Multiple Crystal Forms Research Articles

Structure of a backtracked state reveals conformational changes similar to the state following nucleotide incorporation in human norovirus polymerase.

The RNA-dependent RNA polymerase (RdRP) from norovirus (NV) genogroup II has previously been crystallized as an apoenzyme (APO1) in multiple crystal forms, as well as as a pre-incorporation ternary complex (PRE1) bound to Mn(2+), various nucleoside triphosphates and an RNA primer-template duplex in an orthorhombic crystal form. When crystallized under near-identical conditions with a slightly different RNA primer/template duplex, however, the enzyme-RNA complex forms tetragonal crystals (anisotropic data, dmin ≃ 1.9 Å) containing a complex with the primer/template bound in a backtracked state (BACK1) similar to a post-incorporation complex (POST1) in a step of the enzymatic cycle immediately following nucleotidyl transfer. The BACK1 conformation shows that the terminal nucleotide of the primer binds in a manner similar to the nucleoside triphosphate seen in the PRE1 complex, even though the terminal two phosphoryl groups in the triphosphate moiety are absent and a covalent bond is present between the α-phosphoryl group of the terminal nucleotide and the 3'-oxygen of the penultimate nucleotide residue. The two manganese ions bound at the active site coordinate to conserved Asp residues and the bridging phosphoryl group of the terminal nucleotide. Surprisingly, the conformation of the thumb domain in BACK1 resembles the open APO1 state more than the closed conformation seen in PRE1. The BACK1 complex thus reveals a hybrid state in which the active site is closed while the thumb domain is open. Comparison of the APO1, PRE1 and BACK1 structures of NV polymerase helps to reveal a more complete and complex pathway of conformational changes within a single RdRP enzyme system. These conformational changes lend insight into the mechanism of RNA translocation following nucleotidyl transfer and suggest novel approaches for the development of antiviral inhibitors.

Two-component self-assembly with solvent leading to “wet” and microcrystalline organogel fibers

HypothesisThe microcrystalline fibers of N-(2-aminoethyl)-3α-hydroxy-5β-cholan-24-amide 1 provided a useful model system for studying the complex relationship between morphology, experimental parameters, solvent, and the phenomenon of organogelation. The presence of solvents in the solid forms of 1 along with crystallization behavior suggested solvate formation and polymorphic behavior. ExperimentsForty solid state- and xerogel samples of 1 formed in organic solvents and in three categories of experimental conditions were analyzed with single crystal X-ray diffraction (XRD), powder X-ray diffraction (PXRD), Raman microscopy, and attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR). FindingsTwo polymorphs and four isostructural aromatic solvates of 1 were found among some unknown forms in the samples. Single crystal X-ray structures of one polymorph and bromobenzene solvate were obtained, the latter from a xerogel. Multiple crystal forms could be present in a sample, and their contributions to gelation were estimated taking the experimental conditions into account. Gelator 1 could act as a variable component gelator, either alone or in combination with an aromatic solvent. The research brings new insight into the structures of microcrystalline organogel fibers, linking solvate/inclusion crystal formation with microcrystalline fibers of an organogelator for the first time.

Microseed matrix screening for optimization in protein crystallization: what have we learned?

Protein crystals obtained in initial screens typically require optimization before they are of X-ray diffraction quality. Seeding is one such optimization method. In classical seeding experiments, the seed crystals are put into new, albeit similar, conditions. The past decade has seen the emergence of an alternative seeding strategy: microseed matrix screening (MMS). In this strategy, the seed crystals are transferred into conditions unrelated to the seed source. Examples of MMS applications from in-house projects and the literature include the generation of multiple crystal forms and different space groups, better diffracting crystals and crystallization of previously uncrystallizable targets. MMS can be implemented robotically, making it a viable option for drug-discovery programs. In conclusion, MMS is a simple, time- and cost-efficient optimization method that is applicable to many recalcitrant crystallization problems.

“Predicting” Crystal Forms of Pharmaceuticals Using Hydrogen Bond Propensities: Two Test Cases

The Cambridge Structural Database incorporates an algorithm, based on data mining and statistical analysis of the structures in the database, for evaluating the “risk of polymorphism” in any compound. We have applied that algorithm to a number of pharmaceutically important compounds from the European Pharmacopoeia for which multiple crystal forms have not been reported. The survey suggested the possibility of polymorphism in at least two compounds, bufexamac and meglumine. The statistical analysis and the subsequent experimental search for polymorphism are reported here. While polymorphism was detected and characterized in both cases, the structural results were not necessarily compatible with the basis for the measure of the “risk of polymorphism”.

Competition between Monomeric and Dimeric Crystals in Schematic Models for Globular Proteins

Advances in experimental techniques and in theoretical models have improved our understanding of protein crystallization. However, they have also left open questions regarding the protein phase behavior and self-assembly kinetics, such as why (nearly) identical crystallization conditions can sometimes result in the formation of different crystal forms. Here, we develop a patchy particle model with competing sets of patches that provides a microscopic explanation of this phenomenon. We identify different regimes in which one or two crystal forms can coexist with a low-density fluid. Using analytical approximations, we extend our findings to different crystal phases, providing a general framework for treating protein crystallization when multiple crystal forms compete. Our results also suggest different experimental routes for targeting a specific crystal form, and for reducing the dynamical competition between the two forms, thus facilitating protein crystal assembly.

Crystallographic analysis of TPP riboswitch binding by small-molecule ligands discovered through fragment-based drug discovery approaches.

Riboswitches are structured mRNA elements that regulate gene expression in response to metabolite or second-messenger binding and are promising targets for drug discovery. Fragment-based drug discovery methods have identified weakly binding small molecule "fragments" that bind a thiamine pyrophosphate (TPP) riboswitch. However, these fragments require substantial chemical elaboration into more potent, drug-like molecules. Structure determination of the fragments bound to the riboswitch is the necessary next step. In this chapter, we describe the methods for co-crystallization and structure determination of fragment-bound TPP riboswitch structures. We focus on considerations for screening crystallization conditions across multiple crystal forms and provide guidance for building the fragment into the refined crystallographic model. These methods are broadly applicable for crystallographic analyses of any small molecules that bind structured RNAs.

MAIN software for density averaging, model building, structure refinement and validation.

MAIN is software that has been designed to interactively perform the complex tasks of macromolecular crystal structure determination and validation. Using MAIN, it is possible to perform density modification, manual and semi-automated or automated model building and rebuilding, real- and reciprocal-space structure optimization and refinement, map calculations and various types of molecular structure validation. The prompt availability of various analytical tools and the immediate visualization of molecular and map objects allow a user to efficiently progress towards the completed refined structure. The extraordinary depth perception of molecular objects in three dimensions that is provided by MAIN is achieved by the clarity and contrast of colours and the smooth rotation of the displayed objects. MAIN allows simultaneous work on several molecular models and various crystal forms. The strength of MAIN lies in its manipulation of averaged density maps and molecular models when noncrystallographic symmetry (NCS) is present. Using MAIN, it is possible to optimize NCS parameters and envelopes and to refine the structure in single or multiple crystal forms.

Synthesis and thermal behaviour of new benzo-1,2-thiazine long-chain aryl-piperazine derivatives

A series of potential biologically active 2-[3-(4-phenyl-1-piperazinyl)propyl]-3-(4-substituted-benzoyl)-4-hydroxy-2H-1,2-benzothiazine 1,1-dioxides was synthesized in a straightforward manner by condensation of respective 3-substituted-4-hydroxy-1,2-benzothiazine 1,1-dioxides with 1-(1-chloropropyl)-4-phenylpiperazine. The structures of all of the newly formed compounds were identified by elemental analysis, FTIR and 1H NMR. The synthesized compounds were subjected to preliminary evaluation using differential scanning calorimetry (DSC) to determine the existence of multiple crystal forms. The DSC scans for all compounds show more than one endothermic effect, which might suggest dynamic proton transfer between two or three possible tautomeric forms: O-keto/O-enol, O-enol/O-keto and O-keto/O-keto/CH2 in crystals 2-[3-(4-phenyl-1-piperazinyl)propyl]-3-(4-substituted-benzoyl)-4-hydroxy-2H-1,2-benzothiazine 1,1-dioxides.

OA01.15. Role of X ray powder diffraction (xrpd) in analysis of shodhit tamra

Purpose: XRPD is an analytical technique for phase identification of materials. It determines the difference in the phase presentation as the process goes on progressing and changing. This technique was used in analysis of Samanya Shodhit, Samanya Shodhanottar Vishesh Shodhit and Vishesh Shodhit Tamra. Method: Samanya Shodhana and Vishesh Shodhana of Tamra was performed as described in RasaTarangini. (Tarang15). XRPD was done in PANalytical X'Pert PRO Diffractometer with secondary beam graphite monochromator selecting the Cu anode material. Four samples were prepared and analysed viz. Sample a - Ashodhit Tamra, Sample B -Samanya Shodhit Tamra, Sample C - Samanya Shodhanottara Vishesh Shodhit Tamra, Sample D - Vishesh Shodhit Tamra. Result: The XRPD of all the samples of Shodhita Tamra showed presence of copper and copper oxides in them. However, the peak positions and intensities were varying. Some new unidentifiable peaks were noticed. In all the samples, Cu was present in major or minor phase. Only Sample B showed presence of both CuO and Cu2O in it. Other Shodhit samples only contained Cu2O and Cu. Conclusion: Presence of CuO in only Samany Shodhit Tamra suggested its larger particle size. As, it was subjected to Vishesh Shodhana, it got converted to thermodynamically more stable Cu2O and lesser particle size. Though Sample C & D had copper oxide in major phases, they exhibited different XRPD pattern. This may indicate multiple crystal forms or polymorphism of copper compounds. Thus, XRPD is a crucial analytical tool to show that the structural and chemical transformation of metal into metal compounds started from the step of Shodhana itself. Furthermore, presences of metallic copper in all the samples denoted their partial conversion and evident need of Marana.

The protein common interface database (ProtCID)--a comprehensive database of interactions of homologous proteins in multiple crystal forms

The protein common interface database (ProtCID) is a database that contains clusters of similar homodimeric and heterodimeric interfaces observed in multiple crystal forms (CFs). Such interfaces, especially of homologous but non-identical proteins, have been associated with biologically relevant interactions. In ProtCID, protein chains in the protein data bank (PDB) are grouped based on their PFAM domain architectures. For a single PFAM architecture, all the dimers present in each CF are constructed and compared with those in other CFs that contain the same domain architecture. Interfaces occurring in two or more CFs comprise an interface cluster in the database. The same process is used to compare heterodimers of chains with different domain architectures. By examining interfaces that are shared by many homologous proteins in different CFs, we find that the PDB and the Protein Interfaces, Surfaces, and Assemblies (PISA) are not always consistent in their annotations of biological assemblies in a homologous family. Our data therefore provide an independent check on publicly available annotations of the structures of biological interactions for PDB entries. Common interfaces may also be useful in studies of protein evolution. Coordinates for all interfaces in a cluster are downloadable for further analysis. ProtCiD is available at http://dunbrack2.fccc.edu/protcid.

ChemInform Abstract: The Growing World of Crystal Forms

AbstractReview: 98 refs.

Analysis of multiple crystal forms ofBacillus subtilisBacB suggests a role for a metal ion as a nucleant for crystallization

Bacillus subtilis BacB is an oxidase that is involved in the production of the antibiotic bacilysin. This protein contains two double-stranded beta-helix (cupin) domains fused in a compact arrangement. BacB crystallizes in three crystal forms under similar crystallization conditions. An interesting observation was that a slight perturbation of the crystallization droplet resulted in the nucleation of a different crystal form. An X-ray absorption scan of BacB suggested the presence of cobalt and iron in the crystal. Here, a comparative analysis of the different crystal forms of BacB is presented in an effort to identify the basis for the different lattices. It is noted that metal ions mediating interactions across the asymmetric unit dominate the different packing arrangements. Furthermore, a normalized B-factor analysis of all the crystal structures suggests that the solvent-exposed metal ions decrease the flexibility of a loop segment, perhaps influencing the choice of crystal form. The residues coordinating the surface metal ion are similar in the triclinic and monoclinic crystal forms. The coordinating ligands for the corresponding metal ion in the tetragonal crystal form are different, leading to a tighter packing arrangement. Although BacB is a monomer in solution, a dimer of BacB serves as a template on which higher order symmetrical arrangements are formed. The different crystal forms of BacB thus provide experimental evidence for metal-ion-mediated lattice formation and crystal packing.

The structures of free-base carbahemiporphyrazines

In this report we present an X-ray crystallographic study of two N -deficient hemiporphyrazines: dicarbahemiporphyrazine (1) and benziphthalocyanine (2). Both compounds can be isolated in multiple crystal forms, and five different solvated forms of macrocycle 1 are presented. Four of the five are non-planar, showing the high degree of flexibility and capacity for hydrogen bonding from solvent to N-H groups in the macrocycle. The remaining structure of 1 is a planar dicationic species that represents a tautomeric form of the macrocycle often seen in metal binding. Four structures of macrocycle 2 are also presented in this report. The structure of 2 is also planar due to the tautomerism of the internal protons, and structural elucidation of this macrocycle reveals the presence of three ionizable protons rather than the expected single proton postulated by Elvidge and Golden.

The growing world of crystal forms

Engineering crystal properties by a choice of adequate molecular building blocks has to cope with the fact that a given molecule can be isolated in many, at times very many, solid forms (single entity crystals, solvates, salts, co-crystals and their polymorphs) each with its own characteristics and properties. Preparation of new crystal forms of a same molecule, and investigation of the relationship among new crystal forms represent not only a further challenge to the solid state chemists, but also an opportunity of modifying properties of crystals with applicative implications (pharmaceuticals, agrochemicals, pigments, organic semiconductors, etc.). This feature article elaborates on this concept via a number of examples of multiple crystal forms and test cases.

Particle shape manipulation and optimization in cooling crystallization involving multiple crystal morphological forms

AbstractA population balance model for predicting the dynamic evolution of crystal shape distribution is further developed to simulate crystallization processes in which multiple crystal morphological forms co‐exist and transitions between them can take place. The new model is applied to derive the optimal temperature and supersaturation profiles leading to the desired crystal shape distribution in cooling crystallization. Since tracking an optimum temperature or supersaturation trajectory can be easily implemented by manipulating the coolant flowrate in the reactor jacket, the proposed methodology provides a feasible closed‐loop mechanism for crystal shape tailoring and control. The methodology is demonstrated by applying it to a case study of seeded cooling crystallization of potash alum. © 2009 American Institute of Chemical Engineers AIChE J, 2009

A Novel Insensitive High Explosive 3,4‐Bis (Aminofurazano) Furoxan

AbstractA novel insensitive high explosive 3,4‐bis (aminofurazano) furoxan (BAFF) was prepared using 3‐amino‐4‐acylchloroximinofurazan (ACOF) as a precursor. The molecular and crystal structures of BAFF were characterized by IR, MS, 1H NMR, 13C NMR, elemental analysis, and single crystal X‐ray diffraction. The single crystal structure of BAFF recrystallized from water is monoclinic, space group P 21/c, and ρc=1.745 g cm−3, and that recrystallized from ethanol is triclinic, space group P 1, and ρc=1.737 g cm−3. BAFF has multiple crystal forms. The calculated detonation velocity by BKW code is 8100 m s−1 (ρ=1.795 g cm−3, theoretical density calculated by quantum chemistry) and the experimental value is 7177 m s−1 (ρ=1.530 g cm−3, charge density). The tested values of impact, friction, and electrostatic spark sensitivity show that BAFF is insensitive.

Statistical Analysis of Interface Similarity in Crystals of Homologous Proteins

Many proteins function as homo-oligomers and are regulated via their oligomeric state. For some proteins, the stoichiometry of homo-oligomeric states under various conditions has been studied using gel filtration or analytical ultracentrifugation experiments. The interfaces involved in these assemblies may be identified using cross-linking and mass spectrometry, solution-state NMR, and other experiments. However, for most proteins, the actual interfaces that are involved in oligomerization are inferred from X-ray crystallographic structures using assumptions about interface surface areas and physical properties. Examination of interfaces across different Protein Data Bank (PDB) entries in a protein family reveals several important features. First, similarities in space group, asymmetric unit size, and cell dimensions and angles (within 1%) do not guarantee that two crystals are actually the same crystal form, containing similar relative orientations and interactions within the crystal. Conversely, two crystals in different space groups may be quite similar in terms of all the interfaces within each crystal. Second, NMR structures and an existing benchmark of PDB crystallographic entries consisting of 126 dimers as well as larger structures and 132 monomers were used to determine whether the existence or lack of common interfaces across multiple crystal forms can be used to predict whether a protein is an oligomer or not. Monomeric proteins tend to have common interfaces across only a minority of crystal forms, whereas higher-order structures exhibit common interfaces across a majority of available crystal forms. The data can be used to estimate the probability that an interface is biological if two or more crystal forms are available. Finally, the Protein Interfaces, Surfaces, and Assemblies (PISA) database available from the European Bioinformatics Institute is more consistent in identifying interfaces observed in many crystal forms compared with the PDB and the European Bioinformatics Institute's Protein Quaternary Server (PQS). The PDB, in particular, is missing highly likely biological interfaces in its biological unit files for about 10% of PDB entries.

Selection of Protein Crystal Forms Facilitated by Polymer-Induced Heteronucleation.

Crystallization of biological macromolecules as high quality single crystals is critical for determining their structure and facilitates the rational design of drugs. Because macromolecules often crystallize in multiple phases that have unique diffraction properties, the selective production of phases is desirable. Furthermore, determining multiple structures allows for a greater understanding of the relationship between crystal packing and conformation. With the aim of exploiting the polymer-induced heteronucleation approach to selectively nucleate multiple macromolecule crystal forms, hen egg white lysozyme (HEWL) was chosen as a model. Selective phase production was achieved under conditions that, in the absence of added heteronuclei, result in crystallization of a single crystal form. Moreover, nucleation rate, which in turn affects the size and quality of HEWL crystals, was controlled by various polymer surfaces. Thus, the polymer-induced heteronucleation approach provides an additional diversity element which can be easily implemented to complement standard crystal growth techniques for the selective production of high quality protein crystals.

Structure of the Full-length Human RPA14/32 Complex Gives Insights into the Mechanism of DNA Binding and Complex Formation

Replication protein A (RPA) is the ubiquitous, eukaryotic single-stranded DNA (ssDNA) binding protein and is essential for DNA replication, recombination, and repair. Here, crystal structures of the soluble RPA heterodimer, composed of the RPA14 and RPA32 subunits, have been determined for the full-length protein in multiple crystal forms. In all crystals, the electron density for the N-terminal (residues 1–42) and C-terminal (residues 175–270) regions of RPA32 is weak and of poor quality indicating that these regions are disordered and/or assume multiple positions in the crystals. Hence, the RPA32 N terminus, that is hyperphosphorylated in a cell-cycle-dependent manner and in response to DNA damaging agents, appears to be inherently disordered in the unphosphorylated state. The C-terminal, winged helix-loop-helix, protein–protein interaction domain adopts several conformations perhaps to facilitate its interaction with various proteins. Although the ordered regions of RPA14/32 resemble the previously solved protease-resistant core crystal structure, the quaternary structures between the heterodimers are quite different. Thus, the four-helix bundle quaternary assembly noted in the original core structure is unlikely to be related to the quaternary structure of the intact heterotrimer. An organic ligand binding site between subunits RPA14 and RPA32 was identified to bind dioxane. Comparison of the ssDNA binding surfaces of RPA70 with RPA14/32 showed that the lower affinity of RPA14/32 can be attributed to a shallower binding crevice with reduced positive electrostatic charge.

Concomitant Polymorphism in Confined Environment

The aim of this paper is to demonstrate that multiple crystal forms can be generated on patterned self-assembled monolayers (SAMs) substrates in single experiments in a given solvent system. Functionalized metallic islands are fabricated and utilized as individual templates for crystal formation. Taking advantage of the different wetting properties that patterned surfaces offered, arrays of small solution droplets on the nano- and pico- liter scale were produced on the substrates. Different droplet dimensions were deposited on the substrate. As the solvent evaporates from the droplets, crystals were formed within the constrained volume. Crystal habits were examined with optical microscopy while the solid form was identified with Raman microscopy. With mefenamic acid (MA) and sulfathiazole as model pharmaceutical compounds, two and four different polymorphs, respectively, were observed under identical conditions. Moreover, it is established that the polymorphic distribution is highly dependent on the solvent evaporation rate and the solution concentration. These results imply that multiple crystal forms competitively nucleate in solution, and the probability of each form nucleating is strongly dependent on the supersaturation of the solution. Additionally, solvent was observed to play a role in controlling the solid state outcome. Multiple crystal forms can concomitantly nucleate on patterned substrates. This technique can particularly be attractive to screen for polymorphs as elusive, metastable solid forms are favored with the creation of high supersaturation and can be stabilized due to the minimal volumes generated.