Lessons from crystals grown in the Advanced Protein Crystallisation Facility for conventional crystallisation applied to structural biology

The crystallographic quality of protein crystals that were grown in microgravity has been compared to that of crystals that were grown in parallel on earth gravity under otherwise identical conditions. A goal of this comparison was to assess if a more accurate 3D-structure can be derived from crystallographic analysis of the former crystals. Therefore, the properties of crystals prepared with the Advanced Protein Crystallisation Facility (APCF) on earth and in orbit during the last decade were evaluated. A statistical analysis reveals that about half of the crystals produced under microgravity had a superior X-ray diffraction limit with respect of terrestrial controls. Eleven protein structures could be determined at previously unachieved resolutions using crystals obtained in the APCF. Microgravity induced features of the most relevant structures are reported. A second goal of this study was to identify the cause of the crystal quality enhancement useful for structure determination. No correlations between the effect of microgravity and other system-dependent parameters, such as isoelectric point or crystal solvent content, were found except the reduced convection during the crystallisation process. Thus, crystal growth under diffusive regime appears to be the key parameter explaining the beneficial effect of microgravity on crystal quality. The mimicry of these effects on earth in gels or in capillary tubes is discussed and the practical consequences for structural biology highlighted.     

Atomic force microscopy of three-dimensional membrane protein crystals. Ca-ATPase of sarcoplasmic reticulum.

We have observed three-dimensional crystals of the calcium pump from sarcoplasmic reticulum by atomic force microscopy (AFM). From AFM images of dried crystals, both on graphite and mica, we measured steps in the crystal thickness, corresponding to the unit cell spacing normal to the substrate. It is known from transmission electron microscopy that crystal periodicity in the plane of the substrate is destroyed by drying, and it was therefore not surprising that we were unable to observe this periodicity by AFM. Thus, we were motivated to use the AFM on hydrated crystals. In this case, crystal adsorption appeared to be a limiting factor, and our studies indicate that adsorption is controlled by the composition of the medium and by the physical-chemical properties of the substrate. We used scanning electron microscopy to determine the conditions yielding the highest adsorption of crystals, and, under these conditions, we have obtained AFM images of hydrated crystals with a resolution similar to that observed with dried samples (i.e., relatively poor). In the same preparations, we have observed lipid bilayers with a significantly better resolution, indicating that the poor quality of crystal images was not due to instrumental limitations. Rather, we attribute poor images to the intrinsic flexibility of these multilamellar crystals, which apparently allow movement of one layer relative to another in response to shear forces from the AFM tip. We therefore suggest some general guidelines for future studies of membrane proteins with AFM.     

The electronic structure of Fe2+ in reaction centers from Rhodopseudomonas sphaeroides. I. Static magnetization measurements.

We have measured the static magnetization of unreduced and reduced reaction centers that vary in their quinone content. Measurements were performed in the temperature range 0.7 degrees K less than T less than 200 degrees K and magnetic fields of up to 10 kG. The electronic g-value, crystal field parameters D, E, and the exchange interaction, J, between the quinone spin and Fe2+ were determined using the spin Hamiltonian formalism. The effective moment mu eff/Fe2+ of both reduced and unreduced samples were determined to be 5.35 +/- 0.15 Bohr magnetons. This shows, in agreement with previous findings, that Fe2+ does not change its valence state when the reaction centers are reduced. Typical values of D congruent to +5 cm-1 and E/D congruent to 0.27 are consistent with Fe being in an octahedral environment with rhombic distortion. The values of D and E were approximately the same for reaction centers having one and two quinones. These findings imply that quinone is most likely not a ligand of Fe. The Fe2+ and the spin on the quinone in reduced reaction centers were found to be coupled with an exchange interaction 0 less than /J/ less than 1 cm-1. The validity of the spin Hamiltonian was checked by using an orbital Hamiltonian to calculate energy levels of the 25 states of the S = 2, L = 2 manifold and comparing the magnetization of the lowest five states with those obtained from the spin Hamiltonian. Using the orbital Hamiltonian, we calculated the position of the first excited quintet state to be 340 cm-1 above the ground state quintet. This is in good agreement with the temperature dependence of the quadrupole splitting as determined by Mossbauer spectroscopy.     

Crystallization and preliminary X-ray characterization of branched-chain amino acid aminotransferase from Escherichia coli

The branched-chain amino acid aminotransferase of Escherichia coli was crystallized in two crystal systems, monoclinic and tetragonal, from polyethylene glycol and ammonium sulfate solutions, pH 7.0, respectively. The crystals were of good quality, with diffractions extending beyond 2.8 A. The space group and unit cell dimensions of the monoclinic system crystals were determined from precession photographs to be C2, and a = 93.9, b = 143.6, c = 143.9 A and beta = 134.3 degrees. For the tetragonal system crystals, the possible space group P422 or P4122, and cell dimensions of a = b = 101 A and c = 249 A were determined. Three identical subunits exist per an asymmetric unit in both types of crystals.     

Crystal structure of cyclomaltoheptaose (beta-cyclodextrin) complexes with p-aminobenzoic acid and o-aminobenzoic acid

Crystal structures of cyclomaltoheptaose (beta-cyclodextrin) complexes with p-aminobenzoic acid and o-aminobenzoic acid have been determined by single-crystal X-ray diffraction. The space group of the beta-cyclodextrin-p-aminobenzoic acid complex is P2(1) with a host:guest stoichiometry of 1:1, and that of the beta-cyclodextrin-o-aminobenzoic acid complex is P1 with a stoichiometry of 2:3. The different structures of the guest molecules lead to the different molecular packing structures of the two complexes. Intermolecular hydrogen-bond interactions are the main force that stabilize the supramolecular systems. In both crystals, there are water molecules located near the cavity rims and in interstices between molecules of beta-cyclodextrin participating in formation of intermolecular hydrogen bonds.     

Crystallization and preliminary X-ray crystallographic analysis of Mirabilis antiviral protein.

Mirabilis antiviral protein is a single-chain ribosome-inactivating protein purified from the tuberous root of Mirabilis jalapa L. We obtained several forms of crystals of the protein by the hanging drop vapor diffusion method, but most of these crystals were not suitable for X-ray crystallography. After refining the growth conditions, crystals of crystallographic quality were grown in 20-microliters droplets of an equi-volume mixture of 1.5% (w/v) protein solution and a reservoir solution containing 49 to 50% (w/v) ammonium sulfate and 50 mM-ammonium citrate (pH 5.4) at room temperature. Addition of 2 mM-adenine sulfate reduced twinning and "crystal shower". The resulting trigonal crystals diffract beyond 2.5 A resolution using a rotating anode X-ray generator. The space group was determined to be P3(1)21 or P3(2)21 (a = b = 103.9.A, c = 134.6 A, alpha = beta = 90 degrees, gamma = 120 degrees) based on their precession photography of h0l and hk0 zones. There seems to be three monomers in an asymmetric unit for VM = 2.51 A3/Da.     

Enzymatic degradation of monolayer for poly(lactide) revealed by real-time atomic force microscopy: effects of stereochemical structure, molecular weight, and molecular branches on hydrolysis rates

The influences of the stereochemical structure, the molecular weight, and the number of molecular branches for poly(lactide) (PLA) on enzymatic hydrolysis rates of PLA monolayers were studied by atomic force microscopy (AFM) and the Langmuir-Blodgett (LB) technique. Monolayers of six kinds of PLA with different molecular weights, stereochemical structure, and numbers of molecular branches were prepared by LB techniques and then characterized by AFM in air. The PLA molecules covered homogeneously with a silicon substrate and did not form lamellar crystals in the monolayer. We determined the initial hydrolysis rate of PLA monolayers in presence of proteinase K by volumetric analysis from the continuous AFM height images. The presence of D-lactyl unit reduced the hydrolysis rate of the monolayer. The hydrolysis rate for the linear PLLA samples increased with a decrease in the molecular weight. In contrast, the rates of erosion for branched PLLA monolayers were independent of the molecular weight of samples. The erosion rate of branched PLLA monolayers was found to be dependent on the average molecular weight of PLLA segment in branched molecules, not on the overall molecular weight of samples. From these results, furthermore, the hydrolysis mode of PLAs by proteinase K is discussed.     

Macromolecular crystal growth experiments on International Microgravity Laboratory--1.

Macromolecular crystal growth experiments, using satellite tobacco mosaic virus (STMV) and canavalin from jack beans as samples, were conducted on a US Space Shuttle mission designated International Microgravity Laboratory--1 (IML-1), flown January 22-29, 1992. Parallel experiments using identical samples were carried out in both a vapor diffusion-based device (PCG) and a liquid-liquid diffusion-based instrument (CRYOSTAT). The experiments in each device were run at 20-22 degrees C and at colder temperatures. Crystals were grown in virtually every trial, but the characteristics of the crystals were highly dependent on the crystallization technique employed and the temperature experience of the sample. In general, very good results, based on visual inspection of the crystals, were obtained in both PCG and CRYOSTAT. Unusually impressive results were, however, achieved for STMV in the CRYOSTAT instrument. STMV crystals grown in microgravity by liquid-liquid diffusion were more than 10-fold greater in total volume than any STMV crystals previously grown in the laboratory. X-ray diffraction data collected from eight STMV crystals grown in CRYOSTAT demonstrated a substantial improvement in diffraction quality over the entire resolution range when compared to data from crystals grown on Earth. In addition, the extent of the diffraction pattern for the STMV crystals grown in space extended to 1.8 A resolution, whereas the best crystals that were ever grown under conditions of Earth's gravity produced data limited to 2.3 A resolution. Other observations indicate that the growth of macromolecular crystals is indeed influenced by the presence or absence of gravity. These observations further suggest, consistent with earlier results, that the elimination of gravity provides a more favorable environment for such processes.     

Catalytic activity of rabbit skeletal muscle aldolase in the crystalline state

The monoclinic crystalline form of aldolase from rabbit skeletal muscle grown at 29 degrees C is catalytically active in the direction of aldol cleavage. Activity was assayed for in a crystallization buffer containing 45% saturated ammonium sulfate using chemically unmodified single crystals cut to precise dimensions. Diffusion effects on velocities from assays employing aldolase crystals do not appear to be limiting when cut single crystals are crushed. Assays of crushed crystals are linear with respect to both time and enzyme concentration. Kinetic constants are reported for both substrates fructose 1-phosphate and fructose 1,6-phosphate. Maximal velocities and binding constants determined differ by no more than a factor of 2 between the crystalline and the soluble state of the enzyme. Analysis of the kinetic constants for fructose 1-phosphate as substrate shows that binding of substrate does not change in going to the crystalline state. Release of product is reduced roughly 2-fold in the crystalline state. A similar conclusion can be reached in the case of fructose 1,6-phosphate as substrate provided the "on" steps of substrate and product are only diffusion limited but independent of the physical state of the enzyme. It is not possible to distinguish between a more sluggish conformational change during catalysis or simply tighter product binding in the crystalline state as compared to the soluble enzyme state.     

Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase

Two arsenite-inhibited forms of each of the aldehyde oxidoreductases from Desulfovibrio gigas and Desulfovibrio desulfuricans have been studied by X-ray crystallography and electron paramagnetic resonance (EPR) spectroscopy. The molybdenum site of these enzymes shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. Arsenite addition to active as-prepared enzyme or to a reduced desulfo form yields two different species called A and B, respectively, which show different Mo(V) EPR signals. Both EPR signals show strong hyperfine and quadrupolar couplings with an arsenic nucleus, which suggests that arsenic interacts with molybdenum through an equatorial ligand. X-ray data of single crystals prepared from EPR-active samples show in both inhibited forms that the arsenic atom interacts with the molybdenum ion through an oxygen atom at the catalytic labile site and that the sulfido ligand is no longer present. EPR and X-ray data indicate that the main difference between both species is an equatorial ligand to molybdenum which was determined to be an oxo ligand in species A and a hydroxyl/water ligand in species B. The conclusion that the sulfido ligand is not essential to determine the EPR properties in both Mo–As complexes is achieved through EPR measurements on a substantial number of randomly oriented chemically reduced crystals immediately followed by X-ray studies on one of those crystals. EPR saturation studies show that the electron transfer pathway, which is essential for catalysis, is not modified upon inhibition. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Antibody-unfolding and metastable-state binding in force spectroscopy and recognition imaging

Force spectroscopy and recognition imaging are important techniques for characterizing and mapping molecular interactions. In both cases, an antibody is pulled away from its target in times that are much less than the normal residence time of the antibody on its target. The distribution of pulling lengths in force spectroscopy shows the development of additional peaks at high loading rates, indicating that part of the antibody frequently unfolds. This propensity to unfold is reversible, indicating that exposure to high loading rates induces a structural transition to a metastable state. Weakened interactions of the antibody in this metastable state could account for reduced specificity in recognition imaging where the loading rates are always high. The much weaker interaction between the partially unfolded antibody and target, while still specific (as shown by control experiments), results in unbinding on millisecond timescales, giving rise to rapid switching noise in the recognition images. At the lower loading rates used in force spectroscopy, we still find discrepancies between the binding kinetics determined by force spectroscopy and those determined by surface plasmon resonance—possibly a consequence of the short tethers used in recognition imaging. Recognition imaging is nonetheless a powerful tool for interpreting complex atomic force microscopy images, so long as specificity is calibrated in situ, and not inferred from equilibrium binding kinetics.     

Modelling the mechanical effect of muscles with large attachment sites: Application to the shoulder mechanism

A general theory is described for deriving the mechanical effect of muscles with large attachment sites. In a cadaver experiment the complete attachment sites and bundle distribution of 16 muscles of the shoulder mechanism were recorded. These data were used to calculate the mechanical effect, i.e. the resulting force and moment vector, for a large number (200) and a reduced number (maximal 6) of muscle lines of action. The resulting error between both representations is small. The number of muscle lines of action in the reduced representation depends on the shape of the attachment site and muscle architecture. An important feature of this method is that the necessary number of muscle lines of action is determined afterwards. In the often used centroid line approach the number of muscle lines of action and partitioning of muscles is determined before recording the geometry, leading to unverifiable results.     

Dehydration converts DsbG crystal diffraction from low to high resolution

Diffraction quality crystals are essential for crystallographic studies of protein structure, and the production of poorly diffracting crystals is often regarded as a dead end in the process. Here we show a dramatic improvement of poorly diffracting DsbG crystals allowing high-resolution diffraction data measurement. Before dehydration, the crystals are fragile and the diffraction pattern is streaky, extending to 10 A resolution. After dehydration, there is a spectacular improvement, with the diffraction pattern extending to 2 A resolution. This and other recent results show that dehydration is a simple, rapid, and inexpensive approach to convert poor quality crystals into diffraction quality crystals.     

重组双功能水蛭素质量标准研究

以生色底物法测定抗凝血酶活性,比浊法测定抗血小板聚集活性,还原型SDS-PAGE测定分子量,SDS-PAGE和反相高效液相色谱测定纯度,毛细管电泳法测定等电点,胰蛋白酶酶切后进行肽图分析,其余检测项目按《中国药典》2005版三部规定进行。结果显示,用建立的方法对原液和成品进行了检定,各项指标均符合《人用重组DNA制品质量控制技术指导原则》和《中国药典》2005版三部的要求。建立的质控方法和质量标准具有保证产品安全、有效、质量可控的特点,可用于重组双功能水蛭素产品的常规检定。     

Structure and function of the glucose PTS transporter from Escherichia coli

The glucose transporter IICB of the Escherichia coli phosphotransferase system (PTS) consists of a polytopic membrane domain (IIC) responsible for substrate transport and a hydrophilic C-terminal domain (IIB) responsible for substrate phosphorylation. We have overexpressed and purified a triple mutant of IIC (mut-IIC), which had recently been shown to be suitable for crystallization purposes. Mut-IIC was homodimeric as determined by blue native-PAGE and gel-filtration, and had an eyeglasses-like structure as shown by negative-stain transmission electron microscopy (TEM) and single particle analysis. Glucose binding and transport by mut-IIC, mut-IICB and wildtype-IICB were compared with scintillation proximity and in vivo transport assays. Binding was reduced and transport was impaired by the triple mutation. The scintillation proximity assay allowed determination of substrate binding, affinity and specificity of wildtype-IICB by a direct method. 2D crystallization of mut-IIC yielded highly-ordered tubular crystals and made possible the calculation of a projection structure at 12 Å resolution by negative-stain TEM. Immunogold labeling TEM revealed the sidedness of the tubular crystals, and high-resolution atomic force microscopy the surface structure of mut-IIC. This work presents the structure of a glucose PTS transporter at the highest resolution achieved so far and sets the basis for future structural studies.     

Origin of the limited alpha-amylolysis of debranched maltodextrins crystallized in the A form: a TEM study on model substrates

The detailed ultrastructure of a new type of resistant starch and the way that it is modified during hydrolysis by alpha-amylases were studied by transmission electron microscopy (TEM) on model starch crystals. The selected substrates were waxy maize starch lintners and A-type crystals prepared from low degree of polymerization (DP) amylose. A model describing the stacking of double helices is proposed for A-type low DP amylose crystals. The enzymatic hydrolysis of both lintners and low DP crystals has been shown to occur by the side of double helices and not their ends. The results were transposed to a new type of resistant starch (RS) produced by debranching maltodextrins in concentrated solutions. This product presents A-type crystallinity contrary to all other known classified RS. Moreover it consists of low DP chains similar to the model crystals studied and yields similar electron diffraction patterns to those of A-type low DP crystals. The similarities in the morphology of these substrates with that of the studied RS led us to attribute its resistance to its particularly dense and compact morphology, resulting from the epitaxial growth of elementary crystalline A-type platelets. In the resulting structure, the accessibility of double helices to alpha-amylase is strongly reduced by aggregation.     

In situ observation of elementary growth processes of protein crystals by advanced optical microscopy

To start systematically investigating the quality improvement of protein crystals, the elementary growth processes of protein crystals must be first clarified comprehensively. Atomic force microscopy (AFM) has made a tremendous contribution toward elucidating the elementary growth processes of protein crystals and has confirmed that protein crystals grow layer by layer utilizing kinks on steps, as in the case of inorganic and low-molecular-weight compound crystals. However, the scanning of the AFM cantilever greatly disturbs the concentration distribution and solution flow in the vicinity of growing protein crystals. AFM also cannot visualize the dynamic behavior of mobile solute and impurity molecules on protein crystal surfaces. To compensate for these disadvantages of AFM, in situ observation by two types of advanced optical microscopy has been recently performed. To observe the elementary steps of protein crystals noninvasively, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) was developed. To visualize individual mobile protein molecules, total internal reflection fluorescent (TIRF) microscopy, which is widely used in the field of biological physics, was applied to the visualization of protein crystal surfaces. In this review, recent progress in the noninvasive in situ observation of elementary steps and individual mobile protein molecules on protein crystal surfaces is outlined.     

The role of membrane stiffness and actin turnover on the force exerted by DRG lamellipodia

We used optical tweezers to analyze the effect of jasplakinolide and cyclodextrin on the force exerted by lamellipodia from developing growth cones (GCs) of isolated dorsal root ganglia (DRG) neurons. We found that 25 nM of jasplakinolide, which is known to inhibit actin filament turnover, reduced both the maximal exerted force and maximal velocity during lamellipodia leading-edge protrusion. By using atomic force microscopy, we verified that cyclodextrin, which is known to remove cholesterol from membranes, decreased the membrane stiffness of DRG neurons. Lamellipodia treated with 2.5 mM of cyclodextrin exerted a larger force, and their leading edge could advance with a higher velocity. Neither jasplakinolide nor cyclodextrin affected force or velocity during lamellipodia retraction. The amplitude and frequency of elementary jumps underlying force generation were reduced by jasplakinolide but not by cyclodextrin. The action of both drugs at the used concentration was fully reversible. These results support the notion that membrane stiffness provides a selective pressure that shapes force generation, and confirm the pivotal role of actin turnover during protrusion.     

Conformational analysis of the polar head group in phosphatidylcholine bilayers: a structural change induced by cations

The conformation of the polar head group of phosphatidylcholine in a bilayer in the liquid-crystalline state was deduced by analyzing the deuterium quadrupole splittings of the choline group and the phosphorus chemical shift anisotropy of the phosphate group in combination with the restriction of the choline conformation determined in laser Raman studies. The latter efficiently reduced the number of candidates for the actual conformation. A family of conformations was obtained for both the dynamic-structure and rigid-structure models, respectively. The polar head group is oriented roughly parallel to the membrane surface in both models. Furthermore, they are close to conformation A of the crystal structure of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The dynamic-structure model was concluded to be more reasonable in view of the fact that the polar head-group structures in most crystals comprise two conformations, which are nearly mirror images of each other. Conformational analysis was also carried out for the polar head group in the presence of multivalent cations. A possible conformational change of the polar head group induced by cations is discussed in the light of the present results.     

Quantitative biomolecular sensing station based on magnetoresistive patterned arrays

The combination of magnetoresistive sensors and magnetic labeling of bioanalytes, which are selectively captured by their complementary antibody in the proximity of the sensor is a powerful method in order to attain truly quantitative immunological assays. In this paper we present a technical solution to exploit the existing spin valve technology to readout magnetic signals of bio-functionalized magnetic nanoparticles. The method is simple and reliable, and it is based on a discrete scan of lateral flow strips with a precise control of the contact force between sensor and sample. It is shown that the signal of the sensor is proportional to the local magnetization produced by the nanoparticles in a wide range of concentrations, and the sensitivity thresholds in both calibration samples and real immunorecognition assays of human chorionic gonadotropin hormone are well below the visual inspection limit (5.5 ng/ml). Furthermore the sample scanning approach and the reduced dimensions of the sensors provide unprecedented spatial resolution of the nanoparticle distribution across the supporting nitrocellulose strip, therefore enabling on-stick control references and multi-analyte capability.