Multi-temperature single-crystal and powder diffraction experiments on 1-(2′-aminophenyl)-2-methyl-4-nitroimidazole show that this crystal undergoes an isomorphic phase transition with the coexistence of two phase domains over a wide temperature range. The anharmonic approach was the only way to model the resulting disorder.
The harmonic model of atomic nuclear motions is usually enough for multipole modelling of high-resolution X-ray diffraction data; however, in some molecular crystals, such as 1-(2′-aminophenyl)-2-methyl-4-nitro-1H-imidazole [Paul, Kubicki, Jelsch et al. (2011 ▶). Acta Cryst. B67, 365–378], it may not be sufficient for a correct description of the charge-density distribution. Multipole refinement using harmonic atom vibrations does not lead to the best electron density model in this case and the so-called ‘shashlik-like’ pattern of positive and negative residual electron density peaks is observed in the vicinity of some atoms. This slight disorder, which cannot be modelled by split atoms, was solved using third-order anharmonic nuclear motion (ANM) parameters. Multipole refinement of the experimental high-resolution X-ray diffraction data of 1-(2′-aminophenyl)-2-methyl-4-nitro-1H-imidazole at three different temperatures (10, 35 and 70 K) and a series of powder diffraction experiments (20 ≤ T ≤ 300 K) were performed to relate this anharmonicity observed for several light atoms (N atoms of amino and nitro groups, and O atoms of nitro groups) to an isomorphic phase transition reflected by a change in the b cell parameter around 65 K. The observed disorder may result from the coexistence of domains of two phases over a large temperature range, as shown by low-temperature powder diffraction.
anharmonicity; isomorphic phase transition; experimental charge density; X-ray closed-circuit helium cryostat; Hansen–Coppens model; multiple-temperature powder diffraction
Single-structure models derived from X-ray data do not adequately account for the inherent, functionally important dynamics of protein molecules. We generated ensembles of structures by time-averaged refinement, where local molecular vibrations were sampled by molecular-dynamics (MD) simulation whilst global disorder was partitioned into an underlying overall translation–libration–screw (TLS) model. Modeling of 20 protein datasets at 1.1–3.1 Å resolution reduced cross-validated Rfree values by 0.3–4.9%, indicating that ensemble models fit the X-ray data better than single structures. The ensembles revealed that, while most proteins display a well-ordered core, some proteins exhibit a ‘molten core’ likely supporting functionally important dynamics in ligand binding, enzyme activity and protomer assembly. Order–disorder changes in HIV protease indicate a mechanism of entropy compensation for ordering the catalytic residues upon ligand binding by disordering specific core residues. Thus, ensemble refinement extracts dynamical details from the X-ray data that allow a more comprehensive understanding of structure–dynamics–function relationships.
It has been clear since the early days of structural biology in the late 1950s that proteins and other biomolecules are continually changing shape, and that these changes have an important influence on both the structure and function of the molecules. X-ray diffraction can provide detailed information about the structure of a protein, but only limited information about how its structure fluctuates over time. Detailed information about the dynamic behaviour of proteins is essential for a proper understanding of a variety of processes, including catalysis, ligand binding and protein–protein interactions, and could also prove useful in drug design.
Currently most of the X-ray crystal structures in the Protein Data Bank are ‘snap-shots’ with limited or no information about protein dynamics. However, X-ray diffraction patterns are affected by the dynamics of the protein, and also by distortions of the crystal lattice, so three-dimensional (3D) models of proteins ought to take these phenomena into account. Molecular-dynamics (MD) computer simulations transform 3D structures into 4D ‘molecular movies’ by predicting the movement of individual atoms.
Combining MD simulations with crystallographic data has the potential to produce more realistic ensemble models of proteins in which the atomic fluctuations are represented by multiple structures within the ensemble. Moreover, in addition to improved structural information, this process—which is called ensemble refinement—can provide dynamical information about the protein. Earlier attempts to do this ran into problems because the number of model parameters needed was greater than the number of observed data points. Burnley et al. now overcome this problem by modelling local molecular vibrations with MD simulations and, at the same time, using a course-grain model to describe global disorder of longer length scales.
Ensemble refinement of high-resolution X-ray diffraction datasets for 20 different proteins from the Protein Data Bank produced a better fit to the data than single structures for all 20 proteins. Ensemble refinement also revealed that 3 of the 20 proteins had a ‘molten core’, rather than the well-ordered residues core found in most proteins: this is likely to be important in various biological functions including ligand binding, filament formation and enzymatic function. Burnley et al. also showed that a HIV enzyme underwent an order–disorder transition that is likely to influence how this enzyme works, and that similar transitions might influence the interactions between the small-molecule drug Imatinib (also known as Gleevec) and the enzymes it targets. Ensemble refinement could be applied to the majority of crystallography data currently being collected, or collected in the past, so further insights into the properties and interactions of a variety of proteins and other biomolecules can be expected.
protein; crystallography; structure; function; dynamics; None
In order to overcome the difficulties associated with the ‘classical’ heavy-atom derivatization procedure, an attempt has been made to develop a rational crystal-free heavy-atom-derivative screening method and a quick-soak derivatization procedure which allows heavy-atom compound identification.
Despite the development in recent times of a range of techniques for phasing macromolecules, the conventional heavy-atom derivatization method still plays a significant role in protein structure determination. However, this method has become less popular in modern high-throughput oriented crystallography, mostly owing to its trial-and-error nature, which often results in lengthy empirical searches requiring large numbers of well diffracting crystals. In addition, the phasing power of heavy-atom derivatives is often compromised by lack of isomorphism or even loss of diffraction. In order to overcome the difficulties associated with the ‘classical’ heavy-atom derivatization procedure, an attempt has been made to develop a rational crystal-free heavy-atom derivative-screening method and a quick-soak derivatization procedure which allows heavy-atom compound identification. The method includes three basic steps: (i) the selection of likely reactive compounds for a given protein and specific crystallization conditions based on pre-defined heavy-atom compound reactivity profiles, (ii) screening of the chosen heavy-atom compounds for their ability to form protein adducts using mass spectrometry and (iii) derivatization of crystals with selected heavy-metal compounds using the quick-soak method to maximize diffraction quality and minimize non-isomorphism. Overall, this system streamlines the process of heavy-atom compound identification and minimizes the problem of non-isomorphism in phasing.
heavy-atom derivativization; heavy-atom screening; phasing; structure determination
The temperature dependence of the crystalline phase of (nitrosyl)(tetraphenylporphinato)-cobalt(II), [Co(TPP)(NO)], has been explored over the temperature range of 100–250 K by X-ray diffraction experiments. The crystalline complex is found in the tetragonal crystal system at higher temperatures and in the triclinic crystal system at lower temperatures. In the tetragonal system, the axial ligand is strongly disordered, with the molecule having crystallographically required 4/m symmetry, leading to eight distinct positions of the single nitrosyl oxygen atom. The phase transition to the triclinic crystal system leads to a partial ordering with the molecule now having inversion symmetry and disorder of the axial nitrosyl ligand over only two positions. At an intermediate temperature near the transition point, a transition structure in which the ordering observed at lower temperatures is only partially complete has been characterized. The increase in ordering allows subtle molecular geometry features to be observed. The transition of the reversible phase change begins at about 195 K. This transition has been confirmed by both X-ray diffraction studies and a differential scanning calorimetry study.
A novel sample cell with control of temperature and relative humidity permitted collection of data of excellent quality, enabling unrestrained refinement of all atomic parameters. One of the K atoms in the structure is disordered; very strong anisotropy in three of the four water O atoms indicates partial static disorder which does not involve the bonded H atoms.
The crystal structure of the high-temperature paraelectric phase of Rochelle salt (K+·Na+·C4H4O6
2−·4H2O) at 308 K has been reinvestigated using synchrotron X-ray diffraction with refinement parameters R(int) = 0.0123, final (shift/e.s.d.)max = 0.019, R
1(all) = 0.0371 and wR
2(all) = 0.0608. The application of a new gas-flow sample cell designed to control both temperature and relative humidity permitted collection of data of excellent quality and enabled unrestrained refinement of all parameters, including those of the isotropic hydrogen atoms. A precise description of the structure has ensued. One K atom is disordered between two symmetry-equivalent sites; three O atoms in three of the four water molecules exhibit very strong anisotropy. Refining one O atom as a split atom was successful, yielding small improvements in the bonding parameters of several H atoms. The H atoms of all water molecules behave as single pairs. Their final U values are of moderate magnitude indicating that these atoms do not participate in the anisotropy of the parent O atoms. It is suggested that the three water O atoms are in part statically disordered, while the bonded H atoms are not. Except for the split K atom and the three water O atoms there is no evidence of general disorder in the structure.
single-crystal X-ray study; synchrotron radiation; control of sample environment
The crystal structure of tamarugite [sodium aluminium bis(sulfate) hexahydrate] was redetermined from a single crystal from Mina Alcaparossa, near Cerritos Bayos, southwest of Calama, Chile. In contrast to the previous work [Robinson & Fang (1969 ▶). Am. Mineral.
54, 19–30], all non-H atoms were refined with anisotropic displacement parameters and H-atoms were located by difference Fourier methods and refined from X-ray diffraction data. The structure is built up from nearly regular [Al(H2O)6]3+ octahedra and infinite double-stranded chains [Na(SO4)2]3− that extend parallel to . The Na+ cation has a strongly distorted octahedral coordination by sulfate O atoms [Na—O = 2.2709 (11) – 2.5117 (12) Å], of which five are furnished by the chain-building sulfate group S2O4 and one by the non-bridging sulfate group S1O4. The [Na(SO4)2]3− chain features an unusual centrosymmetric group formed by two NaO6 octahedra and two S2O4 tetrahedra sharing five adjacent edges, one between two NaO6 octahedra and two each between the resulting double octahedron and two S2O4 tetrahedra. These groups are then linked into a double-stranded chain via corner-sharing between NaO6 octahedra and S2O4 tetrahedra. The S1O4 group, attached to Na in the terminal position, completes the chains. The [Al(H2O)6]3+ octahedron (〈Al—O〉 = 1.885 (11) Å) donates 12 comparatively strong hydrogen bonds (O⋯O = 2.6665 (14) – 2.7971 (15) Å) to the sulfate O atoms of three neighbouring [Na(SO4)2]3− chains, helping to connect them in three dimensions, but with a prevalence parallel to (010), the cleavage plane of the mineral. Compared with the previous work on tamarugite, the bond precision of Al—O bond lengths as an example improved from 0.024 to 0.001 Å.
Volcanic cristobalite commonly contains structural substitutions of Al3+ and Na+ for Si4+. Quantifying the effect of these substitutions on the crystal structure may provide insight into volcanic processes and the variable toxicity of crystalline silica.
Cristobalite is a common mineral in volcanic ash produced from dome-forming eruptions. Assessment of the respiratory hazard posed by volcanic ash requires understanding the nature of the cristobalite it contains. Volcanic cristobalite contains coupled substitutions of Al3+ and Na+ for Si4+; similar co-substitutions in synthetic cristobalite are known to modify the crystal structure, affecting the stability of the α and β forms and the observed transition between them. Here, for the first time, the dynamics and energy changes associated with the α–β phase transition in volcanic cristobalite are investigated using X-ray powder diffraction with simultaneous in situ heating and differential scanning calorimetry. At ambient temperature, volcanic cristobalite exists in the α form and has a larger cell volume than synthetic α-cristobalite; as a result, its diffraction pattern sits between ICDD α- and β-cristobalite library patterns, which could cause ambiguity in phase identification. On heating from ambient temperature, volcanic cristobalite exhibits a lower degree of thermal expansion than synthetic cristobalite, and it also has a lower α–β transition temperature (∼473 K) compared with synthetic cristobalite (upwards of 543 K); these observations are discussed in relation to the presence of Al3+ and Na+ defects. The transition shows a stable and reproducible hysteresis loop with α and β phases coexisting through the transition, suggesting that discrete crystals in the sample have different transition temperatures.
cristobalite; phase transitions
The temperature-dependence of the crystalline phase of (nitrosyl)(tetraphenylpor-phinato)iron(II), [Fe(TPP)(NO)], has been explored over the temperature range of 33 to 293 K. The crystalline complex is found in the tetragonal crystal system at higher temperatures and in the triclinic crystal system at lower temperatures. In the tetragonal system, the axial ligand is strongly disordered with the molecule having crystallographically required 4/m symmetry leading to eight distinct positions of the single nitrosyl oxygen atom. The phase transition to the triclinic crystal system leads to a partial ordering with the molecule now having inversion symmetry and disorder of the axial nitrosyl ligand over only two positions. The increase in ordering allows subtle molecular geometry features to be observed, in particular an off-axis tilt of the Fe–NNO bond from the heme normal is apparent. The transition of the reversible phase change begins at about 250 K. This transition has been confirmed by both X-ray diffraction studies and a differential scanning calorimetry study.
The short-range order (SRO) in Pd78Cu6Si16 liquid was studied by high energy x-ray diffraction and ab initio molecular dynamics (MD) simulations. The calculated pair correlation functions at different temperatures agree well with the experimental results. The partial pair correlation functions from ab intio MD simulations indicate that Si atoms prefer to be uniformly distributed while Cu atoms tend to aggregate. By performing structure analysis using Honeycutt-Andersen index, Voronoi tessellation, and atomic cluster alignment method, we show that the icosahedron and face-centered cubic SRO increase upon cooling. The dominant SRO is the Pd-centered Pd9Si2 motif, namely the structure of which motif is similar to the structure of Pd-centered clusters in the Pd9Si2 crystal. The study further confirms the existence of trigonal prism capped with three half-octahedra that is reported as a structural unit in Pd-based amorphous alloys. The majority of Cu-centered clusters are icosahedra, suggesting that the presence of Cu is benefit to promote the glass forming ability.
13 new phases of the inositols, 1,2,3,4,5,6-hexahydroxycyclohexane, were found. Crystal structure determinations and thermal analyses reveal a very complex picture of phases, rotator phases and phase transitions.
Inositol, 1,2,3,4,5,6-hexahydroxycyclohexane, exists in nine stereoisomers with different crystal structures and melting points. In a previous paper on the relationship between the melting points of the inositols and the hydrogen-bonding patterns in their crystal structures [Simperler et al. (2006 ▶). CrystEngComm
8, 589], it was noted that although all inositol crystal structures known at that time contained 12 hydrogen bonds per molecule, their melting points span a large range of about 170 °C. Our preliminary investigations suggested that the highest melting point must be corrected for the effect of molecular symmetry, and that the three lowest melting points may need to be revised. This prompted a full investigation, with additional experiments on six of the nine inositols. Thirteen new phases were discovered; for all of these their crystal structures were examined. The crystal structures of eight ordered phases could be determined, of which seven were obtained from laboratory X-ray powder diffraction data. Five additional phases turned out to be rotator phases and only their unit cells could be determined. Two previously unknown melting points were measured, as well as most enthalpies of melting. Several previously reported melting points were shown to be solid-to-solid phase transitions or decomposition points. Our experiments have revealed a complex picture of phases, rotator phases and phase transitions, in which a simple correlation between melting points and hydrogen-bonding patterns is not feasible.
inositol; X-ray powder diffraction; melting point; rotator phase; polymorphism
X-ray diffraction patterns from human arterial specimens containing atherosclerotic fatty streak lesions exhibited a single sharp reflection, corresponding to a structural spacing of about 35 A. Specimens without lesions did not. When specimens with fatty streaks were heated, an order-to-disorder phase transition was revealed by the disappearance of the sharp reflection. The transition was thermally reversible and its temperature varied from aorta to aorta over a range from 28 degrees to 42 degrees C. Since cholesteryl ester droplets are a major component of fatty streaks, comparison studies were made of the diffraction behavior from pure cholesteryl esters. We found that the diffraction patterns of the fatty streak material could be accounted for by the organization of the cholesteryl esters into a liquid-crystalline smectic phase that melts from the smectic to a less ordered phase upon heating. When combined with the conclusions of others from polarized light microscopy, our study shows that a droplet in the smectic phase has well-defined concentric layers of lipid molecules. In each layer, the long axes of the molecules have a net radial orientation with respect to the droplet, but the side-to-side organization is disordered. We suggest that the accessibility of portions of the lipids for specific binding to enzymes or transport proteins may be restricted when they are in the smectic state, and that exchange of lipids with surrounding membranes or other potential binding sites may likewise be inhibited. The restriction in the smectic phase should be greater than in the less ordered phases that exist at higher temperatures.
The crystal structures of two solid phases of the title compound, C4H5N2
−·H2O, have been determined at 225 and 120 K. In the high-temperature phase, stable above 198 K, the transition temperature of which has been determined by 35Cl nuclear quadrupole resonance and differential thermal analysis measurements, the three components are held together by O—H⋯O, N⋯H⋯O, C—H⋯O and C—H⋯Cl hydrogen bonds, forming a centrosymmetric 2+2+2 aggregate. In the N⋯H⋯O hydrogen bond formed between the pyrimidin-1-ium cation and the water molecule, the H atom is disordered over two positions, resulting in two states, viz. pyrimidin-1-ium–water and pyrimidine–oxonium. In the low-temperature phase, the title compound crystallizes in the same monoclinic space group and has a similar molecular packing, but the 2+2+2 aggregate loses the centrosymmetry, resulting in a doubling of the unit cell and two crystallographically independent molecules for each component in the asymmetric unit. The H atom in one N⋯H⋯O hydrogen bond between the pyrimidin-1-ium cation and the water molecule is disordered, while the H atom in the other hydrogen bond is found to be ordered at the N-atom site with a long N—H distance [1.10 (3) Å].
Phase relations and solidification behavior in the Ge-rich
of the phase diagram have been determined in two isothermal sections
at 700 and 750 °C and in a liquidus projection. A reaction scheme
has been derived in the form of a Schulz–Scheil diagram. Phase
equilibria are characterized by three ternary compounds: τ1-BaRhGe3 (BaNiSn3-type) and two novel
phases, τ2-Ba3Rh4Ge16 and τ3-Ba5Rh15Ge36-x, both forming in peritectic reactions. The crystal structures of
τ2 and τ3 have been elucidated from
single-crystal X-ray intensity data and were found to crystallize
in unique structure types: Ba3Rh4Ge16 is tetragonal (I4/mmm, a = 0.65643(2) nm, c = 2.20367(8) nm, and RF = 0.0273), whereas atoms in Ba5Rh15Ge36–x (x = 0.25) arrange in a large orthorhombic unit cell (Fddd, a = 0.84570(2) nm, b = 1.4725(2) nm, c = 6.644(3) nm, and RF = 0.034). The body-centered-cubic superstructure of
binary Ba8Ge43□3 was observed
to extend at 800 °C to Ba8Rh0.6Ge43□2.4, while the clathrate type I phase, κI-Ba8RhxGe46–x–y□y, reveals a maximum solubility of x = 1.2
Rh atoms in the structure at a vacancy level of y = 2.0. The cubic lattice parameter increases with increasing Rh
content. Clathrate I decomposes eutectoidally at 740 °C: κI ⇔ (Ge) + κIX + τ2. A very small solubility range is observed at 750 °C for the
clathrate IX, κIX-Ba6RhxGe25–x (x ∼ 0.16). Density functional theory calculations have been
performed to derive the enthalpies of formation and densities of states
for various compositions Ba8RhxGe46–x (x = 0–6).
The physical properties have been investigated for the phases κI, τ1, τ2, and τ3, documenting a change from thermoelectric (κI) to superconducting behavior (τ2). The electrical
resistivity of κI-Ba8Rh1.2Ge42.8□2.0 increases almost linearly with the
temperature from room temperature to 730 K, and the Seebeck coefficient
is negative throughout the same temperature range. τ1-BaRhGe3 has a typical metallic electrical resistivity.
A superconducting transition at TC = 6.5
K was observed for τ2-Ba3Rh4Ge16, whereas τ3-Ba5Rh15Ge35.75 showed metallic-like behavior down to
Phase equilibria in the Ba−Rh−Ge system are
characterized by three ternary cage compounds: τ 1-BaRhGe3 (BaNiSn3-type) and two novel phases
with unique structures, τ 2-Ba3Rh4Ge16 and τ 3-Ba5Rh15Ge36−x, besides κI-Ba8RhxGe46−x−y□y (x ≤ 1.2 and y ≥
2.0). Density functional theory calculations for the enthalpies of
formation and density of states for various compositions Ba8RhxGe46−x (x = 0−6) demonstrate a strong stabilizing
influence of Ge/Rh substitution. The physical properties have been
investigated for κI, τ 1, τ 2, and τ 3, documenting a change from n-type
thermoelectric (κI) to superconducting behavior (τ 2; TC = 6.5 K).
Vaults are the largest known cytoplasmic ribonucleoprotein structures and may function in innate immunity. The vault shell self-assembles from 96 copies of major vault protein and encapsulates two other proteins and a small RNA. We crystallized rat liver vaults and several recombinant vaults, all among the largest non-icosahedral particles to have been crystallized. The best crystals thus far were formed from empty vaults built from a cysteine-tag construct of major vault protein (termed cpMVP vaults), diffracting to about 9-Å resolution. The asymmetric unit contains a half vault of molecular mass 4.65 MDa. X-ray phasing was initiated by molecular replacement, using density from cryo-electron microscopy (cryo-EM). Phases were improved by density modification, including concentric 24- and 48-fold rotational symmetry averaging. From this, the continuous cryo-EM electron density separated into domain-like blocks. A draft atomic model of cpMVP was fit to this improved density from 15 domain models. Three domains were adapted from a nuclear magnetic resonance substructure. Nine domain models originated in ab initio tertiary structure prediction. Three C-terminal domains were built by fitting poly-alanine to the electron density. Locations of loops in this model provide sites to test vault functions and to exploit vaults as nanocapsules.
Vaults are large barrel-shaped particles found in the cytoplasm in all mammalian cells, which may function in innate immunity. As naturally occurring nanoscale capsules, vaults may be useful objects to engineer as delivery vehicles. In this study, we propose an atomic structure for the thin outer shell of the vault. Using x-ray diffraction and computer modeling, we have inferred a draft atomic model for the major vault protein, which forms the shell-like enclosure of the vault. The shell is made up of 96 identical protein chains, each of 873 amino acid residues, folded into 14 domains. Each chain forms an elongated stave of half the vault, as well as the cap of the barrel-like shell. Our draft atomic model is essentially an atomic-level model for the entire 9.3-MDa vault shell, which offers a guide for protein engineering to test vault functions and to exploit vault particles as nanocapsules.
A draft atomic structure has been determined for the 9.3-MDa protein shell of the vault cytoplasmic particle, revealing stave-like polypeptides forming the barrel-like structure of the vault.
High-quality, ice-free X-ray diffraction data were continuously collected while cryoprotectant-free thaumatin crystals were cooled at 0.1 K s−1 from 300 to 100 K. This establishes the feasibility of fully temperature controlled studies of protein structure and dynamics, and provides insight into how cooling creates crystal disorder.
Cryoprotectant-free thaumatin crystals have been cooled from 300 to 100 K at a rate of 0.1 K s−1 – 103–104 times slower than in conventional flash cooling – while continuously collecting X-ray diffraction data, so as to follow the evolution of protein lattice and solvent properties during cooling. Diffraction patterns show no evidence of crystalline ice at any temperature. This indicates that the lattice of protein molecules is itself an excellent cryoprotectant, and with sodium potassium tartrate incorporated from the 1.5 M mother liquor ice nucleation rates are at least as low as in a 70% glycerol solution. Crystal quality during slow cooling remains high, with an average mosaicity at 100 K of 0.2°. Most of the mosaicity increase occurs above ∼200 K, where the solvent is still liquid, and is concurrent with an anisotropic contraction of the unit cell. Near 180 K a crossover to solid-like solvent behavior occurs, and on further cooling there is no additional degradation of crystal order. The variation of B factor with temperature shows clear evidence of a protein dynamical transition near 210 K, and at lower temperatures the slope dB/dT is a factor of 3–6 smaller than has been reported for any other protein. These results establish the feasibility of fully temperature controlled studies of protein structure and dynamics between 300 and 100 K.
protein crystallography; cryocrystallography; X-ray diffraction; slow cooling; temperature control
The radiation-induced disordering of selenomethionine (SeMet) side chains represents a significant impediment to protein structure solution. Not only does the increased B-factor of these sites result in a serious drop in phasing power, but some sites decay much faster than others in the same unit cell. These radiolabile SeMet side chains decay faster than high-order diffraction spots with dose, making it difficult to detect this kind of damage by inspection of the diffraction pattern. The selenium X-ray absorbance near-edge spectrum (XANES) from samples containing SeMet was found to change significantly after application of X-ray doses of 10–100 MGy. Most notably, the sharp ‘white line’ feature near the canonical Se edge disappears. The change was attributed to breakage of the Cγ—Se bond in SeMet. This spectral change was used as a probe to measure the decay rate of SeMet with X-ray dose in cryo-cooled samples. Two protein crystal types and 15 solutions containing free SeMet amino acid were examined. The damage rate was influenced by the chemical and physical condition of the sample, and the half-decaying dose for the selenium XANES signal ranged from 5 to 43 MGy. These decay rates were 34- to 3.8-fold higher than the rate at which the Se atoms interacted directly with X-ray photons, so the damage mechanism must be a secondary effect. Samples that cooled to a more crystalline state generally decayed faster than samples that cooled to an amorphous solid. The single exception was a protein crystal where a nanocrystalline cryoprotectant had a protective effect. Lowering the pH, especially with ascorbic or nitric acids, had a protective effect, and SeMet lifetime increased monotonically with decreasing sample temperature (down to 93 K). The SeMet lifetime in one protein crystal was the same as that of the free amino acid, and the longest SeMet lifetime measured was found in the other protein crystal type. This protection was found to arise from the folded structure of the protein molecule. A mechanism to explain observed decay rates involving the damaging species following the electric field lines around protein molecules is proposed.
X-ray dose; protective solutes; pH and temperature effects; protein crystal structure; mechanism of radiation damage
We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1–1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. This proof of principle paves the way for the implementation of a new technique, which we name ‘MicroED’, that may have wide applicability in structural biology.
X-ray crystallography has been used to work out the atomic structure of a large number of proteins. In a typical X-ray crystallography experiment, a beam of X-rays is directed at a protein crystal, which scatters some of the X-ray photons to produce a diffraction pattern. The crystal is then rotated through a small angle and another diffraction pattern is recorded. Finally, after this process has been repeated enough times, it is possible to work backwards from the diffraction patterns to figure out the structure of the protein.
The crystals used for X-ray crystallography must be large to withstand the damage caused by repeated exposure to the X-ray beam. However, some proteins do not form crystals at all, and others only form small crystals. It is possible to overcome this problem by using extremely short pulses of X-rays, but this requires a very large number of small crystals and ultrashort X-ray pulses are only available at a handful of research centers around the world. There is, therefore, a need for other approaches that can determine the structure of proteins that only form small crystals.
Electron crystallography is similar to X-ray crystallography in that a protein crystal scatters a beam to produce a diffraction pattern. However, the interactions between the electrons in the beam and the crystal are much stronger than those between the X-ray photons and the crystal. This means that meaningful amounts of data can be collected from much smaller crystals. However, it is normally only possible to collect one diffraction pattern from each crystal because of beam induced damage. Researchers have developed methods to merge the diffraction patterns produced by hundreds of small crystals, but to date these techniques have only worked with very thin two-dimensional crystals that contain only one layer of the protein of interest.
Now Shi et al. report a new approach to electron crystallography that works with very small three-dimensional crystals. Called MicroED, this technique involves placing the crystal in a transmission electron cryo-microscope, which is a fairly standard piece of equipment in many laboratories. The normal ‘low-dose’ electron beam in one of these microscopes would normally damage the crystal after a single diffraction pattern had been collected. However, Shi et al. realized that it was possible to obtain diffraction patterns without severely damaging the crystal if they dramatically reduced the normal low-dose electron beam. By reducing the electron dose by a factor of 200, it was possible to collect up to 90 diffraction patterns from the same, very small, three-dimensional crystal, and then—similar to what happens in X-ray crystallography—work backwards to figure out the structure of the protein. Shi et al. demonstrated the feasibility of the MicroED approach by using it to determine the structure of lysozyme, which is widely used as a test protein in crystallography, with a resolution of 2.9 Å. This proof-of principle study paves the way for crystallographers to study protein that cannot be studied with existing techniques.
electron crystallography; electron diffraction; electron cryomicroscopy (cryo-EM); microED; protein structure; microcrystals; None
The oxygen radical scavenger activity (ORSA) of [CuII(Pir)2] (HPir = Piroxicam = 4-hydroxy -2- methyl -N-2-
pyridyl -2H- 1,2-benzothiazine -3- carboxamide 1,1-dioxide) was determined by
chemiluminescence of samples obtained by mixing human neutrophils (from healthy subjects) and [CuII(Pir)2(DMF)2] (DMF = N,N -dimethylformammide) in DMSO/GLY/PBS (2:1:2, v/v) solution
(DMSO = dimethylsulfoxide, GLY = 1,2,3-propantriol, PBS = Dulbecco’s buffer salt solution). The ratio of the residual radicals, for the HPir (1.02·10−4M)
(1.08·10−5M)/HPir (8.01·10−−5M) systems was higher than 12 (not stimulated) [excess of piroxicam was added (Cu/Pir molar ratio ≈1:10) in order to have most of the metal complexed as bischelate]. In contrast, the ratio
of residual radicals for the CuCl2 (1.00·10−5M) and [CuII(Pir)2(DMF)2] (1.08·10−5M)/Hpir (8.01·10−5M)system was 5. The [CuII(Pir)2] compound is therefore a stronger radical scavenger than either
HPir or CuCl2. A molecular mechanics (MM) analysis of the gas phase structures of neutral HPir, its
zwitterionic (HPir+-) and anionic (Pir-) forms, and some CuII-piroxicam complexes based on X-ray structures allowed calculation of force constants. The most stable structure for HPir has a ZZZ conformation similar to that found in the CuII (and CdII complexes) in the solid state as well as in the gas phase. The structure is stabilized by a strong H bond which involves the N(amide)-H and O(enolic) groups. The MM simulation for the [CuII(Pir)2(DMF)2] complex showed that two high repulsive intramolecular contacts exist between a pyridyl hydrogen atom of one Pir- molecule with
the O donor of the other ligand. These interactions activate a transition toward a pseudo-tetrahedral
geometry, in the case the apical ligands are removed. On refluxing a suspension of
in acetone a brown microcystalline solid with the Cu(Pir)2·0.5DMF
was in fact prepared. 13C spin-lattice relaxation rates of neutral, zwitterionic and anionic piroxicam,
in DMSO solution are explained by the thermal equilibrium between the three most stable
structures of the three forms, thus confirming the high quality of the force field. The EPR spectrum
of [CuII(Pir)2(DMF)2] (DMSO/GLY, 2:1, v/v, 298 and 110 K) agrees with a N2O2+O2 pseudo-octahedral
coordination geometry. The EPR spectrum of [CuII(Pir)2·0.5DMF
agrees with a pseudo-tetrahedral coordination geometry. The parameters extracted from the room temperature spectra of
the solution phases are in agreement with the data reported for powder and frozen solutions. The
extended-Hückel calculations on minimum energy structures of [CuII(Pir)2(DMF)2] and [CuII(Pir)2]
(square planar) revealed that the HOMOs have a relevant character of dx2−y2. On the other hand
the HOMO of a computer generated structure for [CuII(Pir)2] (pseudo-tetrahedral) has a relevant
character of dxy atomic orbital. A dxy orbital is better suited to allow a dπ-pπ interaction to the O2- anion. Therefore this work shows that the anti-inflammatory activity of piroxicam could be due in part
to the formation of [CuII(Pir)2]
chelates, which can exert a SOD-like activity.
Explorations of phases in the quaternary FeIII–BIII–PV–O system prepared by the high temperature solution growth (HTSG) method led to single-crystal growth of anhydrous diiron(III) borotriphosphate, Fe2[BP3O12]. This phase has been synthesized previously as a microcrystalline material and its structure refined in space group P3 from powder X-ray diffraction data using the Rietveld method [Chen et al. (2004 ▶). J. Inorg. Mater.
19, 429-432]. In the current single-crystal study, it was shown that the correct space group is P63/m. The three-dimensional structure of the title compound is built up from FeO6 octahedra (3.. symmetry), trigonal–planar BO3 groups ( symmetry) and PO4 tetrahedra (m.. symmetry). Two FeO6 octahedra form Fe2O9 dimers via face-sharing, while the anionic BO3 and PO4 groups are connected via corner-sharing to build up the [BP3O12]6− anion. Both units are interconnected via corner-sharing.
The crystal structure of durangite, ideally NaAl(AsO4)F (chemical name sodium aluminium arsenate fluoride), has been determined previously [Kokkoros (1938). Z. Kristallogr.
99, 38–49] using Weissenberg film data without reporting displacement parameters of atoms or a reliability factor. This study reports the redetermination of the structure of durangite using single-crystal X-ray diffraction data from a natural sample with composition (Na0.95Li0.05)(Al0.91Fe3+
0.02)(AsO4)(F0.73(OH)0.27) from the type locality, the Barranca mine, Coneto de Comonfort, Durango, Mexico. Durangite is isostructural with minerals of the titanite group in the space group C2/c. Its structure is characterized by kinked chains of corner-sharing AlO4F2 octahedra parallel to the c axis. These chains are cross-linked by isolated AsO4 tetrahedra, forming a three-dimensional framework. The Na+ cation (site symmetry 2) occupies the interstitial sites and is coordinated by one F− and six O2− anions. The AlO4F2 octahedron has symmetry -1; it is flattened, with the Al—F bond length [1.8457 (4) Å] shorter than the Al—O bond lengths [1.8913 (8) and 1.9002 (9) Å]. Examination of the Raman spectra for arsenate minerals in the titanite group reveals that the position of the band originating from the As—O symmetric stretching vibrations shifts to lower wavenumbers from durangite, maxwellite [ideally NaFe(AsO4)F], to tilasite [CaMg(AsO4)F].
The co-crystallization of cyclic and polymeric isomers in the same crystal in varying ratios with the skeleton frameworks packed in a geometrically compatible and energetically similar fashion gives a chance to rationalize ring-opening isomerization in a crystal growth process.
A rare example is reported in which discrete Ag2
2 ring and (AgL)∞ chain motifs [L = N,N′-bis(3-imidazol-1-yl-propyl)-pyromellitic diimide] co-crystallize in the same crystal lattice with varying ratios and degrees of disorder. Crystal structures obtained from representative crystals reveal compatible packing arrangements of the cyclic and polymeric isomers within the crystal lattice, which enables them to co-exist within a crystalline solid solution. A feasible pathway for transformation between the isomers is suggested via facile rotation of the coordinating imidazolyl groups. This chemical system could provide a chance for direct observation of ring-opening isomerization at the crystal surface. Mass spectrometry and 1H NMR titration show a dynamic equilibrium between cyclic and oligomeric species in solution, and a potential crystallization process is suggested involving alignment of precursors directed by aromatic stacking interactions between pyromellitic diimide units, followed by ring-opening isomerization at the interface between the solid and the solution. Both cyclic and oligomeric species can act as precursors, with interconversion between them being facile due to a low energy barrier for rotation of the imidazole rings. Thermogravimetric analysis and variable-temperature powder X-ray diffraction indicate a transition to a different crystalline phase around 120°C, which is associated with loss of solvent from the crystal lattice.
crystallization; structural transformation; ring-opening isomerism; solid solution; disorder
The 2-hydroxycyclohexane-1,3,5-triaminium (= H3
3+) cation of the title compound, 3C6H18N3O3+·8Cl−·HSO4
−·2H2O, exhibits a cyclohexane chair with three equatorial ammonium groups and one axial hydroxy group in an all-cis configuration. The hydrogen sulfate anion and two water molecules lie on or in proximity to a threefold axis and are disordered. The crystal structure features N—H⋯Cl and O—H⋯Cl hydrogen bonds. Three C
3-symmetric motifs can be identified in the structure: (i) Two chloride ions (on the C
3-axis) together with three H3
3+ cations constitute an [(H3
L)3Cl2]7+ cage. (ii) The lipophilic C6H6-sides of three H3
3+ cations, which are oriented directly towards the C
3-axis, generate a lipophilic void. The void is filled with the disordered water molecules and with the disordered part of the hydrogen sulfate ion. The hydrogen atoms of these disordered moieties were not located. (iii) Three H3
3+ cations together with one HSO4
− and three Cl− counter-ions form an [(HSO4)(H3
L)3Cl3]5+ cage. Looking along the C
3-axis, these three motifs are arranged in the order (cage 1)⋯(lipophilic void)⋯(cage 2). The crystal studied was found to be a racemic twin.
Three fusion proteins were generated in order to resolve the atomic structure of the CFA/I fimbriae of enterotoxigenic E. coli. CfaEB is a fusion of the minor and major CFA/I subunits, while CfaBB and CfaBBB are tandem fusions of two and three repeats, respectively, of the major subunit. Each protein was crystallized and the crystal structures of each of these fusions were determined successively by the molecular-replacement method using the CfaE crystal structure as an initial phasing model.
Enterotoxigenic Escherichia coli (ETEC), a major global cause of diarrhea, initiates the pathogenic process via fimbriae-mediated attachment to the small intestinal epithelium. A common prototypic ETEC fimbria, colonization factor antigen I (CFA/I), consists of a tip-localized minor adhesive subunit CfaE and the stalk-forming major subunit CfaB, both of which are necessary for fimbrial assembly. To elucidate the structure of CFA/I at atomic resolution, three recombinant proteins were generated consisting of fusions of the minor and major subunits (CfaEB) and of two (CfaBB) and three (CfaBBB) repeats of the major subunit. Crystals of CfaEB diffracted X-rays to 2.1 Å resolution and displayed the symmetry of space group P21. CfaBB exhibited a crystal diffraction limit of 2.3 Å resolution and had the symmetry of space group P21212. CfaBBB crystallized in the monoclinic space group C2 and diffracted X-rays to 2.3 Å resolution. These structures were determined using the molecular-replacement method.
colonization factor antigen I fimbriae; CfaB subunit; enterotoxigenic Escherichia coli
Three compounds, each derived from Fentanyl and differing essentially only in the length of a carboxylic acid chain, were synthesized and yielded four crystal structures three of which share several structural similarities, including the length of the chain, while the fourth, with a shorter chain, is quite different. The chain length has a significant influence on the crystal structures formed. The ‘three atom’ chain compounds are all solvated zwitterions which feature a hydrogen-bonded ‘dimer’ between adjacent zwitterions. The formation of this large dimer leaves available a second carboxylate O atom to take part in hydrogen bonding interactions with solvent molecules. The shorter ‘two atom’ chain compound was difficult to crystallize and required the use of synchrotron radiation to measure X-ray diffraction data. It does not form the same dimer motif observed in the ‘three atom’ chain compounds and has not formally formed a zwitterion; although there is evidence of proton sharing or disorder X-ray data are insufficient to create a disordered model, and the compound was modeled as formally neutral based on O–H and N–H distances. Room temperature analyses showed the proton transfer behavior to be independent of crystal temperature, and nuclear magnetic resonance studies show proton transfer behavior in solution. The formation of a zwitterionic hydrogen-bonded dimer is implicated in providing some stability during crystal growth of the easily crystallized ‘three atom’ chain compounds.
Relating three-dimensional fold to function is a central challenge in RNA structural biology. Toward this goal, X-ray crystallography has long been considered the “gold standard” for structure determinations at atomic resolution, although NMR spectroscopy has become a powerhouse in this arena as well. In the area of dynamics, NMR remains the dominant technique to probe the magnitude and timescales of molecular motion. Although the latter area remains largely unassailable by conventional crystallographic methods, inroads have been made on proteins using Laue radiation on timescales of ms to ns. Proposed ‘fourth generation’ radiation sources, such as free-electron X-ray lasers, promise ps- to fs-timescale resolution, and credible evidence is emerging that supports the feasibility of single molecule imaging. At present however, the preponderance of RNA structural information has been derived from timescale and motion insensitive crystallographic techniques. Importantly, developments in computing, automation and high-flux synchrotron sources have propelled the rapidity of ‘conventional’ RNA crystal structure determinations to timeframes of hours once a suitable set of phases is obtained. With a sufficient number of crystal structures, it is possible to create a structural ensemble that can provide insight into global and local molecular motion characteristics that are relevant to biological function. Here we describe techniques to explore conformational changes in the hairpin ribozyme, a representative non-protein-coding RNA catalyst. The approaches discussed include: (i) construct choice and design using prior knowledge to improve X-ray diffraction; (ii) recognition of long-range conformational changes; and (iii) use of single-base or single-atom changes to create ensembles. The methods are broadly applicable to other RNA systems.
RNA crystallography; ribozyme; crystallization; RNA structure; crystallographic ensembles; alternate conformation; long-range motion; fold and function; difference Fourier; non-protein-coding RNA