Project description:Enterobactin is a secondary metabolite produced by Enterobacteriaceae for acquiring iron, an essential metal nutrient. The biosynthesis and utilization of enterobactin permits many Gram-negative bacteria to thrive in environments where low soluble iron concentrations would otherwise preclude survival. Despite extensive work carried out on this celebrated molecule since its discovery over 40 years ago, the ferric enterobactin complex has eluded crystallographic structural characterization. We report the successful growth of single crystals containing ferric enterobactin using racemic crystallization, a method that involves cocrystallization of a chiral molecule with its mirror image. The structures of ferric enterobactin and ferric enantioenterobactin obtained in this work provide a definitive assignment of the stereochemistry at the metal center and reveal secondary coordination sphere interactions. The structures were employed in computational investigations of the interactions of these complexes with two enterobactin-binding proteins, which illuminate the influence of metal-centered chirality on these interactions. This work highlights the utility of small-molecule racemic crystallography for obtaining elusive structures of coordination complexes.
Project description:Racemic crystallography has been used to elucidate the secondary and tertiary structures of peptides and small proteins that are recalcitrant to conventional crystallization. It is unclear, however, whether racemic crystallography can capture native quaternary structure, which could be disrupted by heterochiral associations. We are exploring the use of racemic crystallography to characterize the self-assembly behavior of membrane-associated peptides, very few of which have been crystallized. We report a racemic crystal structure of the membrane-active peptide melittin; the new structure allows comparison with a previously reported crystal structure of L-melittin. The tetrameric assembly observed in crystalline L-melittin has been proposed to represent the tetrameric state detected in solution for this peptide. This tetrameric assembly is precisely reproduced in the racemic crystal, which strengthens the conclusion that the tetramer is biologically relevant. More broadly, these findings suggest that racemic crystallography can provide insight on native quaternary structure.
Project description:Electron crystallography is widespread in material science applications, but for biological samples its use has been restricted to a handful of examples where two-dimensional (2D) crystals or helical samples were studied either by electron diffraction and/or imaging. Electron crystallography in cryoEM, was developed in the mid-1970s and used to solve the structure of several membrane proteins and some soluble proteins. In 2013, a new method for cryoEM was unveiled and named Micro-crystal Electron Diffraction, or MicroED, which is essentially three-dimensional (3D) electron crystallography of microscopic crystals. This method uses truly 3D crystals, that are about a billion times smaller than those typically used for X-ray crystallography, for electron diffraction studies. There are several important differences and some similarities between electron crystallography of 2D crystals and MicroED. In this review, we describe the development of these techniques, their similarities and differences, and offer our opinion of future directions in both fields.
Project description:The ease with which racemic mixtures crystallize compared with the equivalent chiral systems is routinely taken advantage of to produce crystals of small molecules. However, biological macromolecules such as DNA and proteins are naturally chiral, and thus the limited range of chiral space groups available hampers the crystallization of such molecules. Inspiring work over the past 15 years has shown that racemic mixtures of proteins, which were made possible by impressive advances in protein chemical synthesis, can indeed improve the success rate of protein crystallization experiments. More recently, the racemic crystallization approach was extended to include nucleic acids as a possible aid in the determination of enantiopure DNA crystal structures. Here, findings are reported that suggest that the benefits may extend beyond this. Two racemic crystal structures of the DNA sequence d(CCCGGG) are described which were found to fold into A-form DNA. This form differs from the Z-form DNA conformation adopted by the chiral equivalent in the solid state, suggesting that the use of racemates may also favour the emergence of new conformations. Importantly, the racemic mixture forms interactions in the solid state that differ from the chiral equivalent (including the formation of racemic pseudo-helices), suggesting that the use of racemic DNA mixtures could provide new possibilities for the design of precise self-assembled nanomaterials and nanostructures.
Project description:It has previously been shown that the use of racemic mixtures of naturally chiral macromolecules such as protein and DNA can significantly aid the crystallogenesis process, thereby addressing one of the major bottlenecks to structure determination by X-ray crystallographic methods-that of crystal growth. Although previous studies have provided convincing evidence of the applicability of the racemic crystallization technique to DNA through the study of well-characterized DNA structures, we sought to apply this method to a historically challenging DNA sequence. For this purpose we chose a non-self-complementary DNA duplex containing the biologically-relevant Pribnow box consensus sequence 'TATAAT'. Four racemic crystal structures of this previously un-crystallizable DNA target are reported (with resolutions in the range of 1.65-2.3 Å), with further crystallographic studies and structural analysis providing insight into the racemic crystallization process as well as structural details of this highly pertinent DNA sequence.
Project description:Protein 3D structure can be a powerful predictor of function, but it often faces a critical roadblock at the crystallization step. Rv1738, a protein from Mycobacterium tuberculosis that is strongly implicated in the onset of nonreplicating persistence, and thereby latent tuberculosis, resisted extensive attempts at crystallization. Chemical synthesis of the L- and D-enantiomeric forms of Rv1738 enabled facile crystallization of the D/L-racemic mixture. The structure was solved by an ab initio approach that took advantage of the quantized phases characteristic of diffraction by centrosymmetric crystals. The structure, containing L- and D-dimers in a centrosymmetric space group, revealed unexpected homology with bacterial hibernation-promoting factors that bind to ribosomes and suppress translation. This suggests that the functional role of Rv1738 is to contribute to the shutdown of ribosomal protein synthesis during the onset of nonreplicating persistence of M. tuberculosis.
Project description:We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization.
Project description:Racemic protein crystallography, enabled by total chemical synthesis, has allowed us to determine the X-ray structure of native scorpion toxin BmBKTx1; direct methods were used for phase determination. This is the first example of a protein racemate that crystallized in space group I41/a.
Project description:Membrane proteins and macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons four to five orders of magnitude more strongly than they do X-rays. Furthermore, as electron crystallography yields Coulomb potential maps rather than electron density maps, it could provide a unique method to visualize the charged states of amino acid residues and metals. Here we describe an attempt to develop a methodology for electron crystallography of ultrathin (only a few layers thick) 3D protein crystals and present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, obtained from Ca(2+)-ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca(2+)-binding sites of Ca(2+)-ATPase and that of the iron atom in the heme in catalase.