Acoustically mounted microcrystals yield high-resolution X-ray structures

We demonstrate a general strategy for determining structures from showers of microcrystals. It uses acoustic droplet ejection to transfer 2.5 nL droplets from the surface of microcrystal slurries, through the air, onto mounting micromesh pins. Individual microcrystals are located by raster-scanning a several-micrometer X-ray beam across the cryocooled micromeshes. X-ray diffraction data sets merged from several micrometer-sized crystals are used to determine 1.8 ?? resolution crystal structures.     

Incorporation of microcrystals by growing protein and virus crystals

In the course of time-lapse video and atomic force microscopy (AFM) investigations of macromolecular crystal growth, we frequently observed the sedimentation of microcrystals and three-dimensional nuclei onto the surfaces of much larger, growing protein or virus crystals. This was followed by the direct incorporation over time of the smaller crystals into the bulk of the larger crystals. In some cases, clear indications were present that upon absorption of the small crystal onto the surface of the larger, there was proper alignment of the respective lattices, and consolidation proceeded without observable defect formation, i.e., the two lattices knitted together without discontinuity. In the case of at least one virus crystal, cubic satellite tobacco mosaic virus (STMV), addition of three-dimensional nuclei and subsequent expansion provided the principal growth mechanism at high supersaturation. This process has not been reported for growth from solution of conventional crystals. In numerous other instances, the lattices of the small and larger crystals were obviously misaligned, and incorporation occurred with the formation of some defect. This phenomenon of small crystals physically embedded in larger crystals could only degrade the overall diffraction and materials properties of macromolecular crystals.     

Structure of desheptapeptide (B24–B30) insulin in a new crystal form

The structure of desheptapeptide (B24–B30) insulin (DHPI) in a new crystal form (form B) has been determined and refined to 0.2 nm resolution. The crystals were obtained under the same crystallization condition as previously reported crystal form (form A). The overall structures of the two crystal forms are similar but obvious differences can be observed in crystal packing and local conformation. The crystal structures of the two forms show that the two independent molecules in an asymmetric unit from a DHPI dimer, and the dimer formation buries more than 18.20 and 16.95 nm² of solvent accessible surfaces for form A and form B DHPI, respectively, the largest among insulin and insulin analogs ever reported. Close examination at crystal packing shows that the dimer-forming surface of DHPI, namely Surface II, is normally present in the association of insulin and insulin analogs in their crystal structures. The results demonstrate that Surface II is crucially important for the formation of two crystal forms under the same crystallization condition.     

Structure of desheptapeptide (B24-B30) insulin in a new crystal form

The structure of desheptapeptide (B24-B30) insulin (DHPI) in a new crystal form (form B) has been determined and refined to 0.2 nm resolution. The crystals were obtained under the same crystallization condition as previously reported crystal form (form A). The overall structures of the two crystal forms are similar but obvious differences can be observed in crystal packing and local conformation. The crystal structures of the two forms show that the two independent molecules in an asymmetric unit from a DHPI dimer, and the dimer formation buries more than 18.20 and 16.95 nm~2 of solvent accessible surfaces for form A and form B DHPI, respectively, the largest among insulin and insulin analogs ever reported. Close examination at crystal packing shows that the dimer-forming surface of DHPI, namely Surface Ⅱ, is normally present in the association of insulin and insulin analogs in their crystal structures. The results demonstrate that Surface Ⅱ is crucially important for the formation of two crystal form     

Structural and morphological characterization of ultralente insulin crystals by atomic force microscopy: evidence of hydrophobically driven assembly.

Although x-ray crystal structures exist for many forms of insulin, the hormone involved in glucose metabolism and used in the treatment of diabetes, x-ray structural characterization of therapeutically important long-acting crystalline ultralente insulin forms has been elusive because of small crystal size and poor diffraction characteristics. We describe tapping-mode atomic force microscopy (TMAFM) studies, performed directly in crystallization liquor, of ultralente crystals prepared from bovine, human, and porcine insulins. Lattice images obtained from direct imaging of crystal planes are consistent with R3 space group symmetry for each insulin type, but the morphology of the human and porcine crystals observed by AFM differs substantially from that of the bovine insulin crystals. Human and porcine ultralente crystals exhibited large, molecularly flat (001) faces consisting of hexagonal arrays of close packed hexamers. In contrast, bovine ultralente crystals predominantly exhibited faces with cylindrical features assignable to close-packed stacks of insulin hexamers laying in-plane, consistent with the packing motif of the (010) and (011) planes. This behavior is attributed to a twofold increase in the hydrophobic character of the upper and lower surfaces of the donut-shaped insulin hexamer in bovine insulin compared to its human and porcine counterparts that results from minor sequence differences between these insulins. The increased hydrophobicity of these surfaces can promote hexamer-hexamer stacking in precrystalline aggregates or enhance attachment of single hexamers along the c axis at the crystal surface during crystal growth. Both events lead to enhanced growth of ¿hk0¿ planes instead of (001). The insulin hexamers on the (010) and (110) faces are exposed "edge-on" to the aqueous medium, such that solvent access to the center of the hexamer and to solvent channels is reduced compared to the (001) surface, consistent with the slower dissolution and reputed unique basal activity of bovine ultralente insulin. These observations demonstrate that subtle variations in amino acid sequence can dramatically affect the interfacial structure of crystalline proteins.     

X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule. A completely inactive analogue.

A crystal structure of a totally inactive insulin molecule has been determined. For this insulin molecule, the first without detectable activity to be characterized, the A and B-chains are linked by a peptide bond between A1 Gly and B29 Lys. The molecule has retained all its normal self-association properties and it can also accommodate the two different conformations designated T and R, as seen in 4Zn native pig insulin crystals. The hexamers of the crosslinked insulin molecule were crystallized using the 4Zn insulin recipe of Schlichtkrull. The structure has been crystallographically refined with data extending to 2 A using restrained least-square methods. Comparison of the B29-A1 peptide crosslink insulin and the 4Zn native insulin reveals close structural similarities with the native dimer. The analysis of the structure confirms the earlier hypothesis that insulin structures in crystals are not in an active conformation and that a separation of N-terminal A-chain and C-terminal B-chain is required for interaction with the insulin receptor.     

The validity of the cholesterol nucleation assay.

The validity of the cholesterol nucleation assay rests on the assumption that all cholesterol crystals are removed at the start of the assay so that de novo formation of crystals can be studied. In this paper we have tested the validity of this assumption. Cholesterol crystals were added to supersaturated model bile. Subsequently the mixtures were either filtered over a 0.22 micron filter or centrifuged at 37 degrees C for 2 h at 100,000 x g. After ultracentrifugation the isotropic interphase was collected. Using polarized light microscopy no crystals could be visualized in this fraction. However, the nucleation time of the isotropic interphase decreased from 6.8 +/- 1.1 days to 1.8 +/- 0.2 days (mean +/- S.E., P less than 0.01, n = 5) when 10-100 micrograms/ml crystals were added prior to centrifugation. Similar results were observed when instead of centrifugation the mixtures containing crystals were filtered. After filtration over a 0.22 micron filter no crystals could be detected in the filtrate. Yet the nucleation time of the filtrate decreased from 6.4 +/- 0.7 days to 3.1 +/- 0.5 days (mean +/- S.E.) when 10 micrograms/ml cholesterol crystals were added before filtration (n = 10, P less than 0.01). Since no cholesterol crystals could be detected at the start of the assay the reduction in nucleation time must have been brought about by cholesterol microcrystals that passed through the filter. Supplementation of cholesterol crystals to model bile did not accelerate the nucleation time when the samples were passed over a 0.02 micron filter, indicating that the size of the microcrystals was larger than 20 nm. The effect of addition of cholesterol crystals prior to filtration over a 0.22 micron filter was also tested in the crystal growth assay recently developed by Busch et al. ((1990) J. Lipid Res. 31, 1903-1909). Addition of crystals had only a minor effect on the assay. In conclusion, the reduced nucleation time of biles from gallstone patients is probably not only due to the presence of promoting or the absence of inhibiting proteins, but can be caused by the presence of small cholesterol crystals in these biles.     

Insulin replacement therapy for the rat model of streptozotocin-induced diabetes mellitus

OBJECTIVE: This study was conducted to compare various strategies for insulin replacement therapy in the streptozotocin-induced diabetic rat model. METHODS: Control and diabetic Sprague Dawley rats were fed ad libitum, blood glucose concentration was measured twice daily, and outcome was assessed over the final 5 days of a 10-day treatment period, with adjustment of insulin dosage toward the goal of normal glucose values. RESULTS: All insulin regimens induced weight gain at least comparable to that of controls, but glucose regulation differed. It was not possible to normalize glucose values by use of protamine zinc insulin (PZI) or Ultralente insulin given once daily. In contrast, PZI and neutral protamine Hagedorn (NPH) insulin given twice daily provided glucose values comparable to those in controls, whereas glucose values were modestly higher in response to a 70% human insulin isophane suspension and 30% soluble human insulin solution (70/ 30 insulin) given twice daily. Attempted normalization of glucose values was limited by hypoglycemia, which was most common after administration of PZI once daily, and least common after 70/30 insulin given twice daily. Dosage requirements for Ultralente insulin were four- to fivefold higher than those for all other insulins. CONCLUSION: In streptozotocin-diabetic rats, normal weight gain can be achieved by treatment with PZI insulin once daily, but attainment of near-normal glucose values requires administration of PZI, NPH, or 70/ 30 insulin twice daily. Ultralente insulin may have reduced bioeffectiveness in this animal model.     

High-efficiency screening of monoclonal antibodies for membrane protein crystallography

Determination of crystal structures of membrane proteins is often limited by difficulties obtaining crystals diffracting to high resolution. Co-crystallization with Fab fragments of monoclonal antibodies has been reported to improve diffraction of membrane proteins crystals. However, it is not simple to generate useful monoclonal antibodies for membrane protein crystallography. In this report, we present an optimized process for efficient screening from immunization to final validation of monoclonal antibody for membrane protein crystallography.     

Directional degradation of β-chitin by chitinase A1 revealed by a novel reducing end labelling technique

A novel procedure for labelling the molecular ends of β-chitin crystals has been established. By introducing a hydrazide derivative of biotin at the reducing end of a chitin chain, followed by a specific interaction between biotin and streptavidin coupled with a colloidal gold particle, the chain directionality of β-chitin microcrystals could be directly visualized by transmission electron microscopy. This method allowed to certify the parallelism of the chitin chains in the β-chitin microcrystals, and also to label the reducing tips of β-chitin microcrystals degraded by Bacillus circulans chitinase A1. With these substrates, the labelling occurred only at their tapered tip, which indicates that the digestion of these crystals proceeded from their reducing end. The generalization of this new labelling method to other polysaccharide crystals is discussed.     

Three-dimensional crystals of CaATPase from sarcoplasmic reticulum. Symmetry and molecular packing.

Structural studies of CaATPase from sarcoplasmic reticulum have so far been restricted to low resolution due to the poor order of two-dimensional crystal forms. However, we report that three-dimensional microcrystals of detergent-solubilized CaATPase diffract to 7.2 A in x-ray powder patterns and may therefore provide an opportunity to study CaATPase structure at higher resolutions. In the present study, we have characterized the symmetry and molecular packing of negatively stained crystals by electron microscopy (em). By altering the detergent-to-lipid ratio, different sized crystals were produced, which adhere to an em grid in different orientations. Thus, we obtained micrographs of three different projections and from these determined unit cell dimensions to be 151 X 51 X 158 A and the three-dimensional space group to be C2 with an angle beta very close to 90 degrees; x-ray powder patterns of hydrated, unstained crystals yielded dimensions of 166 X 58 X 164 A. Micrographs from each of two principal projections were averaged to produce two-dimensional density maps. Based on these maps and on the previously determined low-resolution structure of CaATPase, a packing diagram for these three-dimensional crystals is presented and major intermolecular contacts are proposed.     

Microcrystals of tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium are catalytically active

An improved and efficient method has been developed for the purification of the tryptophan synthase alpha 2 beta 2 complex (EC 4.2.1.20) from Salmonella typhimurium containing a multicopy plasmid. Microcrystals prepared in 12% poly(ethylene glycol) 8000 containing 2.5 mM spermine are shown by scanning electron microscopy to have the same crystal habit as the larger crystals that are being used for structural analysis by X-ray crystallography. The average dimensions of the crystals are 33 microns (length) X 9 microns (width) X 3 microns (maximum thickness). Our finding that suspensions of microcrystals are active in several reactions catalyzed by the active sites of the alpha and beta 2 subunits demonstrates that both active sites are functional in the crystal and accessible to substrates. Thus the larger crystals being used for X-ray crystallographic studies should form complexes with substrates and analogues at both active sites and should yield functionally relevant structural information. A comparison of the reaction rates of suspensions of microcrystals with those of the soluble enzyme shows that the maximum rate of the crystalline enzyme is 0.8 that of the soluble enzyme in the cleavage of indole-3-glycerol phosphate (alpha reaction), 0.3 that of the soluble enzyme in the synthesis of L-tryptophan by the beta reaction or the coupled alpha beta reaction, and 2.7 that of the soluble enzyme in the serine deaminase reaction. These small differences in rates probably reflect functional differences between the crystalline and soluble enzymes since the reaction rates of the microcrystals are calculated to be virtually free of diffusional limitation under these reaction conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure of 2Zn pig insulin crystals at 1.5 A resolution

The paper describes the arrangement of the atoms within rhombohedral crystals of 2Zn pig insulin as seen in electron density maps calculated from X-ray data extending to 1.5 A (1 A = 10(-10) m = 10(-1) nm) at room temperature and refined to R = 0.153. The unit cell contains 2 zinc ions, 6 insulin molecules and about 3 x 283 water molecules. The atoms in the protein molecules appear well defined, 7 of the 102 side chains in the asymmetric unit have been assigned alternative disordered positions. The electron density over the water molecules has been interpreted in terms of 349 sites, 217 weighted 1.0, 126 weighted 0.5, 5 at 0.33 and 1 at 0.25 giving ca. 282 molecules. The positions and contacts of all the residues belonging to the two A and B chains of the asymmetric unit are shown first and then details of their arrangement in the two insulin molecules, 1 and 2, which are different. The formation from these molecules of a compact dimer and the further aggregation of three dimers to form a hexamer around two zinc ions, follows. It appears that in the packing of the hexamers in the crystal there are conflicting influences; too-close contacts between histidine B5 residues in neighbouring hexamers are probably responsible for movements of atoms at the beginning of the A chain of one of the two molecules of the dimer that initiate movements in other parts, particularly near the end of the B chain. At every stage of the building of the protein structure, residues to chains of definite conformation, molecules, dimers, hexamers and crystals, we can trace the effect of the packing of like groups to like, aliphatic groups together, aromatic groups together, hydrogen-bonded structures, positive and negative ions. Between the protein molecules, the water is distributed in cavities and channels that are continuous throughout the crystals. More than half the water molecules appear directly hydrogen bonded to protein atoms. These are generally in contact with other water molecules in chains and rings of increasing disorder, corresponding with their movement through the crystals. Within the established crystal structure we survey next the distribution of hydrogen bonds within the protein molecules and between water and protein and water and water; all but eight of the active atoms in the protein form at least one hydrogen bond.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystals of modified fibrinogen: Size,shape and packing of molecules

We have produced several new macroscopic crystal forms and a variety of microcrystals from modified flbrinogens. Bovine and rabbit flbrinogens crystallize after limited digestion by a bacterial protease or α-chymotrypsin. The fibrinogens making up these crystals are largely intact and highly clottable. Tentative molecular packing arrangements for two crystal forms have been deduced. The crystal morphology and cleavage planes were used in this analysis. The characteristic α-helical coiled-coil reflections arid spikes of intensity in certain directions in the crystal X-ray patterns serve as markers for the orientation of the fibrinogen molecules. Changes that occur in one of the forms during preparation for electron microscopy, as shown by comparison with X-ray experiments on crystals in various stains and solvents, support this packing model. These studies provide preliminary evidence that fibrinogen is about 450 Å in length and that the molecules bond end-to-end to form filaments making up the crystals.     

Epitaxial crystallization of alkane chain lipids for electron diffraction analysis

Thin microcrystals of a wide variety of polymethylene chain materials, including n-alkanes, linear waxes, glycerides, a detergent, phospholipids and phospholipid analogs based on cyclopentane-1,2,3-triol, are epitaxially grown on naphthalene to give an orientation with long chain axes parallel to the best developed crystal face. These crystals, which represent a different orientation than those grown from solution, facilitate ab initio quantitative crystal structure analysis from electron diffraction intensity data from the projection yielding the most crystallographic information.     

Calcite microcrystals in the pineal gland of the human brain: first physical and chemical studies

A new form of biomineralization has been studied in the pineal gland of the human brain. It consists of small crystals that are less than 20 microm in length and that are completely distinct from the often observed mulberry-type hydroxyapatite concretions. A special procedure was developed for isolation of the crystals from the organic matter in the pineal gland. Cubic, hexagonal, and cylindrical morphologies have been identified using scanning electron microscopy. The crystal edges were sharp whereas their surfaces were very rough. Energy dispersive spectroscopy showed that the crystals contained only the elements calcium, carbon, and oxygen. Selected area electron diffraction and near infrared Raman spectroscopy established that the crystals were calcite. With the exception of the otoconia structure of the inner ear, this is the only known nonpathological occurrence of calcite in the human body. The calcite microcrystals are probably responsible for the previously observed second harmonic generation in pineal tissue sections. The complex texture structure of the microcrystals may lead to crystallographic symmetry breaking and possible piezoelectricity, as is the case with otoconia. It is believed that the presence of two different crystalline compounds in the pineal gland is biologically significant, suggesting two entirely different mechanisms of formation and biological functions. Studies directed toward the elucidation of the formation and functions, and possible nonthermal interaction with external electromagnetic fields are currently in progress.     

Conformational correlation and coupled motion between residue A21 and B25 side chain observed in crystal structures of insulin mutants at position A21

The C-terminal residue of the insulin A chain is invariant and kept as asparagine in all known insulin molecules from hagfish through birds to mammals. To get information on the role of this conserved residue, which is still unclear, the three-dimensional structures of four human insulin mutants, A21 Asn-->Gly, A21 Asn-->Asp, A21 Asn-->Ala, and A21 Asn-->Gln DesB30, were determined by X-ray crystallography. The four mutants crystallize separately into two kinds (rhombohedral and cubic) of crystals. In the refined structures, conformational correlation and coupled motion between the A chain C-terminal residue A21 and the B25 side chain was observed, in contrast to the nearly unchanged general structures as compared with the native insulin structures in their respective crystals. A detailed analysis suggests that residue A21 can affect insulin receptor binding by interaction with the B25 side chain and the B chain C-terminal segment to assist the B25 side chain rearranging into the 'active' conformation.     

Serial femtosecond crystallography at the SACLA: breakthrough to dynamic structural biology

X-ray crystallography visualizes the world at the atomic level. It has been used as the most powerful technique for observing the three-dimensional structures of biological macromolecules and has pioneered structural biology. To determine a crystal structure with high resolution, it was traditionally required to prepare large crystals (> 200 μm). Later, synchrotron radiation facilities, such as SPring-8, that produce powerful X-rays were built. They enabled users to obtain good quality X-ray diffraction images even with smaller crystals (ca. 200–50 μm). In recent years, one of the most important technological innovations in structural biology has been the development of X-ray free electron lasers (XFELs). The SPring-8 Angstrom Compact free electron LAser (SACLA) in Japan generates the XFEL beam by accelerating electrons to relativistic speeds and directing them through in-vacuum, short-period undulators. Since user operation started in 2012, we have been involved in the development of serial femtosecond crystallography (SFX) measurement systems using XFEL at the SACLA. The SACLA generates X-rays a billion times brighter than SPring-8. The extremely bright XFEL pulses enable data collection with microcrystals (ca. 50–1 μm). Although many molecular analysis techniques exist, SFX is the only technique that can visualize radiation-damage-free structures of biological macromolecules at room temperature in atomic resolution and fast time resolution. Here, we review the achievements of the SACLA-SFX Project in the past 5 years. In particular, we focus on: (1) the measurement system for SFX; (2) experimental phasing by SFX; (3) enzyme chemistry based on damage-free room-temperature structures; and (4) molecular movie taken by time-resolved SFX.     

Interaction of l‐leucyl‐l‐leucyl‐l‐leucine thin film with water and organic vapors: receptor properties and related morphology

The ability of highly ordered tripeptide structures to keep or change their morphology in contact with organic vapors was studied. A thin film of tripeptide l ‐leucyl‐l ‐leucyl‐l ‐leucine (LLL) was prepared having microcrystals and nanocrystals on its surface, which are stable upon vacuum drying but become objects of selective morphology change after a contact with vapors of organic solvents. Fine separate LLL crystals and their agglomerates of submicron and larger dimensions were observed by atomic force microscopy and scanning electron microscopy. After saturation with guest vapors, these crystals can remain intact or change their morphology with the increase in size or complete destruction depending on the guest molecular structure. The crystals completely lose their shape after the binding of pyridine vapors. The other studied guests produce much smaller transformations or have no effect on crystal morphology despite being sorbed by solid LLL, which was shown using quartz crystal microbalance sensor. The observed size‐exclusion effect for guest sorption by LLL was found to be broken by the same guests that can change the initial crystal shape. This helps to explain the morphology changes of LLL crystals after the guest sorption and release. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Quantifying anisotropic solute transport in protein crystals using 3-D laser scanning confocal microscopy visualization

The diffusion of a solute, fluorescein into lysozyme protein crystals has been studied by confocal laser scanning microscopy (CLSM). Confocal laser scanning microscopy makes it possible to non-invasively obtain high-resolution three-dimensional (3-D) images of spatial distribution of fluorescein in lysozyme crystals at various time steps. Confocal laser scanning microscopy gives the fluorescence intensity profiles across horizontal planes at several depths of the crystal representing the concentration profiles during diffusion into the crystal. These intensity profiles were fitted with an anisotropic model to determine the diffusivity tensor. Effective diffusion coefficients obtained range from 6.2 x 10(-15) to 120 x 10(-15) m2/s depending on the lysozyme crystal morphology. The diffusion process is found to be anisotropic, and the level of anisotropy depends on the crystal morphology. The packing of the protein molecules in the crystal seems to be the major factor that determines the anisotropy.