Electron tunneling in respiratory complex I

NADH:ubiquinone oxidoreductase (complex I) plays a central role in the respiratory electron transport chain by coupling the transfer of electrons from NADH to ubiquinone to the creation of the proton gradient across the membrane necessary for ATP synthesis. Here the atomistic details of electronic wiring of all Fe/S clusters in complex I are revealed by using the tunneling current theory and computer simulations; both density functional theory and semiempirical electronic structure methods were used to examine antiferromagnetically coupled spin states and corresponding tunneling wave functions. Distinct electron tunneling pathways between neighboring Fe/S clusters are identified; the pathways primarily consist of two cysteine ligands and one additional key residue. Internal water between protein subunits is identified as an essential mediator enhancing the overall electron transfer rate by almost three orders of magnitude to achieve a physiologically significant value. The identified key residues are further characterized by sensitivity of electron transfer rates to their mutations, examined in simulations, and their conservation among complex I homologues. The unusual electronic structure properties of Fe(4)S(4) clusters in complex I explain their remarkable efficiency of electron transfer.     

Electron crystallography of ultrathin 3D protein crystals: Atomic model with charges

Membrane proteins and macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons four to five orders of magnitude more strongly than they do X-rays. Furthermore, as electron crystallography yields Coulomb potential maps rather than electron density maps, it could provide a unique method to visualize the charged states of amino acid residues and metals. Here we describe an attempt to develop a methodology for electron crystallography of ultrathin (only a few layers thick) 3D protein crystals and present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, obtained from Ca²⁺-ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca²⁺-binding sites of Ca²⁺-ATPase and that of the iron atom in the heme in catalase.Protein atoms scatter electrons four to five orders of magnitude more strongly than they do X-rays, thus allowing individual protein molecules to be imaged by electron microscopy (1). Although not fully exploited so far, electron protein crystallography has great potential and indeed has yielded superb high-resolution (∼2.0-Å resolution) atomic structures from 2D crystals (2). However, electron crystallography of 3D crystals is problematic, as stacking of even a few layers makes diffraction patterns discrete in all directions, and methods developed for conventional electron crystallography of 2D crystals (3) are not useful (SI Appendix, Fig. S1A, Left). This problem can be overcome, however, as Gonen and coworkers demonstrated (4, 5), by rotating the crystal to spatially integrate the intensities of diffraction spots as in X-ray crystallography (SI Appendix, Fig. S1A, Right) or, in certain cases, even combining simple tilt series.Another important feature of electron scattering is that the diffraction pattern formed by elastically scattered electrons is directly related to the distribution of Coulomb potential. This is in marked contrast to X-rays, which, because they are scattered by electrons, yield an electron density map. Coulomb potential maps may be more difficult to interpret, compared with electron density maps by X-ray crystallography, as the appearance of the same residues may differ depending on their charged state, resolution, and surrounding environment (Fig. 1 and SI Appendix, Fig. S2), but they provide unique information, not attainable by X-rays (6). Theoretical potential maps (F_calc maps; Fig. 1 B–E) calculated from an atomic model of Ca²⁺-ATPase (7) using scattering factors for 300-keV electrons highlight these features. For instance, densities of acidic residues are absent or weak when lower-resolution data are included in the map calculation (Fig. 1 B and C). In contrast, when calculated using scattering factors for X-rays, the densities of the same residues appear more or less identical irrespective of the resolution range (Fig. 1 F and G). These features of Coulomb potential maps result from the fact that atomic scattering factors for electrons vary considerably over a range of spatial frequency depending on the charged state (Fig. 1A) and can become close to zero or even negative (e.g., for O⁻, Fig. 1A). An advantageous consequence is that it is possible to determine experimentally the charged states of protein residues and metals. As proteins use metals of different ionic states for many purposes, notably for catalysis and electron transfer, information on the charged state of metals and amino acid residues can be critical in understanding protein function.Open in a separate windowFig. 1.Atomic scattering factors and theoretical maps. (A) Atomic scattering factors for 300-keV electrons based on values from International Tables for Crystallography (14), except for H⁺ (taken from ref. 15). Scattering factors for X-rays are provided in SI Appendix, Fig. S2. (B–G) Theoretical Coulomb potential (B–E) and electron density (F and G) maps around the Ca²⁺-binding site of Ca²⁺-ATPase, calculated for 8- to 3.4-Å (B, D, and F) and 5- to 3.4-Å (C, E, and G) resolution and contoured at 1.0 σ (B–E) or 1.2 σ (F and G). Viewed from the cytoplasmic side approximately perpendicular to the membrane. Superimposed is the atomic model of Ca²⁺-ATPase derived from that determined by X-ray (PDB ID code 1SU4) (7) and refined in this study. Cyan spheres represent bound Ca²⁺ (I and II). For the potential maps (B–E), standard charges are assigned to all titratable residues (except for Asp-800 in D and E, treated as a neutral residue) and calculated using the scattering factors for 300-keV electrons. Note that appearances of some charged residues vary substantially depending on the resolution range in the Coulomb potential maps (compare B and C or D and E), but are nearly identical in the electron density maps (F and G).Here we present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, of Ca²⁺-ATPase and catalase obtained from ultrathin (just a few layers thick) crystals using a new electron diffractometer (SI Appendix, Fig. S1B). These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca²⁺-binding sites of Ca²⁺-ATPase and that of the iron atom in the heme in catalase.     

Electron tunneling through alkanedithiol self-assembled monolayers in large-area molecular junctions

The electrical transport through self-assembled monolayers of alkanedithiols was studied in large-area molecular junctions and described by the Simmons model [Simmons JG (1963) J Appl Phys 34:1793-1803 and 2581-2590] for tunneling through a practical barrier, i.e., a rectangular barrier with the image potential included. The strength of the image potential depends on the value of the dielectric constant. A value of 2.1 was determined from impedance measurements. The large and well defined areas of these molecular junctions allow for a simultaneous study of the capacitance and the tunneling current under operational conditions. Electrical transport for octanedithiol through tetradecanedithiol self-assembled monolayers up to 1 V can simultaneously be described by a single effective mass and a barrier height. There is no need for additional fit constants. The barrier heights are in the order of 4-5 eV and vary systematically with the length of the molecules. Irrespective of the length of the molecules, an effective mass of 0.28 was determined, which is in excellent agreement with theoretical predictions.     

Electron tunneling process and the segment mobility of macromolecules.

New experimental data [Berg, A. I., Noks, P. P., Kononenko, A. A., Frolov, E. N., Khrymova, I. N., Rubin A. B., Likhtenstein, G. I., Goldanskii, V. I., Parak, F., Bukl, M. & Mössbauer, R. (1979) Mol. Biol. (USSR) 13, 81-89; Berg, A. I., Noks, P. P., Kononenko, A. A., Frolov, E. N., Uspenskaya, N. Y., Khrymova, I. N., Rubin, A. B., Likhtenstein, G. I. & Hideg, K. (1979) Mol. Biol (USSR) 13, 469-477] provide evidence that the electron tunneling process is connected to a special type of conformational transition (segmental transition) protein macromolecules in photosynthetic membranes. This problem is investigated with a simple mechanical model. It is shown that the segmental degree of freedom can play the role of the strongly interacting accepting mode for the electron tunneling process. The temperature dependences of the electron tunneling rate and the recoilless gamma-ray absorption of membrane-bound 57Fe, as an indicator of the intramolecular mobility, are calculated. The problem of energy storage in proteins is also discussed.     

Electron self-exchange in azurin: calculation of the superexchange electron tunneling rate.

Electronic coupling between the copper atoms in an azurin dimer has been calculated in this conformationally well-defined system by using many-electronic wave functions. When one of the two water molecules forming intermolecular hydrogen bonds between the copper-ligating His-117 of the two azurins is removed, the calculated coupling element is reduced from 2.5 x 10(-6) to 1.1 x 10(-7) eV (1 eV = 1.602 x 10(-19) J). Also, the effects of the relative orientations of the two water molecules have been analyzed. The results show that water molecules may play an important role as switches for biological electron transfer. The rate of electron self-exchange between two azurins has been calculated, and the result is in very good agreement with the rate found experimentally.     

Liquid-solid transition in nuclei of protein crystals

It is generally assumed that crystallization begins with a small, crystalline nucleus. For proteins this paradigm may not be valid. Our numerical simulations show that under conditions typically used to produce protein crystals, small clusters of model proteins (particles with short-range, attractive interactions) cannot maintain a crystalline structure. Protein crystal nucleation is therefore an indirect, two-step process. A nucleus first forms and grows as a disordered, liquid-like aggregate. Once the aggregate grows beyond a critical size (about a few hundred particles) crystal nucleation becomes possible.     

Cross-linked protein crystals for vaccine delivery

The progress toward subunit vaccines has been limited by their poor immunogenicity and limited stability. To enhance the immune response, subunit vaccines universally require improved adjuvants and delivery vehicles. In the present paper, we propose the use of cross-linked protein crystals (CLPCs) as antigens. We compare the immunogenicity of CLPCs of human serum albumin with that of soluble protein and conclude that there are marked differences in the immune response to the different forms of human serum albumin. Relative to the soluble protein, crystalline forms induce and sustain over almost a 6-month study a 6- to 10-fold increase in antibody titer for highly cross-linked crystals and an approximately 30-fold increase for lightly cross-linked crystals. We hypothesize that the depot effect, the particulate structure of CLPCs, and highly repetitive nature of protein crystals may play roles in the enhanced production of circulating antibodies. Several features of CLPCs, such as their remarkable stability, purity, biodegradability, and ease of manufacturing, make them highly attractive for vaccine formulations. This work paves the way for a systematic study of protein crystallinity and cross-linking on enhancement of humoral and T cell responses.     

Atomic-level accuracy in simulations of large protein crystals.

Proper treatment of long-range Coulombic forces presents a major obstacle to providing realistic molecular dynamics simulations of macromolecules. Traditional approximations made to lessen computational cost ultimately lead to unrealistic behavior. The particle mesh Ewald method accommodates long-range Coulombic forces accurately and efficiently by use of fast Fourier transform techniques. We report a 1-ns simulation of bovine pancreatic trypsin inhibitor in a crystal unit cell using the particle mesh Ewald methodology. We find an rms backbone deviation from the x-ray structure (0.33 A) that is lower than that observed between bovine pancreatic trypsin inhibitor in different crystal forms and much lower than those of previous simulations. These results bridge the gap between structures obtained from molecular simulation and those from experiment.     

Predicting the X-ray lifetime of protein crystals

Radiation damage is a major cause of failure in macromolecular crystallography experiments. Although it is always best to evenly illuminate the entire volume of a homogeneously diffracting crystal, limitations of the available equipment and imperfections in the sample often require a more sophisticated targeting strategy, involving microbeams smaller than the crystal, and translations of the crystal during data collection. This leads to a highly inhomogeneous distribution of absorbed X-rays (i.e., dose). Under these common experimental conditions, the relationship between dose and time is nonlinear, making it difficult to design an experimental strategy that optimizes the radiation damage lifetime of the crystal, or to assign appropriate dose values to an experiment. We present, and experimentally validate, a predictive metric diffraction-weighted dose for modeling the rate of decay of total diffracted intensity from protein crystals in macromolecular crystallography, and hence we can now assign appropriate “dose” values to modern experimental setups. Further, by taking the ratio of total elastic scattering to diffraction-weighted dose, we show that it is possible to directly compare potential data-collection strategies to optimize the diffraction for a given level of damage under specific experimental conditions. As an example of the applicability of this method, we demonstrate that by offsetting the rotation axis from the beam axis by 1.25 times the full-width half maximum of the beam, it is possible to significantly extend the dose lifetime of the crystal, leading to a higher number of diffracted photons, better statistics, and lower overall radiation damage.Given an adequately diffracting crystal, radiation damage is the dominant cause of failure for macromolecular crystallography (MX) experiments (1), and overcoming this problem has been one of the major motivations for the development of new methods. By way of illustration: over the last 11 years at beamline 8.3.1 of the Advanced Light Source (ALS), more than 1,000 structures have been solved and deposited into the Protein Data Bank, but more than 25,000 datasets were collected. Similar dataset-to-deposition ratios have been reported elsewhere (2, 3). A retrospective analysis of the ALS 8.3.1 data reveals that radiation damage played a dominant role in the failure to obtain phases for structure solution by anomalous dispersion methods. Indeed, if radiation damage did not exist, investigators could simply keep collecting data until any desired signal-to-noise ratio was attained.Much of the recent excitement over serial femtosecond crystallography with X-ray Free Electron Lasers (XFELs) has been due to the vast gains in the diffraction/damage ratios demonstrated (4). Despite these major advances, the technology for these systems is not yet mature, and the linear nature of the XFEL facilities limits the number of end stations, greatly reducing capacity compared with a traditional synchrotron source. Synchrotron-based MX is thus likely to remain the dominant method for structural determination in the coming years, and there is a pressing need to improve how we deal with radiation damage to maintain the maximum utility of synchrotron based MX in the XFEL era.Even at XFELs, there appears to be some kind of radiation damage present (5). Radiation damage is unavoidable, but it is also not the ultimate problem: the challenge is that radiation damage remains difficult to predict. Most experienced investigators know that subjecting a protein crystal to a lower dose will give them less radiation damage, but it will also give them less diffraction, and striking the appropriate balance is the key to success. This paper presents a method for optimizing this ratio, allowing the best data to be gained from a given diffracting crystal volume.Once macromolecular crystals have been obtained, structural biologists working on challenging samples are often presented with a handful of well-diffracting crystals among a much larger population of poorly diffracting ones. Under these common conditions, it is vital to collect data as efficiently as possible from this available crystal volume. A prerequisite to optimizing the experimental protocol is knowing the effective crystal lifetime available for data collection. Diffracted intensity is known to decay with dose (6, 7), relative B factor is known to increase (8), and protocols exist for determining dose tolerance under carefully controlled conditions (9). However, when collecting data with the goal of solving challenging new structures, these results are often of limited use (10), because the optimally efficient even-dose case (11) is often not experimentally achievable. The reason for this is the absence of a suitable model that takes into account the effect of the uneven distribution of dose throughout the crystal volume during an experiment, where the maximum dose at the intersection of the beam and the rotation axes can often be more than an order of magnitude greater than the average dose within the diffracting volume (11).The recently developed program raddose-3D (12) allows users to simulate the progression of the absorbed energy that eventually leads to damage during data collection. The raw output of this calculation is a dose field, which reports the dose at each point in the crystal, at each time step during data collection: typically 10⁶ volume elements, and hundreds of time steps. This raw output is, in itself, of limited use, because it does not directly inform the experimenter about the likely damage state of his or her crystal. To achieve this, aggregate metrics describing the dose state of the crystal in a succinct way must be calculated. Two intuitive metrics are the average dose for the whole crystal volume (AD-WC); i.e., the total absorbed energy divided by the mass of the whole crystal, and the maximum dose: the highest dose reached at any point in the crystal volume. Maximum dose, assuming a well-aligned beam and rotation axis, is the metric output by previous versions of raddose (13–15) when using the Gaussian beam profile (GAUSS) keyword to define the beam profile, a worst-case estimate for the dose.     

An approach to membrane protein structure without crystals

The lactose permease of Escherichia coli catalyzes coupled translocation of galactosides and H(+) across the cell membrane. It is the best-characterized member of the Major Facilitator Superfamily, a related group of membrane proteins with 12 transmembrane domains that mediate transport of various substrates across cell membranes. Despite decades of effort and their functional importance in all kingdoms of life, no high-resolution structures have been solved for any member of this family. However, extensive biochemical, genetic, and biophysical studies on lactose permease have established its transmembrane topology, secondary structure, and numerous interhelical contacts. Here we demonstrate that this information is sufficient to calculate a structural model at the level of helix packing or better.     

Significance of bound water to local chain conformations in protein crystals

We examine how the polypeptide chain in protein crystal structures exploits the multivalent hydrogen-bonding potential of bound water molecules. This shows that multiple interactions with a single water molecule tend to occur locally along the chain. A distinctive internal-coordinate representation of the local water-binding segments reveals several consensus conformations. The fractional water occupancy of each was found by comparison of the total number of conformations in the database regardless of the presence or absence of bound water. The water molecule appears particularly frequently in type II beta-turn geometries and an N-terminal helix feature. This work constitutes a first step into assessing not only the generality but also the significance of specific water binding in globular proteins.     

Role of clusters in nonclassical nucleation and growth of protein crystals

The development of multistep nucleation theory has spurred on experimentalists to find intermediate metastable states that are relevant to the solidification pathway of the molecule under interest. A great deal of studies focused on characterizing the so-called “precritical clusters” that may arise in the precipitation process. However, in macromolecular systems, the role that these clusters might play in the nucleation process and in the second stage of the precipitation process, i.e., growth, remains to a great extent unknown. Therefore, using biological macromolecules as a model system, we have studied the mesoscopic intermediate, the solid end state, and the relationship that exists between them. We present experimental evidence that these clusters are liquid-like and stable with respect to the parent liquid and metastable compared with the emerging crystalline phase. The presence of these clusters in the bulk liquid is associated with a nonclassical mechanism of crystal growth and can trigger a self-purifying cascade of impurity-poisoned crystal surfaces. These observations demonstrate that there exists a nontrivial connection between the growth of the macroscopic crystalline phase and the mesoscopic intermediate which should not be ignored. On the other hand, our experimental data also show that clusters existing in protein solutions can significantly increase the nucleation rate and therefore play a relevant role in the nucleation process.The process of crystallization is generally considered to occur in two consecutive but very different stages: nucleation and growth. The first stage was already studied more than two centuries ago by Gibbs, who considered the nucleation of water droplets from a supersaturated vapor through the formation of globulae. He was the first to develop a thermodynamic formalism of nucleation by considering the generation of nuclei of a liquid phase as a density fluctuation of the parent phase (1, 2). Since then, Gibbs’ nucleation theory has been extended to the nucleation of solid phases from solution and gaseous phases from which the current paradigm (based on the capillary approximation) for nucleation emerged, i.e., the classical nucleation theory [CNT (3–8)]. There is, however, an increasing body of evidence that shows that CNT can fail drastically when used in cases where the implicit and explicit assumptions of CNT are poorly justified (for a full dissection of the limitations, see refs. 9–11). An obvious situation where CNT will have limited applicability is in cases where the old and the new phases differ by at least two order parameters, e.g., density and structure (12).Recent theoretical, computational, and experimental efforts have demonstrated that densification and local increase in crystallinity need not occur simultaneously (13–20). These results have inspired the development of a new approach that considers nucleation from solution as a multistep process attributing key roles to metastable intermediate states, coined “multistep nucleation theory” (MNT). Although initially conceived for proteins by ten Wolde and Frenkel to describe nucleation close to the critical point, the operational range of MNT was later expanded to also include regions in the phase diagram close to the liquid–liquid binodal––the rationale being that macroscopic droplets are formed that are enriched in protein, in which (due to the reduced surface tension) crystal nucleation is greatly facilitated. Even later, the discovery of long-lived mesoscopic clusters (10⁵–10⁶ monomers) in regions of the phase diagram distant of both the critical point and the liquid–liquid binodal makes it tantalizing to conclude that MNT may also dominate these regions of phase space. These clusters, which exist in both super- and undersaturated protein solutions, are hypothesized to be precursors of nuclei of crystals, irrespective of the vicinity of the critical point in the phase diagram.However, contrary to what is sometimes suggested, no direct experimental evidence has been presented that demonstrates that these clusters are in fact prenucleation clusters taking part in a multistep nucleation scenario (9, 21). The occurrence of crystals within macroscopic dense protein droplets formed by liquid–liquid phase separation is an often-cited pro-MNT argument for protein crystallization. However, for the specific proteins for which it has been observed, liq–liq phase separation is certainly not a prerequisite for crystal nucleation––for these systems, there are numerous observations of nucleation well above the liq–liq binodal curve. Secondly, such qualitative observations give no information on the relative rates of the possible CNT and MNT crystallization routes. Subsequently, no obvious relevance to any MNT pathways can be attributed. Ultimately, these issues need to be addressed if the MNT concept is to be elevated from an interesting, albeit academic exercise to a fully developed model of wide applicability. As such, to date, MNT remains a plausible theory, but a theory nonetheless.Notwithstanding the undeniable advances discussed above, many questions specific to protein condensation remain open. Do nuclei form within the mesoscopic protein clusters or are these clusters (merely) involved in a heterogeneous nucleation pathway? Or, even more boldly, are they at all involved in the nucleation process or is cluster formation simply a dead end in the many conceivable pathways toward the crystalline state? In this contribution, we aim to start formulating answers to such questions using a number of different protein model systems. We begin by characterizing the protein clusters in liquid as well as the crystal–cluster interaction using both conventional and newer methods, i.e., static–dynamic light scattering, brownian microscopy (BM), and laser confocal microscopy enhanced by differential interference contrast. By combining both approaches we demonstrate that the cluster presence in solution is directly correlated to a well-known growth mechanism, i.e., instantaneous multilayer formation. Secondly, we show that cluster removal from the bulk liquid can have a drastic effect on nucleation kinetics, demonstrating that these clusters might be a preferred intermediate in protein crystallization––a strong argument in favor of MNT-like scenarios. Thirdly, and quite surprisingly, we reveal that cluster assimilation by the crystal can trigger a self-purifying cascade of impurity-poisoned crystal surfaces. Stated differently, the coalescence of a cluster with an impurity-poisoned crystal surface can lead to a rapid and complete cleansing of the entire surface. From these observations we conclude that protein clusters can have a strong positive impact on protein crystallization during both the nucleation as well as the growth stage.     

Radiation damage in protein crystals is reduced with a micron-sized X-ray beam

Radiation damage is a major limitation in crystallography of biological macromolecules, even for cryocooled samples, and is particularly acute in microdiffraction. For the X-ray energies most commonly used for protein crystallography at synchrotron sources, photoelectrons are the predominant source of radiation damage. If the beam size is small relative to the photoelectron path length, then the photoelectron may escape the beam footprint, resulting in less damage in the illuminated volume. Thus, it may be possible to exploit this phenomenon to reduce radiation-induced damage during data measurement for techniques such as diffraction, spectroscopy, and imaging that use X-rays to probe both crystalline and noncrystalline biological samples. In a systematic and direct experimental demonstration of reduced radiation damage in protein crystals with small beams, damage was measured as a function of micron-sized X-ray beams of decreasing dimensions. The damage rate normalized for dose was reduced by a factor of three from the largest (15.6 μm) to the smallest (0.84 μm) X-ray beam used. Radiation-induced damage to protein crystals was also mapped parallel and perpendicular to the polarization direction of an incident 1-μm X-ray beam. Damage was greatest at the beam center and decreased monotonically to zero at a distance of about 4 μm, establishing the range of photoelectrons. The observed damage is less anisotropic than photoelectron emission probability, consistent with photoelectron trajectory simulations. These experimental results provide the basis for data collection protocols to mitigate with micron-sized X-ray beams the effects of radiation damage.     

Electron nuclear double resonance spectroscopy of a hen egg-white lysozyme--Cu2+ complex in tetragonal single crystals.

We have used the electron paramagnetic resonance of Cu2+ bound in a tetragonal single crystal of hen egg-white lysozyme to obtain the electron nuclear double resonance spectra of protons in the vicinity of the Cu2+ at the site designated as B by Teichberg et al. [Teichberg, V. I., Sharon, N., Moult, J., Smilansky, A. & Yonath, A. (1974) J. Mol. Biol. 87, 357-368]. The values of the hyperfine interaction parameters and the coordinates of eight protons are reported. The configuration of the H2O molecules coordinated to the Cu2+ and their relationships to the protein molecule structure are discussed.     

Experiment and theory for heterogeneous nucleation of protein crystals in a porous medium

The determination of high-resolution structures of proteins requires crystals of suitable quality. Because of the new impetus given to structural biology by structural genomics/proteomics, the problem of crystallizing proteins is becoming increasingly acute. There is therefore an urgent requirement for the development of new efficient methods to aid crystal growth. Nucleation is the crucial step that determines the entire crystallization process. Hence, the holy grail is to design a "universal nucleant," a substrate that induces the nucleation of crystals of any protein. We report a theory for nucleation on disordered porous media and its experimental testing and validation using a mesoporous bioactive gel-glass. This material induced the crystallization of the largest number of proteins ever crystallized using a single nucleant. The combination of the model and the experimental results opens up the scope for the rational design of nucleants, leading to alternative means of controlling crystallization.     

Experimental determination of the radiation dose limit for cryocooled protein crystals

Radiation damage to cryocooled protein crystals during x-ray structure determination has become an inherent part of macromolecular diffraction data collection at third-generation synchrotrons. Generally, radiation damage is an undesirable component of the experiment and can result in erroneous structural detail in the final model. The characterization of radiation damage thus has become an important area for structural biologists. The calculated dose limit of 2 x 10(7) Gy for the diffracting power of cryocooled protein crystals to drop by half has been experimentally evaluated at a third-generation synchrotron source. Successive data sets were collected from four holoferritin and three apoferritin crystals. The absorbed dose for each crystal was calculated by using the program raddose after measurement of the incident photon flux and determination of the elemental crystal composition by micro-particle-induced x-ray emission. Degradation in diffraction quality and specific structural changes induced by synchrotron radiation then could be compared directly with absorbed dose for different dose/dose rate regimes: a 10% lifetime decrease for a 10-fold dose rate increase was observed. Remarkable agreement both between different crystals of the same type and between apoferritin and holoferritin was observed for the dose required to reduce the diffracted intensity by half (D(1/2)). From these measurements, a dose limit of D(1/2) = 4.3 (+/-0.3) x10(7) Gy was obtained. However, by considering other data quality indicators, an intensity reduction to I(ln2) = ln2 x I(0), corresponding to an absorbed dose of 3.0 x 10(7) Gy, is recommended as an appropriate dose limit for typical macromolecular crystallography experiments.     

Electron microscopic visualization of the tetrodotoxin-binding protein from Electrophorus electricus.

Preparations of highly purified tetrodotoxin-binding protein (sodium channel) from the electric organ of the eel Electrophorus electricus were examined in negatively stained preparations. Structures observed in preparations exhibiting the highest tetrodotoxin binding tended to aggregate into ordered clusters with a unique ribbon-like conformation. The individual particles of these aggregates are elongated or rod-shaped, approximately 40 A wide and 170 A long. Stereoscopic imaging of the three-dimensional aspects of the structures revealed that the rod-like image is not an edge view of a flattened disc but represents a cylindrical structure. Individual rods in nonclustered forms were also observed but with greater frequency in preparations with lower specific activity. The dimensions of the particles suggest that they represent a protein core formed by perhaps one copy of the large glycopeptide previously identified as being part of the sodium channel. The structure of the sodium channel component visualized by negative staining is discussed in the context of the excitable properties it contributes to biological membranes.