Fotini Karavassili

Structural studies of human insulin cocrystallized with phenol or resorcinol via powder diffraction.

The effects of the ligands phenol and resorcinol on the crystallization of human insulin have been investigated as a function of pH. Powder diffraction data were used to characterize several distinct polymorphic forms. A previously unknown polymorph with monoclinic symmetry (P2(1)) was identified for both types of ligand with similar characteristics [the unit-cell parameters for the insulin-resorcinol complex were a = 114.0228 (8), b = 335.43 (3), c = 49.211 (6) Å, β = 101.531 (8)°].

Macromolecular Powder Diffraction: Ready for genuine biological problems.

Knowledge of 3D structures of biological molecules plays a major role in both understanding important processes of life and developing pharmaceuticals. Among several methods available for structure determination, macromolecular X-ray powder diffraction (XRPD) has transformed over the past decade from an impossible dream to a respectable method. XRPD can be employed in biosciences for various purposes such as observing phase transitions, characterizing bulk pharmaceuticals, determining structures via the molecular replacement method, detecting ligands in protein-ligand complexes, as well as combining micro-sized single crystal crystallographic data and powder diffraction data. Studies using synchrotron and laboratory sources in some standard configuration setups are reported in this review, including their respective advantages and disadvantages. Methods presented here provide an alternative, complementary set of tools to resolve structural problems. A variety of already existing software packages for powder diffraction data processing and analysis, some of which have been adapted to large unit cell studies, are briefly described. This review aims to provide necessary elements of theory and current methods, along with practical explanations, available software packages and highlighted case studies.

Human insulin polymorphism upon ligand binding and pH variation: the case of 4-ethylresorcinol

This study focuses on the effects of the organic ligand 4-ethylresorcinol on the crystal symmetry and lattice dimensions of human insulin using powder X-ray crystallography.

Solid solution along the synthetic LiAlSi2O6-LiFeSi2O6 (spodumene-ferri-spodumene) join: A general picture of solid solutions, bond lengths, lattice strains, steric effects, symmetries, and chemical compositions of Li clinopyroxenes

Abstract Seven clinopyroxene compositions along the join M2LiM1AlTSi2O6 (spodumene) to M2LiM1Fe3+TSi2O6 (ferri-spodumene) were synthesized at 2 GPa, 800 °C under highly oxidizing conditions (using H2O2 fluid) in an end-loaded piston cylinder. In addition, the LiFe3+Si2O6 composition was also synthesized under the intrinsically reducing conditions in a piston cylinder, to check the effect of fO2 on iron speciation. The run products were characterized by field emission scanning electron microscope (FE-SEM), Rietveld refinements on XRPD synchrotron data, and space groups were assigned using SAED-TEM patterns. Run products are composed mainly of lithium clinopyroxene (Li-Cpx), plus minor amounts of hematite (magnetite under reducing condition) and corundum, as independently detected by image analysis (area%) and Rietveld refinements (wt%); moreover, Rietveld results were used to derive cell parameters, M1-site occupancy (Al vs. Fe3+), atomic positions, and average bond lengths of all these Li-Cpx indexed in the C2/c space groups according to SAED-TEM. Li-Cpx with Al and Fe3+ amounts close to 50:50 are actually slightly richer in Al apfu than nominal; the LiFe3+Si2O6 grown under very oxidized and reducing conditions have very similar cell parameters, indicating that fO2 is unable to induce a significant incorporation of Fe2+ in these Li-Cpx. The replacement of Al with Fe3+ induces a linear (%) increase of the cell edges following b > a > c, whereas β is roughly constant and the cell volume increases linearly. Furthermore, the substitution of Al with Fe3+ only weakly affects the T-O average length (<1%), whereas M2-O and M1-O bonds increase linearly of 2.3 and 5.0%, respectively. These new experimental data have been compared with other available on Li-, Na-, and Ca-Cpx, i.e., M2(Li,Na,Ca,Mg,Fe2+)M1(Mg,Fe2+Al,Ni,Cr,Ga,V,Fe3+,Mn,Sc,In)TSi2O6, to model lattice strain, bond lengths, steric effects, and phase transitions behaviors. The replacement of Al with progressively larger cations in LiM3+Si2O6 Cpx (M3+: Ni, Cr, Ga, V, Fe3+, Ti, Sc, and In) results in a linear increase following V > b > a > c, whereas β is roughly constant except for Ti-end-member and P21/c compositions. Lattice strains induced by X, T, and P for Li-Cpx in the C2/c stability field show that when the M1 site is progressively filled with a large cation, ε1 axis (ε1 > ε2 > ε3) increases along b, whereas ε2 and ε3 are nearly parallel to a and at about 30° from c. Conversely, T will provoke a similar enlargement of ε1 and ε2 along b and a edges, respectively, whereas ε3 is again oriented at about 30° from c; the increasing of P will instead shorten all strain tensor components (ε1, ε2, and ε3) with a similar percentage amount; notably, high-P is the only stress that induces a strain component to be almost parallel to c edge. Moreover, finite lattice strains and orientation in C2/c LiMe3+Si2O6 Li-Cpx induced by Me3+: Al-Fe3+, Fe3+-Sc, Sc-In are slightly different, with ε1 invariably lying along b; conversely, Li-Na cation substitution is completely different with the highest and lowest deformations on the ac plane and ε2 along b; ε3 vector is negative and oriented at about 30° from T-chains. The ideal replacement of Al with larger cations up to In in Li-Cpx induces the M1-O, M2-O, and T-O average bond lengths to increase by 10.6, 4.3, and <0.5%. Steric effects in LiM1Me3+Si2O6 and NaM1Me3+Si2O6 Cpx are significant and very similar, whereas several other Me1+ and Me2+ substitutions in Cpx at both the M1 and M2 site, keeping fixed the other site, display less or even the absence of steric effects. Our new data also better elucidate relationships between Li-Cpx composition, symmetry at room and non-ambient conditions and Tc. The aggregate cation radii at the M1 site does not exclusively control the stability of C2/c and P21/c polymorphs; instead valence electrons can profoundly favor the stabilization of a polymorph.

Dengue virus 3 NS5 methyltransferase domain: expression, purification, crystallization and first structural data from microcrystalline specimens

Abstract Flavivirus infections often provoke life-threatening diseases of epidemic magnitudes, thus extensive research is currently directed towards the development of efficient vaccines and approved antiviral compounds. We present here the expression, purification, crystallization and preliminary X-ray diffraction analysis of one of the components of the flavivirus replication complex, the non-structural protein 5 (NS5) mRNA methyltransferase (MTase) domain, from an emerging pathogenic flavivirus, dengue virus 3 (DEN3). Polycrystalline precipitates of DEN3 NS5 MTase, suitable for X-ray powder diffraction (XRPD) measurements, were produced in the presence of PEG 8000 (25–32.5% (w/v)), 0.1 M Tris-Amino, in a pH range from 7.0 to 8.0. A polymorph of orthorhombic symmetry (space group: P21212, a=61.9 Å, b=189.6 Å, c=52.4 Å) was identified via XRPD. These results are the first step towards the complete structural determination of this molecule via XRPD and a parallel demonstration of the applicability of the method.

Humidity-induced phase transitions of HEW lysozyme investigated by powder diffraction

The simplicity of XRPD data collection and the uniqueness of the diffraction pattern that each polymorph shows, marks powder diffraction as the most suitable technique for quick and accurate characterization of microcrystalline suspensions. However, for protein samples, the intense synchrotron beam for ultrahigh-resolution XRPD data collection, typically causes radiation-damage effects. The damage rapidly appears during the measurement, affecting both angular (FWHM) and d-spacing resolution. As radiation damage can be a serious obstacle for collecting high quality data, measurements on a laboratory system have a lot to offer. Here, we present our analysis results of well known tetragonal and of a new monoclinic HEWL polymoph on a laboratory X-ray powder diffractometer including in situ measurements under variable relative humidity conditions for both polymorphs. Proteins often crystallize in microcrystalline precipitates. The protein molecules are then surrounded by solvent and their packing arrangement is retained by limited intermolecular contacts. A change in the crystal environment first affects the bulk solvent that fills the intermolecular space, with resulting changes in the crystal structure. In literature it is reported that protein crystals in controlled humidity environments show a large change in unit-cell parameters when the humidity is decreased [1-2]. When a protein crystal is carefully dehydrated, it is in a metastable state in which the crystal initially still retains the original packing structure [2]. Further dehydration may cause the collapse of the crystal lattice: the crystal no longer maintains its packing structure because of the loss of a large amount of bulk solvent. However in some crystals, the dehydration induces a molecular arrangement change resulting in a new crystal structure. This has been already reported for hen egg-white (HEW) lysozyme [3]. While dehydration can induce structural changes, this is also likely to happen upon hydration of the same crystals. The observed gradual structural changes during our experiments as well as phase transitions upon dehydration and hydration of HEWL are analyzed in the relative humidity range 50% 95%. Dehydration and hydration processes are reversible in humidity cycles in the range of 95% rH to 75% rH. Without stabilizing PEG the lower limit for dehydration of tetragonal HEWL is around 75% rH. With PEG the tetragonal HEWL samples remain crystalline below 75% rH, but show phase transitions and larger variations of the cell parameters. Below 75% rH another new tetragonal polymorph was discovered.

Humidity induced phase transitions of HEW lysozyme investigated by microcrystalline powder diffraction

Pro teins of ten crys tal lize in microcrystalline pre cip i tates. The pro tein mol e cules are then sur rounded by sol vent and their pack ing ar range ment is re tained by lim ited inter molecular con tacts. A change in the crys tal en vi ron ment first af fects the bulk sol vent that fills the inter molecular space, with re sult ing changes in the crys tal struc ture. In lit er a ture it is re ported that pro tein crys tals in con trolled hu mid ity en vi ron ments show a large change in unit-cell pa ram e ters when the hu mid ity is de creased [1-2]. When a pro tein crys tal is care fully de hy drated, it is in a metastable state in which the crys tal ini tially still re tains the orig i nal pack ing struc ture [2]. Fur ther de hy dra tion may cause the col lapse of the crys tal lat tice: the crys tal no lon ger main tains its pack ing struc ture be cause of the loss of a large amount of bulk sol vent. How ever in some crys tals, the de hy dra tion in duces a mo lec u lar ar range ment change re sult ing in a new crys tal struc ture. This has been al ready re ported for hen egg-white (HEW) lysozyme [3]. While de hy dra tion can in duce struc tural changes, this is also likely to hap pen upon hydration of the same crys tals. Here, we pre sent our re sults of micro crys tal li ne tetra go nal and mo noc li nic HEWL sam ples on a la bo ra to ry X-ray pow der diffrac to me ter inclu ding in situ me a su re ments un der va ri a ble re la ti ve hu mi di ty con di ti ons. The ob ser ved gra du al structu ral chan ges as well as phase tran si ti ons upon de hyd rati on and hyd rati on of HEWL are ana ly zed in the relative humidity range 50% 95%. (Figure 1.) De hy dra tion and hydration pro cesses are re vers ible in hu mid ity cy cles in the range of 95% rH to 75% rH. With out sta bi liz ing PEG the lower limit for de hy dra tion of tetragonal HEWL is around 75% rH. With PEG the tetragonal HEWL sam ples re main crys tal line be low 75% rH, but show phase tran si tions and larger vari a tions of the cell pa ram e ters. Be low 75% rH a new tetragonal polymorph was dis cov ered.