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1.  Slow cooling of protein crystals 
Journal of Applied Crystallography  2009;42(Pt 5):944-952.
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.
doi:10.1107/S0021889809023553
PMCID: PMC2746722  PMID: 19798409
protein crystallography; cryocrystallography; X-ray diffraction; slow cooling; temperature control
2.  The effect of temperature and pressure on the crystal structure of piperidine 
Background
The response of molecular crystal structures to changes in externally applied conditions such as temperature and pressure are the result of a complex balance between strong intramolecular bonding, medium strength intermolecular interactions such as hydrogen bonds, and weaker intermolecular van der Waals contacts. At high pressure the additional thermodynamic requirement to fill space efficiently becomes increasingly important.
Results
The crystal structure of piperidine-d11 has been determined at 2 K and at room temperature at pressures between 0.22 and 1.09 GPa. Unit cell dimensions have been determined between 2 and 255 K, and at pressures up to 2.77 GPa at room temperature. All measurements were made using neutron powder diffraction. The crystal structure features chains of molecules formed by NH…N H-bonds with van der Waals interactions between the chains. Although the H-bonds are the strongest intermolecular contacts, the majority of the sublimation enthalpy may be ascribed to weaker but more numerous van der Waals interactions.
Conclusions
Analysis of the thermal expansion data in the light of phonon frequencies determined in periodic DFT calculations indicates that the expansion at very low temperature is governed by external lattice modes, but above 100 K the influence of intramolecular ring-flexing modes also becomes significant. The principal directions of thermal expansion are determined by the sensitivity of different van der Waals interactions to changes in distance. The principal values of the strain developed on application of pressure are similarly oriented to those determined in the variable-temperature study, but more isotropic because of the need to minimise volume by filling interstitial voids at elevated pressure.
Graphical AbstractThough H-bonds are important interactions in the crystal structure of piperidine, the response to externally-applied conditions are determined by van der Waals interactions.
Electronic supplementary material
The online version of this article (doi:10.1186/s13065-015-0086-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s13065-015-0086-3
PMCID: PMC4403828  PMID: 25897321
3.  The effect of temperature and pressure on the crystal structure of piperidine 
Background
The response of molecular crystal structures to changes in externally applied conditions such as temperature and pressure are the result of a complex balance between strong intramolecular bonding, medium strength intermolecular interactions such as hydrogen bonds, and weaker intermolecular van der Waals contacts. At high pressure the additional thermodynamic requirement to fill space efficiently becomes increasingly important.
Results
The crystal structure of piperidine-d11 has been determined at 2 K and at room temperature at pressures between 0.22 and 1.09 GPa. Unit cell dimensions have been determined between 2 and 255 K, and at pressures up to 2.77 GPa at room temperature. All measurements were made using neutron powder diffraction. The crystal structure features chains of molecules formed by NH…N H-bonds with van der Waals interactions between the chains. Although the H-bonds are the strongest intermolecular contacts, the majority of the sublimation enthalpy may be ascribed to weaker but more numerous van der Waals interactions.
Conclusions
Analysis of the thermal expansion data in the light of phonon frequencies determined in periodic DFT calculations indicates that the expansion at very low temperature is governed by external lattice modes, but above 100 K the influence of intramolecular ring-flexing modes also becomes significant. The principal directions of thermal expansion are determined by the sensitivity of different van der Waals interactions to changes in distance. The principal values of the strain developed on application of pressure are similarly oriented to those determined in the variable-temperature study, but more isotropic because of the need to minimise volume by filling interstitial voids at elevated pressure.
Graphical AbstractThough H-bonds are important interactions in the crystal structure of piperidine, the response to externally-applied conditions are determined by van der Waals interactions.
Electronic supplementary material
The online version of this article (doi:10.1186/s13065-015-0086-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s13065-015-0086-3
PMCID: PMC4403828  PMID: 25897321
4.  Purification, crystallization and preliminary X-ray analysis of Enterococcus casseliflavus aminoglycoside-2′′-phosphotransferase-IVa 
Aminoglycoside-2′′-phosphotransferase-IVa [APH(2′′)-IVa] is an enzyme that is responsible for high-level gentamicin resistance in E. casseliflavus isolates. Three different crystals of wild-type substrate-free APH(2′′)-IVa have been prepared and preliminary X-ray diffraction experiments have been undertaken on all three crystal forms.
The deactivation of aminoglycoside antibiotics by chemical modification is one of the major sources of bacterial resistance to this family of therapeutic compounds, which includes the clinically relevant drugs streptomycin, kanamycin and gentamicin. The aminoglycoside phosphotransferases (APHs) form one such family of enzymes responsible for this resistance. The gene encoding one of these enzymes, aminoglycoside-2′′-phosphotransferase-IVa [APH(2′′)-IVa] from Enterococcus casseliflavus, has been cloned and the protein (comprising 306 amino-acid residues) has been expressed in Escherichia coli and purified. The enzyme was crystallized in three substrate-free forms. Two of the crystal forms belonged to the orthorhombic space group P212121 with similar unit-cell parameters, although one of the crystal forms had a unit-cell volume that was approximately 13% smaller than the other and a very low solvent content of around 38%. The third crystal form belonged to the monoclinic space group P21 and preliminary X-ray diffraction analysis was consistent with the presence of two molecules in the asymmetric unit. The orthorhombic crystal forms of apo APH(2′′)-IVa both diffracted to 2.2 Å resolution and the monoclinic crystal form diffracted to 2.4 Å resolution; synchrotron diffraction data were collected from these crystals at SSRL (Stanford, California, USA). Structure determination by molecular replacement using the structure of the related enzyme APH(2′′)-IIa is proceeding.
doi:10.1107/S1744309109050039
PMCID: PMC2805544  PMID: 20057078
aminoglycoside-2′′-phosphotransferase-IVa; Enterococcus casseliflavus; antibiotic resistance
5.  Use of a novel microtitration protocol to obtain diffraction-quality crystals of 4-hydroxy-2-oxoglutarate aldolase from Bos taurus  
A novel vapour-diffusion crystallization approach was utilized to produce crystals of the enzyme 4-hydroxy-2-oxoglutarate aldolase. This ‘microtitration’ method incorporated ammonium sulfate titration, a miniaturization of the working volume (<10 µl) and a controlled rate of evaporation by utilizing a large reservoir, and yielded crystals with favourable diffraction parameters compared with crystals obtained using the conventional hanging-drop method.
The enzyme 4-hydroxy-2-oxoglutarate aldolase (HOGA) catalyses the retro-aldol degradation of 4-hydroxy-2-oxoglutarate to pyruvate and glyoxylate as part of the hydroxyproline catabolic pathway in mammals. Mutations in the coding region of the human HOGA gene are associated with primary hyperoxaluria type 3, a disease characterized by excessive oxalate production and ultimately stone deposition. Native HOGA was purified from bovine kidney using an improved and streamlined purification protocol from which two crystal forms were obtained using two different approaches. Vapour diffusion using PEG 3350 as a precipitant produced monoclinic crystals that belonged to space group C2 and diffracted to 3.5 Å resolution. By comparison, orthorhombic crystals belonging to space group I222 or I212121 and diffracting to beyond 2.25 Å resolution were obtained using a novel microtitration protocol with ammonium sulfate. The latter crystal form displayed superior diffraction quality and was suitable for structural determination by X-ray crystallography.
doi:10.1107/S2053230X14021463
PMCID: PMC4231863  PMID: 25372828
primary hyperoxaluria; DHDPSL; 4-hydroxy-2-oxoglutarate aldolase; Bos taurus
6.  Imperfect pseudo-merohedral twinning in crystals of fungal fatty acid synthase 
A case of imperfect pseudo-merohedral twinning in monoclinic crystals of fungal fatty acid synthase is discussed. A space-group transition during crystal dehydration resulted in a Moiré pattern-like interference of the twinned diffraction patterns.
The recent high-resolution structures of fungal fatty acid synthase (FAS) have provided new insights into the principles of fatty acid biosynthesis by large multifunctional enzymes. The crystallographic phase problem for the 2.6 MDa fungal FAS was initially solved to 5 Å resolution using two crystal forms from Thermomyces lanuginosus. Monoclinic crystals in space group P21 were obtained from orthorhombic crystals in space group P212121 by dehydration. Here, it is shown how this space-group transition induced imperfect pseudo-merohedral twinning in the monoclinic crystal, giving rise to a Moiré pattern-like interference of the two twin-related reciprocal lattices. The strategy for processing the twinned diffraction images and obtaining a quantitative analysis is presented. The twinning is also related to the packing of the molecules in the two crystal forms, which was derived from self-rotation function analysis and molecular-replacement solutions using a low-resolution electron microscopy map as a search model.
doi:10.1107/S0907444909000778
PMCID: PMC2631638  PMID: 19171964
imperfect pseudo-merohedral twinning; fungal fatty acid synthase
7.  The use of trimethylamine N-oxide as a primary precipitating agent and related methylamine osmolytes as cryoprotective agents for macromolecular crystallography 
The stabilizing osmolyte trimethylamine N-oxide (TMAO) is shown to be an efficient primary precipitant for protein crystal growth. In addition to TMAO, two other methylamine osmolytes, sarcosine and betaine, are shown to be effective cryoprotective agents for protein crystal cooling.
Both crystallization and cryoprotection are often bottlenecks for high-resolution X-ray structure determination of macromolecules. Methylamine osmolytes are known stabilizers of protein structure. One such osmolyte, trimethylamine N-oxide (TMAO), has seen occasional use as an additive to improve macromolecular crystal quality and has recently been shown to be an effective cryoprotective agent for low-temperature data collection. Here, TMAO and the related osmolytes sarcosine and betaine are investigated as primary precipitating agents for protein crystal growth. Crystallization experiments were undertaken with 14 proteins. Using TMAO, seven proteins crystallized in a total of 13 crystal forms, including a new tetragonal crystal form of trypsin. The crystals diffracted well, and eight of the 13 crystal forms could be effectively cryo­­cooled as grown with TMAO as an in situ cryoprotective agent. Sarcosine and betaine produced crystals of four and two of the 14 proteins, respectively. In addition to TMAO, sarcosine and betaine were effective post-crystallization cryoprotective agents for two different crystal forms of thermolysin. Precipitation reactions of TMAO with several transition-metal ions (Fe3+, Co2+, Cu2+ and Zn2+) did not occur with sarcosine or betaine and were inhibited for TMAO at lower pH. Structures of proteins from TMAO-grown crystals and from crystals soaked in TMAO, sarcosine or betaine were determined, showing osmolyte binding in five of the 12 crystals tested. When an osmolyte was shown to bind, it did so near the protein surface, interacting with water molecules, side chains and backbone atoms, often at crystal contacts.
doi:10.1107/S0907444911050360
PMCID: PMC3245723  PMID: 22194335
crystallization; cryoprotection; osmolytes; trimethylamine N-oxide; sarcosine; betaine
8.  Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments 
A new crystal-mounting method has been developed that involves a combination of controlled humid air and polymer glue for crystal coating. This method is particularly useful when applied to fragile protein crystals that are known to be sensitive to subtle changes in their physicochemical environment.
Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection.
doi:10.1107/S0907444913018027
PMCID: PMC3760132  PMID: 23999307
cryocrystallography; macromolecular crystallography; crystal mounting
9.  Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH 
Background
Insulin is a therapeutic protein that is widely used for the treatment of diabetes. Its biological function was discovered more than 80 years ago and it has since then been characterized extensively. Crystallization of the insulin molecule has always been a key activity since the protein is often administered by subcutaneous injections of crystalline insulin formulations. Over the years, insulin has been crystallized and characterized in a number of crystal systems.
Results
Interestingly, we have now discovered two new crystal forms of human insulin. The crystals were obtained when the two chaotropic agents, urea and thiocyanate were present in the crystallization experiments, and their structures were determined by X-ray crystallography. The crystals belong to the orthorhombic and monoclinic crystal systems, with space groups C2221 and C2 respectively. The orthorhombic crystals were obtained at pH 6.5 and contained three insulin hexamers in R6 conformation in the asymmetric unit whilst the monoclinic C2 crystals were obtained at pH 7.0 and contained one R6 hexamer in the asymmetric unit. Common for the two new crystals is a hexamer-hexamer interaction that has not been found in any of the previous crystal forms of insulin. The contacts involve a tight glutamate-glutamate interaction with a distance of 2.3 Å between groups. The short distance suggests a low barrier hydrogen bond. In addition, two tyrosine-tyrosine interactions occupying a known phenol binding pocket contribute to the stabilization of the contacts. Within the crystals, distinct binding sites for urea were found, adding further to the discussion on the role of urea in protein denaturation.
Conclusion
The change in space group from C2221 to C2 was primarily caused by an increase in pH. The fewer number of hexamer-hexamer interactions comprising the short hydrogen bond in the C2 space group suggest that pH is the driving force. In addition, the distance between the two glutamates increases from 2.32 Å in the C2221 crystals to 2.4 Å in the C2 crystals. However, in both cases the low barrier hydrogen bond and the tyrosine-tyrosine interaction should contribute to the stability of the crystals which is crucial when used in pharmaceutical formulations.
doi:10.1186/1472-6807-7-83
PMCID: PMC2241603  PMID: 18093308
10.  Crystallization and preliminary X-ray diffraction analysis of biotin acetyl-CoA carboxylase ligase (BirA) from Mycobacterium tuberculosis  
The cloning, purification, crystallization and preliminary X-ray characterization of the crystals of biotin acetyl-CoA carboxylase ligase from M. tuberculosis are reported.
The gene encoding biotin acetyl-CoA carboxylase ligase (BirA) from Myco­bacterium tuberculosis was cloned and expressed in Escherichia coli with a C-­terminal Strep-tag. PEG 4000 as well as PEG 8000 were used as precipitants at pH 7.5 to crystallize the protein using the vapour-diffusion technique. X-ray characterization of crystals at room temperature indicated that the crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 79.7, b = 62.8, c = 105.8 Å. Assuming the presence of two BirA molecules in the asymmetric unit, the solvent content of the crystals was 44% (V M = 2.2 Å3 Da−1). When transferred to a cryoprotectant, crystals grown in the same drop exhibited a difference in one unit-cell parameter, with a = 60.1, b = 64.0, c = 103.6 Å, but belonged to the same P212121 space group. These crystals, with two molecules of BirA present per asymmetric unit, appeared to have a very low solvent content of 28% (V M = 1.7 Å3 Da−1).
doi:10.1107/S1744309108012475
PMCID: PMC2496853  PMID: 18540066
biotin; biotin protein ligase; Mycobacterium tuberculosis
11.  Crystal structure of 8-hy­droxy­quinoline: a new monoclinic polymorph 
In an attempt to grow 8-hy­droxy­quinoline–acetamino­phen co-crystals from equimolar amounts of conformers in a chloro­form–ethanol solvent mixture at room temperature, the title compound, C9H7NO, was obtained. The mol­ecule is planar, with the hy­droxy H atom forming an intra­molecular O—H⋯N hydrogen bond. In the crystal, mol­ecules form centrosymmetric dimers via two O—H⋯N hydrogen bonds. Thus, the hy­droxy H atoms are involved in bifurcated O—H⋯N hydrogen bonds, leading to the formation of a central planar four-membered N2H2 ring. The dimers are bound by inter­molecular π–π stacking [the shortest C⋯C distance is 3.2997 (17) Å] and C—H⋯π inter­actions into a three-dimensional framework. The crystal grown represents a new monoclinic polymorph in the space group P21/n. The mol­ecular structure of the present monoclinic polymorph is very similar to that of the ortho­rhom­bic polymorph (space group Fdd2) studied previously [Roychowdhury et al. (1978 ▶). Acta Cryst. B34, 1047–1048; Banerjee & Saha (1986 ▶). Acta Cryst. C42, 1408–1411]. The structures of the two polymorphs are distinguished by the different geometries of the hydrogen-bonded dimers, which in the crystal of the ortho­rhom­bic polymorph possess twofold axis symmetry, with the central N2H2 ring adopting a butterfly conformation.
doi:10.1107/S1600536814016110
PMCID: PMC4186174  PMID: 25309256
8-hy­droxy­quinoline; hydrogen bonds; polymorphism; crystal structure
12.  High-Pressure Synthesis and Characterization of New Actinide Borates, AnB4O8 (An=Th, U) 
New actinide borates ThB4O8 and UB4O8 were synthesized under high-pressure, high-temperature conditions (5.5 GPa/1100 °C for thorium borate, 10.5 GPa/1100 °C for the isotypic uranium borate) in a Walker-type multianvil apparatus from their corresponding actinide oxide and boron oxide. The crystal structure was determined on basis of single-crystal X-ray diffraction data that were collected at room temperature. Both compounds crystallized in the monoclinic space group C2/c (Z=4). Lattice parameters for ThB4O8: a=1611.3(3), b=419.86(8), c=730.6(2) pm; β=114.70(3)°; V=449.0(2) Å3; R1=0.0255, wR2=0.0653 (all data). Lattice parameters for UB4O8: a=1589.7(3), b=422.14(8), c=723.4(2) pm; β=114.13(3)°; V=443.1(2) Å3; R1=0.0227, wR2=0.0372 (all data). The new AnB4O8 (An=Th, U) structure type is constructed from corner-sharing BO4 tetrahedra, which form layers in the bc plane. One of the four independent oxygen atoms is threefold-coordinated. The actinide cations are located between the boron–oxygen layers. In addition to Raman spectroscopic investigations, DFT calculations were performed to support the assignment of the vibrational bands.
doi:10.1002/chem.201302378
PMCID: PMC4068220  PMID: 24123698
actinides; borates; density functional theory; high-pressure chemistry; Raman spectroscopy
13.  Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate 
The Trypanosoma cruzi dihydroorotate dehydrogenase, a key enzyme in pyrimidine de novo biosynthesis and redox homeostasis, was crystallized in complex with its first reaction product, orotate.
Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD–orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 Å resolution using synchrotron radiation (λ = 0.900 Å). X-ray diffraction data were collected at 100 K and processed to 1.9 Å resolution with 98.2% completeness and an overall R merge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 Å. The presence of two molecules in the asymmetric unit (2 × 34 kDa) gives a crystal volume per protein weight (V M) of 2.2 Å3 Da−1 and a solvent content of 44%.
doi:10.1107/S174430910502659X
PMCID: PMC1991314  PMID: 16511183
dihydroorotate dehydrogenase; pyrimidine biosynthesis; Trypanosoma cruzi; fumarate reductase; redox homeostasis; structure-based drug design
14.  Expression, purification and crystallization of the phosphate-binding PstS protein from Pseudomonas aeruginosa  
The PstS protein from Pseudomonas aeruginosa (PA) has a dual role in bacterial phosphate uptake and extracellular assembly of adhesion fibers. Here the subcloning, expression, isolation, crystallization and crystallographic analysis of native PA PstS are presented.
Pseudomonas aeruginosa (PA) infections pose a serious threat to human health. PA is a leading cause of fatal lung infections in cystic fibrosis and immune-suppressed patients, of sepsis in burn victims and of nosocomial infections. An important element in PA virulence is its ability to establish biofilms that evade suppression by the host’s immune system and antibiotics. PstS, a periplasmic subunit of the Pst phosphate-transport system of PA, plays a critical role in the establishment of biofilms. In some drug-resistant PA strains, PstS is secreted in large quantities from the bacteria, where it participates in the assembly of adhesion fibres that enhance bacterial virulence. In order to understand the dual function of PstS in biofilm formation and phosphate transport, the crystal structure of PA PstS was determined. Here, the overexpression in Escherichia coli and purification of PA PstS in the presence of phosphate are described. Two crystal forms were obtained using the vapour-diffusion method at 20°C and X-ray diffraction data were collected. The first crystal form belonged to the centred orthorhombic space group C2221, with unit-cell parameters a = 67.5, b = 151.3, c = 108.9 Å. Assuming the presence of a dimer in the asymmetric unit gives a crystal volume per protein weight (V M) of 2.09 Å3 Da−1 and a solvent content of 41%. The second crystal form belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 35.4, b = 148.3, c = 216.7 Å. Assuming the presence of a tetramer in the asymmetric unit gives a crystal volume per protein weight (V M) of 2.14 Å3 Da−1 and a solvent content of 42.65%. A pseudo-translational symmetry is present in the P212121 crystal form which is consistent with a filamentous arrangement of PstS in the crystal lattice.
doi:10.1107/S2053230X14010279
PMCID: PMC4089529  PMID: 25005086
PstS; Pseudomonas aeruginosa
15.  Structural disorder and transformation in crystal growth: direct observation of ring-opening isomerization in a metal–organic solid solution 
IUCrJ  2014;1(Pt 5):318-327.
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 L 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.
doi:10.1107/S2052252514015966
PMCID: PMC4174874  PMID: 25295173
crystallization; structural transformation; ring-opening isomerism; solid solution; disorder
16.  X-RAY AND CRYSTALLOGRAPHIC STUDIES OF PLANT VIRUS PREPARATIONS. III 
The Journal of General Physiology  1941;25(1):147-165.
These papers give an account of an optical and x-ray examination of preparations of plant virus substances isolated by Bawden and Pirie, in particular of those of tobacco mosaic disease. They open with a historical survey of the work, indicating the order in which new phenomena were discovered. The subsequent treatment is divided into three parts: I. Introduction and preparation of specimens. II. Modes of aggregation of virus particles. III. (1) The structure of the particles. (2) Biological implications. Part I, after an historical introduction, describes the method of preparation, from solutions of the virus, of optically oriented specimens of different concentrations. For their examination special x-ray apparatus was developed, in particular cameras working with very low angles and capable of indicating spacings up to 1000 Å. In Part III, Section 1 deals with the x-ray evidence on the internal structure of the particles. Even in solution, they have an inner regularity like that of a crystal. Virus preparations are thus in a sense doubly crystalline. Closer analysis reveals that the x-ray patterns are not directly comparable to those of a crystal as many of the reflections do not obey Bragg's law, but can be understood on the theory of gratings of limited size. The structure seems to consist of sub-units of the dimensions of approximately 11 Å cube, fitted together in a hexagonal or pseudohexagonal lattice of dimensions—a = 87 Å, c = 68 Å. Contrary to what earlier observations seemed to indicate, the particle seems to be virtually unchanged by drying and must therefore contain little water. There are marked resemblances with the structure of both crystalline and fibrous protein, but the virus structure does not belong to any of the classes hitherto studied. There are indications that the inner structure is of a simpler character than that of the molecules of crystalline proteins. Part III, Section 2 contains a comparative study of the optical and x-ray examinations of three strains of tobacco mosaic virus, two of cucumber disease virus, two of potato virus X, and the virus of bushy stunt disease of tomato. In the last case x-ray measurement confirmed the deduction from its cubic crystal habit that it was composed of spherical rather than long particles, and showed that these had a diameter when dry of 276 Å and were arranged in a body-centred cubic close packing. This single example is sufficient to show that the elongated particle form which gives rise to all the anomalous physical properties of the other viruses studied is of no essential biological importance. The similarity and differences observed between the physical properties of these preparations run closely parallel to their clinical and serological classification. Finally, the biological implications of these results are discussed together with possible applications of the new methods of examination to the study of colloid and biological problems.
PMCID: PMC2142028  PMID: 19873256
17.  Comparative analysis of anti-polyglutamine Fab crystals grown on Earth and in microgravity 
Microgravity was used in an attempt to define the crystal structure of the polyQ repeat of huntingtin alone or bound to MW1, an anti-polyQ Fab. While huntingtin was not crystallized in the experiments, analysis of microgravity-grown and Earth-grown MW1 Fab crystals showed that, on average, microgravity-grown crystals of MW1 Fab showed an increase in size and an improvement in resolution and mosaicity when compared with Earth-grown crystals in one space group, in agreement with data published for other proteins, although the highest overall resolution X-ray data in our experiments were obtained from a crystal grown on Earth.
Huntington’s disease is one of nine neurodegenerative diseases caused by a polyglutamine (polyQ)-repeat expansion. An anti-polyQ antigen-binding fragment, MW1 Fab, was crystallized both on Earth and on the International Space Station, a microgravity environment where convection is limited. Once the crystals returned to Earth, the number, size and morphology of all crystals were recorded, and X-ray data were collected from representative crystals. The results generally agreed with previous microgravity crystallization studies. On average, microgravity-grown crystals were 20% larger than control crystals grown on Earth, and microgravity-grown crystals had a slightly improved mosaicity (decreased by 0.03°) and diffraction resolution (decreased by 0.2 Å) compared with control crystals grown on Earth. However, the highest resolution and lowest mosaicity crystals were formed on Earth, and the highest-quality crystal overall was formed on Earth after return from microgravity.
doi:10.1107/S2053230X16014011
PMCID: PMC5053161  PMID: 27710941
Huntington’s disease; International Space Station; microgravity; polyglutamine; X-ray crystallography; huntingtin; crystallization
18.  Improved X-ray diffraction from Bacillus megaterium penicillin G acylase crystals through long cryosoaking dehydration 
Penicillin G acylase from the Gram-positive bacterium B. megaterium was crystallized and X-ray diffraction from these crystals could be substantially improved by slight dehydration through a long cryo-soak.
Penicillin G acylase from Bacillus megaterium (BmPGA) is currently used in the pharmaceutical industry as an alternative to PGA from Escherichia coli (EcPGA) for the hydrolysis of penicillin G to produce 6-aminopenicillanic acid (6-APA), a penam nucleus for semisynthetic penicillins. Despite the significant differences in amino-acid sequence between PGAs from Gram-positive and Gram-negative bacteria, a representative PGA structure of Gram-positive origin has never been reported. In this study, crystallization and diffraction studies of BmPGA are described. Poor diffraction patterns with blurred spots at higher resolution were typical for BmPGA crystals cryocooled after a brief immersion in cryoprotectant solution. Overnight soaking in the same cryo-solution substantially improved both the mosaicity and resolution limit through the establishment of a new crystal-packing equilibrium. A crystal of BmPGA diffracted X-rays to 2.20 Å resolution and belonged to the monoclinic space group P21 with one molecule of BmPGA in the asymmetric unit.
doi:10.1107/S1744309111040462
PMCID: PMC3232142  PMID: 22139169
Bacillus megaterium; crystal dehydration; long cryo-soaking; penicillin G acylase
19.  Improved crystal orientation and physical properties from single-shot XFEL stills 
X-ray free-electron laser crystallography relies on the collection of still-shot diffraction patterns. New methods are developed for optimal modeling of the crystals’ orientations and mosaic block properties.
X-ray diffraction patterns from still crystals are inherently difficult to process because the crystal orientation is not uniquely determined by measuring the Bragg spot positions. Only one of the three rotational degrees of freedom is directly coupled to spot positions; the other two rotations move Bragg spots in and out of the reflecting condition but do not change the direction of the diffracted rays. This hinders the ability to recover accurate structure factors from experiments that are dependent on single-shot exposures, such as femtosecond diffract-and-destroy protocols at X-ray free-electron lasers (XFELs). Here, additional methods are introduced to optimally model the diffraction. The best orientation is obtained by requiring, for the brightest observed spots, that each reciprocal-lattice point be placed into the exact reflecting condition implied by Bragg’s law with a minimal rotation. This approach reduces the experimental uncertainties in noisy XFEL data, improving the crystallographic R factors and sharpening anomalous differences that are near the level of the noise.
doi:10.1107/S1399004714024134
PMCID: PMC4257623  PMID: 25478847
X-ray free-electron lasers; single-shot exposures
20.  A generic protocol for protein crystal dehydration using the HC1b humidity controller 
A generic protocol for investigating crystal dehydration is presented and tested with a set of protein crystal systems using the HC1b high-precision crystal humidifier/dehumidifier.
Dehydration may change the crystal lattice and affect the mosaicity, resolution and quality of X-ray diffraction data. A dehydrating environment can be generated around a crystal in several ways with various degrees of precision and complexity. This study uses a high-precision crystal humidifier/dehumidifier to provide an airstream of known relative humidity in which the crystals are mounted: a precise yet hassle-free approach to altering crystal hydration. A protocol is introduced to assess the impact of crystal dehydration systematically applied to nine experimental crystal systems. In one case, that of glucose isomerase, dehydration triggering a change of space group from I222 to P21212 was observed. This observation is supported by an extended study of the behaviour of the glucose isomerase crystal structure during crystal dehydration.
doi:10.1107/S2059798316003065
PMCID: PMC4854313  PMID: 27139626
macromolecular crystallization; crystal dehydration; humidity control; cryoprotection; generic protocol; room temperature
21.  Expression, purification, crystallization and preliminary X-ray crystallographic analysis of fructose-1,6-bisphosphate aldolase from Escherichia coli  
FBPA I from E. coli was expressed, purified and crystallized. The crystals diffracted to 2.0 Å resolution and belonged to space group C2.
Fructose-1,6-bisphosphate aldolase is one of the most important enzymes in the glycolytic pathway and catalyzes the reversible cleavage of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The full-length fbaB gene encoding fructose-1,6-bisphosphate aldolase class I (FBPA I) was cloned from Escherichia coli strain BL21. FBPA I was overexpressed in E. coli and purified. Biochemical analysis found that the optimum reaction temperature of FBPA I is 330.5 K and that the enzyme has a high temperature tolerance. Crystals of recombinant FBPA I were obtained by the sitting-drop vapour-diffusion technique in a condition consisting of 19 mg ml−1 FBPA I in 0.1 M Tris pH 9.0, 10%(w/v) polyethylene glycol 8000 and diffracted to 2.0 Å resolution. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 217.7, b = 114.9, c = 183.9 Å, β = 124.6°. The asymmetric unit of these crystals may contain ten molecules, giving a Matthews coefficient of 2.48 Å3 Da−1 and a solvent content of 50.5%.
doi:10.1107/S2053230X14018408
PMCID: PMC4188083  PMID: 25286943
thermostability; Ec-FBPA I; fructose-1,6-bisphosphate aldolase
22.  An evaluation of adhesive sample holders for advanced crystallographic experiments 
Commercially available adhesives have been evaluated for crystal mounting when undertaking complex macromolecular crystallography experiments. Here, their use as tools for advanced sample mounting and cryoprotection is assessed and their suitability for room-temperature data-collection and humidity-controlled studies is investigated.
The hydration state of macromolecular crystals often affects their overall order and, ultimately, the quality of the X-ray diffraction pattern that they produce. Post-crystallization techniques that alter the solvent content of a crystal may induce rearrangement within the three-dimensional array making up the crystal, possibly resulting in more ordered packing. The hydration state of a crystal can be manipulated by exposing it to a stream of air at controlled relative humidity in which the crystal can equilibrate. This approach provides a way of exploring crystal hydration space to assess the diffraction capabilities of existing crystals. A key requirement of these experiments is to expose the crystal directly to the dehydrating environment by having the minimum amount of residual mother liquor around it. This is usually achieved by placing the crystal on a flat porous support (Kapton mesh) and removing excess liquid by wicking. Here, an alternative approach is considered whereby crystals are harvested using adhesives that capture naked crystals directly from their crystallization drop, reducing the process to a one-step procedure. The impact of using adhesives to ease the harvesting of different types of crystals is presented together with their contribution to background scattering and their usefulness in dehydration experiments. It is concluded that adhesive supports represent a valuable tool for mounting macromolecular crystals to be used in humidity-controlled experiments and to improve signal-to-noise ratios in diffraction experiments, and how they can protect crystals from modifications in the sample environment is discussed.
doi:10.1107/S1399004714014370
PMCID: PMC4157448  PMID: 25195752
macromolecular crystallography; humidity control; Crystal Catcher; resolution; crystal packing
23.  Four crystal forms of a Bence-Jones protein 
Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 y ago. The trigonal crystal form may shed some light on the formation of fibrils common to certain storage diseases.
Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 years ago. Closely related tetragonal and orthorhombic forms belonging to space groups P43212 and P212121, with unit-cell parameters a = b = 68.7, c = 182.1 and a = 67.7, b = 69.4, c = 87.3 Å, diffract to 1.5 and 1.9 Å, respectively. Two closely related trigonal forms, both belonging to space group P3121 with unit-cell parameters a = b = 154.3 Å but differing by a doubling of the c axis, one 46.9 Å and the other 94.0 Å, diffract to 2.9 and 2.6 Å resolution, respectively. The trigonal crystal of short c-axis length shows a positive indication of twinning. The trigonal crystal of longer c axis, which appeared only after eight months of incubation at room temperature, is likely to be composed of proteolytically degraded molecules and unlike the other crystal forms contains two entire Bence-Jones dimers in the asymmetric unit. This latter crystal form may shed some light on the formation of fibrils common to certain storage diseases.
doi:10.1107/S1744309104028532
PMCID: PMC1952397  PMID: 16508097
storage diseases; multiple myeloma; immunoglobulins; proteolysis; twinning
24.  A look inside epitaxial cobalt-on-fluorite nanoparticles with three-dimensional reciprocal space mapping using GIXD, RHEED and GISAXS 
Journal of Applied Crystallography  2013;46(Pt 4):874-881.
Three-dimensional reciprocal space mapping by X-ray and electron diffraction [namely grazing-incidence X-ray diffraction (GIXD), reflection high-energy electron diffraction (RHEED) and grazing-incidence small-angle X-ray scattering (GISAXS)] was used to explore the internal structure and shape of differently oriented epitaxial Co/CaF2 facetted nanoparticles.
In this work epitaxial growth of cobalt on CaF2(111), (110) and (001) surfaces has been extensively studied. It has been shown by atomic force microscopy that at selected growth conditions stand-alone faceted Co nanoparticles are formed on a fluorite surface. Grazing-incidence X-ray diffraction (GIXD) and reflection high-energy electron diffraction (RHEED) studies have revealed that the particles crystallize in the face-centered cubic lattice structure otherwise non-achievable in bulk cobalt under normal conditions. The particles were found to inherit lattice orientation from the underlying CaF2 layer. Three-dimensional reciprocal space mapping carried out using X-ray and electron diffraction has revealed that there exist long bright 〈111〉 streaks passing through the cobalt Bragg reflections. These streaks are attributed to stacking faults formed in the crystal lattice of larger islands upon coalescence of independently nucleated smaller islands. Distinguished from the stacking fault streaks, crystal truncation rods perpendicular to the {111} and {001} particle facets have been observed. Finally, grazing-incidence small-angle X-ray scattering (GISAXS) has been applied to decouple the shape-related scattering from that induced by the crystal lattice defects. Particle faceting has been verified by modeling the GISAXS patterns. The work demonstrates the importance of three-dimensional reciprocal space mapping in the study of epitaxial nanoparticles.
doi:10.1107/S0021889813008777
PMCID: PMC3769055  PMID: 24046491
cobalt-on-fluorite nanoparticles; grazing-incidence X-ray diffraction (GIXD); reflection high-energy electron diffraction (RHEED); grazing-incidence small-angle X-ray scattering (GISAXS); epitaxial growth; three-dimensional reciprocal space mapping
25.  Macromolecular diffractive imaging using imperfect crystals 
Nature  2016;530(7589):202-206.
The three-dimensional structures of macromolecules and their complexes are predominantly elucidated by X-ray protein crystallography. A major limitation is access to high-quality crystals, to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields sufficiently high-resolution information that the crystal structure can be solved. The observation that crystals with shrunken unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks1,2 hints that crystallographic resolution for some macromolecules may be limited not by their heterogeneity but rather by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern, equal to the incoherent sum of diffraction from rigid single molecular complexes aligned along several discrete crystallographic orientations and hence with an increased information content3. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins4 —they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5 Å limit of measurable Bragg peaks, which allows us to directly phase5 the pattern. With the molecular envelope conventionally determined at 4.5 Å as a constraint, we then obtain a static image of the photosystem II dimer at 3.5 Å resolution. This result shows that continuous diffraction can be used to overcome long-supposed resolution limits of macromolecular crystallography, with a method that puts great value in commonly encountered imperfect crystals and opens up the possibility for model-free phasing6,7.
doi:10.1038/nature16949
PMCID: PMC4839592  PMID: 26863980

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