conditions to produce these crystals is one of the major bottlenecks encountered in 2D crystallography. Many reviews address 2D crystallization techniques in attempts to provide guidelines for crystallographers. Several techniques including new approaches to remove detergent like the biobeads technique and the development of dedicated devices have been described (dialysis and dilution machines). In addition, 2D crystallization at interfaces has been studied, the most prominent method being the 2D crystallization at the lipid monolayer. A new approach based on detergent complexation by cyclodextrins is presented in this paper. To prove the ability of cyclodextrins to remove detergent from ternary mixtures (lipid, detergent and protein) in order to get 2D crystals, this method has been tested with OmpF, a typical beta-barrel protein, and with SoPIP2;1, a typical alpha-helical protein. Experiments over different time ranges were performed to analyze the kinetic effects of detergent removal with cyclodextrins on the formation of 2D crystals. The quality of the produced crystals was assessed with negative stain electron microscopy, cryo-electron microscopy and diffraction. Both proteins yielded crystals comparable in quality to previous crystallization reports.