Determination of the X-ray structure of the snake venom protein omwaprin by total chemical synthesis and racemic protein crystallography

The 50‐residue snake venom protein L ‐omwaprin and its enantiomer D ‐omwaprin were prepared by total chemical synthesis. Radial diffusion assays were performed against Bacillus megaterium and Bacillus anthracis; both L ‐ and D ‐omwaprin showed antibacterial activity against B. megaterium. The native protein enantiomer, made of L ‐amino acids, failed to crystallize readily. However, when a racemic mixture containing equal amounts of L ‐ and D ‐omwaprin was used, diffraction quality crystals were obtained. The racemic protein sample crystallized in the centrosymmetric space group P2₁/c and its structure was determined at atomic resolution (1.33 Å) by a combination of Patterson and direct methods based on the strong scattering from the sulfur atoms in the eight cysteine residues per protein. Racemic crystallography once again proved to be a valuable method for obtaining crystals of recalcitrant proteins and for determining high‐resolution X‐ray structures by direct methods.     

X‐ray radiation‐induced addition of oxygen atoms to protein residues

The additions of oxygen and peroxide to residues that result when proteins are exposed to the free radicals produced using the Fenton reaction or X‐rays have been studied for over a century. Nevertheless little is known about the impact these modifications have on protein crystal structures. Here evidence is presented that both kinds of modifications occur in protein crystals on a significant scale during the collection of X‐ray diffraction data. For example, at least 538 of the 5,351 residues of protein molecules in the crystal used to obtain the structure for photosystem II described by the PDB accession number 3ARC became oxygenated during data collection.     

Determination of protein oligomeric structure from small‐angle X‐ray scattering

Small‐angle X‐ray scattering (SAXS) is useful for determining the oligomeric states and quaternary structures of proteins in solution. The average molecular mass in solution can be calculated directly from a single SAXS curve collected on an arbitrary scale from a sample of unknown protein concentration without the need for beamline calibration or protein standards. The quaternary structure in solution can be deduced by comparing the experimental SAXS curve to theoretical curves calculated from proposed models of the oligomer. This approach is especially robust when the crystal structure of the target protein is known, and the candidate oligomer models are derived from the crystal lattice. When SAXS data are obtained at multiple protein concentrations, this analysis can provide insight into dynamic self‐association equilibria. Herein, we summarize the computational methods that are used to determine protein molecular mass and quaternary structure from SAXS data. These methods are organized into a workflow and demonstrated with four case studies using experimental SAXS data from the published literature.     

On interpretation of protein X‐ray structures: Planarity of the peptide unit

Pauling's mastery of peptide stereochemistry—based on small molecule crystal structures and the theory of chemical bonding—led to his realization that the peptide unit is planar and then to the Pauling–Corey–Branson model of the α‐helix. Similarly, contemporary protein structure refinement is based on experimentally determined diffraction data together with stereochemical restraints. However, even an X‐ray structure at ultra‐high resolution is still an under‐determined model in which the linkage among refinement parameters is complex. Consequently, restrictions imposed on any given parameter can affect the entire structure. Here, we examine recent studies of high resolution protein X‐ray structures, where substantial distortions of the peptide plane are found to be commonplace. Planarity is assessed by the ω‐angle, a dihedral angle determined by the peptide bond (C? N) and its flanking covalent neighbors; for an ideally planar trans peptide, ω = 180°. By using a freely available refinement package, Phenix [Afonine et al. (2012) Acta Cryst. D, 68:352–367], we demonstrate that tightening default restrictions on the ω‐angle can significantly reduce apparent deviations from peptide unit planarity without consequent reduction in reported evaluation metrics (e.g., R‐factors). To be clear, our result does not show that substantial non‐planarity is absent, only that an equivalent alternative model is possible. Resolving this disparity will ultimately require improved understanding of the deformation energy. Meanwhile, we urge inclusion of ω‐angle statistics in new structure reports in order to focus critical attention on the usual practice of assigning default values to ω‐angle constraints during structure refinement. Proteins 2015; 83:1687–1692. © 2015 Wiley Periodicals, Inc.     

X‐ray crystal structure of anhydrous chitosan at atomic resolution

We determined the crystal structure of anhydrous chitosan at atomic resolution, using X‐ray fiber diffraction data extending to 1.17 Å resolution. The unit cell [a = 8.129(7) Å, b = 8.347(6) Å, c = 10.311(7) Å, space group P2₁2₁2₁] of anhydrous chitosan contains two chains having one glucosamine residue in the asymmetric unit with the primary hydroxyl group in the gt conformation, that could be directly located in the Fourier omit map. The molecular arrangement of chitosan is very similar to the corner chains of cellulose II implying similar intermolecular hydrogen bonding between O6 and the amine nitrogen atom, and an intramolecular bifurcated hydrogen bond from O3 to O5 and O6. In addition to the classical hydrogen bonds, all the aliphatic hydrogens were involved in one or two weak hydrogen bonds, mostly helping to stabilize cohesion between antiparallel chains. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 361–368, 2016.     

X‐ray structure of Danio rerio secretagogin: A hexa‐EF‐hand calcium sensor

Many essential physiological processes are regulated by the modulation of calcium concentration in the cell. The EF‐hand proteins represent a superfamily of calcium‐binding proteins involved in calcium signaling and homeostasis. Secretagogin is a hexa‐EF‐hand protein that is highly expressed in pancreatic islet of Langerhans and neuroendocrine cells and may play a role in the trafficking of secretory granules. We present the X‐ray structure of Danio rerio secretagogin, which is 73% identical to human secretagogin, in calcium‐free form at 2.1‐Å resolution. Secretagogin consists of the three globular domains each of which contains a pair of EF‐hand motifs. The domains are arranged into a V‐shaped molecule with a distinct groove formed at the interface of the domains. Comparison of the secretagogin structure with the solution structure of calcium‐loaded calbindin D_28K revealed a striking difference in the spatial arrangement of their domains, which involves ～180° rotation of the first globular domain with respect to the module formed by the remaining domains. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

X‐ray crystallographic structure of BshB,the zinc‐dependent deacetylase involved in bacillithiol biosynthesis

Many gram‐positive bacteria produce bacillithiol to aid in the maintenance of redox homeostasis and degradation of toxic compounds, including the antibiotic fosfomycin. Bacillithiol is produced via a three‐enzyme pathway that includes the action of the zinc‐dependent deacetylase BshB. Previous studies identified conserved aspartate and histidine residues within the active site that are involved in metal binding and catalysis, but the enzymatic mechanism is not fully understood. Here we report two X‐ray crystallographic structures of BshB from Bacillus subtilis that provide insight into the BshB catalytic mechanism.     

Direct imaging electron microscopy (EM) methods in modern structural biology: Overview and comparison with X‐ray crystallography and single‐particle cryo‐EM reconstruction in the studies of large macromolecules

Determining the structure of macromolecules is important for understanding their function. The fine structure of large macromolecules is currently studied primarily by X‐ray crystallography and single‐particle cryo‐electron microscopy (EM) reconstruction. Before the development of these techniques, macromolecular structure was often examined by negative‐staining, rotary‐shadowing and freeze‐etching EM, which are categorised here as ‘direct imaging EM methods’. In this review, the results are summarised by each of the above techniques and compared with respect to four macromolecules: the ryanodine receptor, cadherin, rhodopsin and the ribosome–translocon complex (RTC). The results of structural analysis of the ryanodine receptor and cadherin are consistent between each technique. The results obtained for rhodopsin vary to some extent within each technique and between the different techniques. Finally, the results for RTC are inconsistent between direct imaging EM and other analytical techniques, especially with respect to the space within RTC, the reasons for which are discussed. Then, the role of direct imaging EM methods in modern structural biology is discussed. Direct imaging methods should support and verify the results obtained by other analytical methods capable of solving three‐dimensional molecular architecture, and they should still be used as a primary tool for studying macromolecule structure in vivo.     

Measurement of protein size in concentrated solutions by small angle X‐ray scattering

By simulations on the distance distribution function (DDF) derived from small angle X‐ray scattering (SAXS) theoretical data of a dense monodisperse system, we found a quantitative mathematical correlation between the apparent size of a spherically symmetric (or nearly spherically symmetric) homogenous particle and the concentration of the solution. SAXS experiments on protein solutions of human hemoglobin and horse myoglobin validated the correlation. This gives a new method to determine, from the SAXS DDF, the size of spherically symmetric (or nearly spherically symmetric) particles of a dense monodisperse system, specifically for protein solutions with interference effects.     

qFit 3: Protein and ligand multiconformer modeling for X‐ray crystallographic and single‐particle cryo‐EM density maps

New X‐ray crystallography and cryo‐electron microscopy (cryo‐EM) approaches yield vast amounts of structural data from dynamic proteins and their complexes. Modeling the full conformational ensemble can provide important biological insights, but identifying and modeling an internally consistent set of alternate conformations remains a formidable challenge. qFit efficiently automates this process by generating a parsimonious multiconformer model. We refactored qFit from a distributed application into software that runs efficiently on a small server, desktop, or laptop. We describe the new qFit 3 software and provide some examples. qFit 3 is open‐source under the MIT license, and is available at https://github.com/ExcitedStates/qfit-3.0 .     

The structure of a putative S‐formylglutathione hydrolase from Agrobacterium tumefaciens

The structure of the Atu1476 protein from Agrobacterium tumefaciens was determined at 2 Å resolution. The crystal structure and biochemical characterization of this enzyme support the conclusion that this protein is an S-formylglutathione hydrolase (AtuSFGH). The three-dimensional structure of AtuSFGH contains the α/β hydrolase fold topology and exists as a homo-dimer. Contacts between the two monomers in the dimer are formed both by hydrogen bonds and salt bridges. Biochemical characterization reveals that AtuSFGH hydrolyzes C—O bonds with high affinity toward short to medium chain esters, unlike the other known SFGHs which have greater affinity toward shorter chained esters. A potential role for Cys54 in regulation of enzyme activity through S-glutathionylation is also proposed.     

Relative stability of protein structures determined by X-ray crystallography or NMR spectroscopy: a molecular dynamics simulation study

The relative stability of protein structures determined by either X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy has been investigated by using molecular dynamics simulation techniques. Published structures of 34 proteins containing between 50 and 100 residues have been evaluated. The proteins selected represent a mixture of secondary structure types including all alpha, all beta, and alpha/beta. The proteins selected do not contain cysteine-cysteine bridges. In addition, any crystallographic waters, metal ions, cofactors, or bound ligands were removed before the systems were simulated. The stability of the structures was evaluated by simulating, under identical conditions, each of the proteins for at least 5 ns in explicit solvent. It is found that not only do NMR-derived structures have, on average, higher internal strain than structures determined by X-ray crystallography but that a significant proportion of the structures are unstable and rapidly diverge in simulations.     

Structure of full‐length class I chitinase from rice revealed by X‐ray crystallography and small‐angle X‐ray scattering

The rice class I chitinase OsChia1b, also referred to as RCC2 or Cht‐2, is composed of an N‐terminal chitin‐binding domain (ChBD) and a C‐terminal catalytic domain (CatD), which are connected by a proline‐ and threonine‐rich linker peptide. Because of the ability to inhibit fungal growth, the OsChia1b gene has been used to produce transgenic plants with enhanced disease resistance. As an initial step toward elucidating the mechanism of hydrolytic action and antifungal activity, the full‐length structure of OsChia1b was analyzed by X‐ray crystallography and small‐angle X‐ray scattering (SAXS). We determined the crystal structure of full‐length OsChia1b at 2.00‐Å resolution, but there are two possibilities for a biological molecule with and without interdomain contacts. The SAXS data showed an extended structure of OsChia1b in solution compared to that in the crystal form. This extension could be caused by the conformational flexibility of the linker. A docking simulation of ChBD with tri‐N‐acetylchitotriose exhibited a similar binding mode to the one observed in the crystal structure of a two‐domain plant lectin complexed with a chitooligosaccharide. A hypothetical model based on the binding mode suggested that ChBD is unsuitable for binding to crystalline α‐chitin, which is a major component of fungal cell walls because of its collisions with the chitin chains on the flat surface of α‐chitin. This model also indicates the difference in the binding specificity of plant and bacterial ChBDs of GH19 chitinases, which contribute to antifungal activity. Proteins 2010. © 2010 Wiley‐Liss,Inc.     

Conformational variability of the stationary phase survival protein E from Xylella fastidiosa revealed by X‐ray crystallography,small‐angle X‐ray scattering studies,and normal mode analysis

Xylella fastidiosa is a xylem‐limited bacterium that infects a wide variety of plants. Stationary phase survival protein E is classified as a nucleotidase, which is expressed when bacterial cells are in the stationary growth phase and subjected to environmental stresses. Here, we report four refined X‐ray structures of this protein from X. fastidiosa in four different crystal forms in the presence and/or absence of the substrate 3′‐AMP. In all chains, the conserved loop verified in family members assumes a closed conformation in either condition. Therefore, the enzymatic mechanism for the target protein might be different of its homologs. Two crystal forms exhibit two monomers whereas the other two show four monomers in the asymmetric unit. While the biological unit has been characterized as a tetramer, differences of their sizes and symmetry are remarkable. Four conformers identified by Small‐Angle X‐ray Scattering (SAXS) in a ligand‐free solution are related to the low frequency normal modes of the crystallographic structures associated with rigid body‐like protomer arrangements responsible for the longitudinal and symmetric adjustments between tetramers. When the substrate is present in solution, only two conformers are selected. The most prominent conformer for each case is associated to a normal mode able to elongate the protein by moving apart two dimers. To our knowledge, this work was the first investigation based on the normal modes that analyzed the quaternary structure variability for an enzyme of the SurE family followed by crystallography and SAXS validation. The combined results raise new directions to study allosteric features of XfSurE protein.     

X‐ray and Cryo‐EM structures reveal mutual conformational changes of Kinesin and GTP‐state microtubules upon binding

The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin‐1/KIF5C preferentially binds to the GTP‐state microtubules over GDP‐state microtubules to selectively enter an axon among many processes; however, because the atomic structure of nucleotide‐free KIF5C is unavailable, its molecular mechanism remains unresolved. Here, the crystal structure of nucleotide‐free KIF5C and the cryo‐electron microscopic structure of nucleotide‐free KIF5C complexed with the GTP‐state microtubule are presented. The structures illustrate mutual conformational changes induced by interaction between the GTP‐state microtubule and KIF5C. KIF5C acquires the ‘rigor conformation’, where mobile switches I and II are stabilized through L11 and the initial portion of the neck‐linker, facilitating effective ADP release and the weak‐to‐strong transition of KIF5C microtubule affinity. Conformational changes to tubulin strengthen the longitudinal contacts of the GTP‐state microtubule in a similar manner to GDP‐taxol microtubules. These results and functional analyses provide the molecular mechanism of the preferential binding of KIF5C to GTP‐state microtubules.     

Different speciation for bromine in brown and red algae,revealed by in vivo X‐ray absorption spectroscopic studies

Members of various algal lineages are known to be strong producers of atmospherically relevant halogen emissions, that is a consequence of their capability to store and metabolize halogens. This study uses a noninvasive, synchrotron‐based technique, X‐ray absorption spectroscopy, for addressing in vivo bromine speciation in the brown algae Ectocarpus siliculosus, Ascophyllum nodosum, and Fucus serratus, the red algae Gracilaria dura, G. gracilis, Chondrus crispus, Osmundea pinnatifida, Asparagopsis armata, Polysiphonia elongata, and Corallina officinalis, the diatom Thalassiosira rotula, the dinoflagellate Lingulodinium polyedrum and a natural phytoplankton sample. The results highlight a diversity of fundamentally different bromine storage modes: while most of the stramenopile representatives and the dinoflagellate store mostly bromide, there is evidence for Br incorporated in nonaromatic hydrocarbons in Thalassiosira. Red algae operate various organic bromine stores – including a possible precursor (by the haloform reaction) for bromoform in Asparagopsis and aromatically bound Br in Polysiphonia and Corallina. Large fractions of the bromine in the red algae G. dura and C. crispus and the brown alga F. serratus are present as Br^? defects in solid KCl, similar to what was reported earlier for Laminaria parts. These results are discussed according to different defensive strategies that are used within algal taxa to cope with biotic or abiotic stresses.     

Exploring the dynamic information content of a protein NMR structure: Comparison of a molecular dynamics simulation with the NMR and X‐ray structures of Escherichia coli ribonuclease HI

The multiconformer nature of solution nuclear magnetic resonance (NMR) structures of proteins results from the effects of intramolecular dynamics, spin diffusion and an uneven distribution of structural restraints throughout the molecule. A delineation of the former from the latter two contributions is attempted in this work for an ensemble of 15 NMR structures of the protein Escherichia coli ribonuclease HI (RNase HI). Exploration of the dynamic information content of the NMR ensemble is carried out through correlation with data from two crystal structures and a 1.7‐ns molecular dynamics (MD) trajectory of RNase HI in explicit solvent. Assessment of the consistency of the crystal and mean MD structures with nuclear Overhauser effect (NOE) data showed that the NMR ensemble is overall more compatible with the high‐resolution (1.48 Å) crystal structure than with either the lower‐resolution (2.05 Å) crystal structure or the MD simulation. Furthermore, the NMR ensemble is found to span more conformational space than the MD simulation for both the backbone and the sidechains of RNase HI. Nonetheless, the backbone conformational variability of both the NMR ensemble and the simulation is especially consistent with NMR relaxation measurements of two loop regions that are putative sites of substrate recognition. Plausible side‐chain dynamic information is extracted from the NMR ensemble on the basis of (i) rotamericity and syn‐pentane character of variable torsion angles, (ii) comparison of the magnitude of atomic mean‐square fluctuations (msf) with those deduced from crystallographic thermal factors, and (iii) comparison of torsion angle conformational behavior in the NMR ensemble and the simulation. Several heterogeneous torsion angles, while adopting non‐rotameric/syn‐pentane conformations in the NMR ensemble, exist in a unique conformation in the simulation and display low X‐ray thermal factors. These torsions are identified as sites whose variability is likely to be an artifact of the NMR structure determination procedure. A number of other torsions show a close correspondence between the conformations sampled in the NMR and MD ensembles, as well as significant correlations among crystallographic thermal factors and atomic msf calculated from the NMR ensemble and the simulation. These results indicate that a significant amount of dynamic information is contained in the NMR ensemble. The relevance of the present findings for the biological function of RNase HI, protein recognition studies, and previous investigations of the motional content of protein NMR structures are discussed. Proteins 1999;36:87–110. © 1999 Wiley‐Liss, Inc.     

Crystal structure of deltarhodopsin‐3 from Haloterrigena thermotolerans

Deltarhodopsin, a new member of the microbial rhodopsin family, functions as a light‐driven proton pump. Here, we report the three‐dimensional structure of deltarhodopsin (dR3) from Haloterrigena thermotolerans at 2.7 Å resolution. A crystal belonging to space group R32 (a, b = 111.71 Å, c = 198.25 Å) was obtained by the membrane fusion method. In this crystal, dR3 forms a trimeric structure as observed for bacteriorhodopsin (bR). Structural comparison of dR with bR showed that the inner part (the proton release and uptake pathways) is highly conserved. Meanwhile, residues in the protein–protein contact region are largely altered so that the diameter of the trimeric structure at the cytoplasmic side is noticeably larger in dR3. Unlike bR, dR3 possesses a helical segment at the C‐terminal region that fills the space between the AB and EF loops. A significant difference is also seen in the FG loop, which is one residue longer in dR3. Another peculiar property of dR3 is a highly crowded distribution of positively charged residues on the cytoplasmic surface, which may be relevant to a specific interaction with some cytoplasmic component.Proteins 2013; © 2013 Wiley Periodicals, Inc.     

Crystal structure of secretory abundant heat soluble protein 4 from one of the toughest “water bears” micro‐animals Ramazzottius Varieornatus

Though anhydrobiotic tardigrades (micro‐animals also known as water bears) possess many genes of secretory abundant heat soluble (SAHS) proteins unique to Tardigrada, their functions are unknown. A previous crystallographic study revealed that a SAHS protein (RvSAHS1) from one of the toughest tardigrades, Ramazzottius varieornatus, has a β‐barrel architecture similar to fatty acid binding proteins (FABPs) and two putative ligand binding sites (LBS1 and LBS2) where fatty acids can bind. However, some SAHS proteins such as RvSAHS4 have different sets of amino acid residues at LBS1 and LBS2, implying that they prefer other ligands and have different functions. Here RvSAHS4 was crystallized and analyzed under a condition similar to that for RvSAHS1. There was no electron density corresponding to a fatty acid at LBS1 of RvSAHS4, where a putative fatty acid was observed in RvSAHS1. Instead, LBS2 of RvSAHS4, which was composed of uncharged residues, captured a putative polyethylene glycol molecule. These results suggest that RvSAHS4 mainly uses LBS2 for the binding of uncharged molecules.