Showing papers in "Acta Crystallographica Section D-biological Crystallography in 2014"

Processing of X-ray snapshots from crystals in random orientations.

Abstract: A functional expression is introduced that relates scattered X-ray intensities from a still or a rotation snapshot to the corresponding structure-factor amplitudes. The new approach was implemented in the program nXDS for processing monochromatic diffraction images recorded by a multi-segment detector where each exposure could come from a different crystal. For images containing indexable spots, the intensities of the expected reflections and their variances are obtained by profile fitting after mapping the contributing pixel contents to the Ewald sphere. The varying intensity decline owing to the angular distance of the reflection from the surface of the Ewald sphere is estimated using a Gaussian rocking curve. This decline is dubbed `Ewald offset correction', which is well defined even for still images. Together with an image-scaling factor and other corrections, an explicit expression is defined that predicts each recorded intensity from its corresponding structure-factor amplitude. All diffraction parameters, scaling and correction factors are improved by post-refinement. The ambiguous case of a lower point group than the lattice symmetry is resolved by a method reminiscent of the technique of `selective breeding'. It selects the indexing alternative for each image that yields, on average, the highest correlation with intensities from all other images. Processing a test set of rotation images by XDS and treating the same images by nXDS as snapshots of crystals in random orientations yields data of comparable quality, clearly indicating an anomalous signal from Se atoms.

Conformation-independent structural comparison of macromolecules with ProSMART

Abstract: The identification and exploration of (dis)similarities between macromolecular structures can help to gain biological insight, for instance when visualizing or quantifying the response of a protein to ligand binding. Obtaining a residue alignment between compared structures is often a prerequisite for such comparative analysis. If the conformational change of the protein is dramatic, conventional alignment methods may struggle to provide an intuitive solution for straightforward analysis. To make such analyses more accessible, the Procrustes Structural Matching Alignment and Restraints Tool (ProSMART) has been developed, which achieves a conformation-independent structural alignment, as well as providing such additional functionalities as the generation of restraints for use in the refinement of macromolecular models. Sensible comparison of protein (or DNA/RNA) structures in the presence of conformational changes is achieved by enforcing neither chain nor domain rigidity. The visualization of results is facilitated by popular molecular-graphics software such as CCP4mg and PyMOL, providing intuitive feedback regarding structural conservation and subtle dissimilarities between close homologues that can otherwise be hard to identify. Automatically generated colour schemes corresponding to various residue-based scores are provided, which allow the assessment of the conservation of backbone and side-chain conformations relative to the local coordinate frame. Structural comparison tools such as ProSMART can help to break the complexity that accompanies the constantly growing pool of structural data into a more readily accessible form, potentially offering biological insight or influencing subsequent experiments.

Latent and active abPPO4 mushroom tyrosinase cocrystallized with hexatungstotellurate(VI) in a single crystal.

Abstract: Tyrosinases, bifunctional metalloenzymes, catalyze the oxidation of monophenols and o-diphenols to o-quinones, the precursor compounds of the brown-coloured pigment melanin. In eukaryotic organisms, tyrosinases are expressed as latent zymogens that have to be proteolytically cleaved in order to form highly active enzymes. This activation mechanism, known as the tyrosinase maturation process, has scientific and industrial significance with respect to biochemical and technical applications of the enzyme. Here, not only the first crystal structure of the mushroom tyrosinase abPPO4 is presented in its active form (Ser2–Ser383) and in its 21 kDa heavier latent form (Ser2–Thr545), but furthermore the simultaneous presence of both forms within one single-crystal structure is shown. This allows for a simple approach to investigate the transition between these two forms. Isoform abPPO4 was isolated and extensively purified from the natural source (Agaricus bisporus), which contains a total of six polyphenol oxidases (PPOs). The enzyme formed crystals (diffracting to a resolution of 2.76 A) owing to the employment of the 6-tungstotellurate(VI) salt (Na6[TeW6O24]·22H2O) as a cocrystallization agent. Two of these disc-shaped Anderson-type polyoxoanions [TeW6O24]6− separate two asymmetric units comprising one crystallographic heterodimer of abPPO4, thus resulting in very interesting crystal packing.

Structural basis for glucose tolerance in GH1 β-glucosidases

Abstract: Product inhibition of β-glucosidases (BGs) by glucose is considered to be a limiting step in enzymatic technologies for plant-biomass saccharification. Remarkably, some β-glucosidases belonging to the GH1 family exhibit unusual properties, being tolerant to, or even stimulated by, high glucose concentrations. However, the structural basis for the glucose tolerance and stimulation of BGs is still elusive. To address this issue, the first crystal structure of a fungal β-glucosidase stimulated by glucose was solved in native and glucose-complexed forms, revealing that the shape and electrostatic properties of the entrance to the active site, including the +2 subsite, determine glucose tolerance. The aromatic Trp168 and the aliphatic Leu173 are conserved in glucose-tolerant GH1 enzymes and contribute to relieving enzyme inhibition by imposing constraints at the +2 subsite that limit the access of glucose to the −1 subsite. The GH1 family β-glucosidases are tenfold to 1000-fold more glucose tolerant than GH3 BGs, and comparative structural analysis shows a clear correlation between active-site accessibility and glucose tolerance. The active site of GH1 BGs is located in a deep and narrow cavity, which is in contrast to the shallow pocket in the GH3 family BGs. These findings shed light on the molecular basis for glucose tolerance and indicate that GH1 BGs are more suitable than GH3 BGs for biotechnological applications involving plant cell-wall saccharification.

Breaking the indexing ambiguity in serial crystallography.

Abstract: In serial crystallography, a very incomplete partial data set is obtained from each diffraction experiment (a `snapshot'). In some space groups, an indexing ambiguity exists which requires that the indexing mode of each snapshot needs to be established with respect to a reference data set. In the absence of such re-indexing information, crystallographers have thus far resorted to a straight merging of all snapshots, yielding a perfectly twinned data set of higher symmetry which is poorly suited for structure solution and refinement. Here, two algorithms have been designed for assembling complete data sets by clustering those snapshots that are indexed in the same way, and they have been tested using 15 445 snapshots from photosystem I [Chapman et al. (2011), Nature (London), 470, 73–77] and with noisy model data. The results of the clustering are unambiguous and enabled the construction of complete data sets in the correct space group P63 instead of (twinned) P6322 that researchers have been forced to use previously in such cases of indexing ambiguity. The algorithms thus extend the applicability and reach of serial crystallo­graphy.

Structure of BamA, an essential factor in outer membrane protein biogenesis

Abstract: Outer membrane protein (OMP) biogenesis is an essential process for maintaining the bacterial cell envelope and involves the β-barrel assembly machinery (BAM) for OMP recognition, folding and assembly. In Escherichia coli this function is orchestrated by five proteins: the integral outer membrane protein BamA of the Omp85 superfamily and four associated lipoproteins. To unravel the mechanism underlying OMP folding and insertion, the structure of the E. coli BamA β-barrel and P5 domain was determined at 3 A resolution. These data add information beyond that provided in the recently published crystal structures of BamA from Haemophilus ducreyi and Neisseria gonorrhoeae and are a valuable basis for the interpretation of pertinent functional studies. In an `open' conformation, E. coli BamA displays a significant degree of flexibility between P5 and the barrel domain, which is indicative of a multi-state function in substrate transfer. E. coli BamA is characterized by a discontinuous β-barrel with impaired β1–β16 strand interactions denoted by only two connecting hydrogen bonds and a disordered C-terminus. The 16-stranded barrel surrounds a large cavity which implies a function in OMP substrate binding and partial folding. These findings strongly support a mechanism of OMP biogenesis in which substrates are partially folded inside the barrel cavity and are subsequently released laterally into the lipid bilayer.

Space-group and origin ambiguity in macromolecular structures with pseudo-symmetry and its treatment with the program Zanuda.

Abstract: The presence of pseudo-symmetry in a macromolecular crystal and its interplay with twinning may lead to an incorrect space-group (SG) assignment. Moreover, if the pseudo-symmetry is very close to an exact crystallographic symmetry, the structure can be solved and partially refined in the wrong SG. Typically, in such incorrectly determined structures all or some of the pseudo-symmetry operations are, in effect, taken for crystallographic symmetry operations and vice versa. A mistake only becomes apparent when the Rfree ceases to decrease below 0.39 and further model rebuilding and refinement cannot improve the refinement statistics. If pseudo-symmetry includes pseudo-translation, the uncertainty in SG assignment may be associated with an incorrect choice of origin, as demonstrated by the series of examples provided here. The program Zanuda presented in this article was developed for the automation of SG validation. Zanuda runs a series of refinements in SGs compatible with the observed unit-cell parameters and chooses the model with the highest symmetry SG from a subset of models that have the best refinement statistics.

The structure of allophycocyanin B from Synechocystis PCC 6803 reveals the structural basis for the extreme redshift of the terminal emitter in phycobilisomes.

Abstract: Allophycocyanin B (AP-B) is one of the two terminal emitters in phycobilisomes, the unique light-harvesting complexes of cyanobacteria and red algae. Its low excitation-energy level and the correspondingly redshifted absorption and fluorescence emission play an important role in funnelling excitation energy from the hundreds of chromophores of the extramembraneous phycobilisome to the reaction centres within the photosynthetic membrane. In the absence of crystal structures of these low-abundance terminal emitters, the molecular basis for the extreme redshift and directional energy transfer is largely unknown. Here, the crystal structure of trimeric AP-B [(ApcD/ApcB)3] from Synechocystis sp. PCC 6803 at 1.75 A resolution is reported. In the crystal lattice, eight trimers of AP-B form a porous, spherical, 48-subunit assembly of 193 A in diameter with an internal cavity of 1.1 × 106 A3. While the overall structure of trimeric AP-B is similar to those reported for many other phycobiliprotein trimers, the chromophore pocket of the α-subunit, ApcD, has more bulky residues that tightly pack the phycocyanobilin (PCB). Ring D of the chromophores is further stabilized by close interactions with ApcB from the adjacent monomer. The combined contributions from both subunits render the conjugated rings B, C and D of the PCB in ApcD almost perfectly coplanar. Together with mutagenesis data, it is proposed that the enhanced planarity effectively extends the conjugation system of PCB and leads to the redshifted absorption (λmax = 669 nm) and fluorescence emission (679 nm) of the ApcD chromophore in AP-B, thereby enabling highly efficient energy transfer from the phycobilisome core to the reaction centres.

Exploiting fast detectors to enter a new dimension in room-temperature crystallography

Abstract: A departure from a linear or an exponential intensity decay in the diffracting power of protein crystals as a function of absorbed dose is reported. The observation of a lag phase raises the possibility of collecting significantly more data from crystals held at room temperature before an intolerable intensity decay is reached. A simple model accounting for the form of the intensity decay is reintroduced and is applied for the first time to high frame-rate room-temperature data collection.

Small-angle scattering gives direct structural information about a membrane protein inside a lipid environment.

Abstract: Monomeric bacteriorhodopsin (bR) reconstituted into POPC/POPG-containing nanodiscs was investigated by combined small-angle neutron and X-ray scattering. A novel hybrid approach to small-angle scattering data analysis was developed. In combination, these provided direct structural insight into membrane-protein localization in the nanodisc and into the protein–lipid interactions. It was found that bR is laterally decentred in the plane of the disc and is slightly tilted in the phospholipid bilayer. The thickness of the bilayer is reduced in response to the incorporation of bR. The observed tilt of bR is in good accordance with previously performed theoretical predictions and computer simulations based on the bR crystal structure. The result is a significant and essential step on the way to developing a general small-angle scattering-based method for determining the low-resolution structures of membrane proteins in physiologically relevant environments.

Stealth carriers for low-resolution structure determination of membrane proteins in solution

Abstract: Structural studies of membrane proteins remain a great experimental challenge. Functional reconstitution into artificial nanoscale bilayer disc carriers that mimic the native bilayer environment allows the handling of membrane proteins in solution. This enables the use of small-angle scattering techniques for fast and reliable structural analysis. The difficulty with this approach is that the carrier discs contribute to the measured scattering intensity in a highly nontrivial fashion, making subsequent data analysis challenging. Here, an elegant solution to circumvent the intrinsic complexity brought about by the presence of the carrier disc is presented. In combination with small-angle neutron scattering (SANS) and the D2O/H2O-based solvent contrast-variation method, it is demonstrated that it is possible to prepare specifically deuterated carriers that become invisible to neutrons in 100% D2O at the length scales relevant to SANS. These `stealth' carrier discs may be used as a general platform for low-resolution structural studies of membrane proteins using well established data-analysis tools originally developed for soluble proteins.

Structure of human Bloom's syndrome helicase in complex with ADP and duplex DNA

Abstract: Bloom's syndrome is an autosomal recessive genome-instability disorder associated with a predisposition to cancer, premature aging and developmental abnormalities. It is caused by mutations that inactivate the DNA helicase activity of the BLM protein or nullify protein expression. The BLM helicase has been implicated in the alternative lengthening of telomeres (ALT) pathway, which is essential for the limitless replication of some cancer cells. This pathway is used by 10–15% of cancers, where inhibitors of BLM are expected to facilitate telomere shortening, leading to apoptosis or senescence. Here, the crystal structure of the human BLM helicase in complex with ADP and a 3′-overhang DNA duplex is reported. In addition to the helicase core, the BLM construct used for crystallization (residues 640–1298) includes the RecQ C-terminal (RQC) and the helicase and ribo­nuclease D C-terminal (HRDC) domains. Analysis of the structure provides detailed information on the interactions of the protein with DNA and helps to explain the mechanism coupling ATP hydrolysis and DNA unwinding. In addition, mapping of the missense mutations onto the structure provides insights into the molecular basis of Bloom's syndrome.

Ten years of probabilistic estimates of biocrystal solvent content: new insights via nonparametric kernel density estimate.

Abstract: The probabilistic estimate of the solvent content (Matthews probability) was first introduced in 2003. Given that the Matthews probability is based on prior information, revisiting the empirical foundation of this widely used solvent-content estimate is appropriate. The parameter set for the original Matthews probability distribution function employed in MATTPROB has been updated after ten years of rapid PDB growth. A new nonparametric kernel density estimator has been implemented to calculate the Matthews probabilities directly from empirical solvent-content data, thus avoiding the need to revise the multiple parameters of the original binned empirical fit function. The influence and dependency of other possible parameters determining the solvent content of protein crystals have been examined. Detailed analysis showed that resolution is the primary and dominating model parameter correlated with solvent content. Modifications of protein specific density for low molecular weight have no practical effect, and there is no correlation with oligomerization state. A weak, and in practice irrelevant, dependency on symmetry and molecular weight is present, but cannot be satisfactorily explained by simple linear or categorical models. The Bayesian argument that the observed resolution represents only a lower limit for the true diffraction potential of the crystal is maintained. The new kernel density estimator is implemented as the primary option in the MATTPROB web application at http://www.ruppweb.org/mattprob/.

New methods for indexing multi-lattice diffraction data

Abstract: A new indexing method is presented which is capable of indexing multiple crystal lattices from narrow wedges of diffraction data. The method takes advantage of a simplification of Fourier transform-based methods that is applicable when the unit-cell dimensions are known a priori. The efficacy of this method is demonstrated with both semi-synthetic multi-lattice data and real multi-lattice data recorded from crystals of ∼1 µm in size, where it is shown that up to six lattices can be successfully indexed and subsequently integrated from a 1° wedge of data. Analysis is presented which shows that improvements in data-quality indicators can be obtained through accurate identification and rejection of overlapping reflections prior to scaling.

Structure-based characterization and antifreeze properties of a hyperactive ice-binding protein from the Antarctic bacterium Flavobacterium frigoris PS1.

Abstract: Ice-binding proteins (IBPs) inhibit ice growth through direct interaction with ice crystals to permit the survival of polar organisms in extremely cold environments. FfIBP is an ice-binding protein encoded by the Antarctic bacterium Flavobacterium frigoris PS1. The X-ray crystal structure of FfIBP was determined to 2.1 A resolution to gain insight into its ice-binding mechanism. The refined structure of FfIBP shows an intramolecular disulfide bond, and analytical ultracentrifugation and analytical size-exclusion chromatography show that it behaves as a monomer in solution. Sequence alignments and structural comparisons of IBPs allowed two groups of IBPs to be defined, depending on sequence differences between the α2 and α4 loop regions and the presence of the disulfide bond. Although FfIBP closely resembles Leucosporidium (recently re-classified as Glaciozyma) IBP (LeIBP) in its amino-acid sequence, the thermal hysteresis (TH) activity of FfIBP appears to be tenfold higher than that of LeIBP. A comparison of the FfIBP and LeIBP structures reveals that FfIBP has different ice-binding residues as well as a greater surface area in the ice-binding site. Notably, the ice-binding site of FfIBP is composed of a T-A/G-X-T/N motif, which is similar to the ice-binding residues of hyperactive antifreeze proteins. Thus, it is proposed that the difference in TH activity between FfIBP and LeIBP may arise from the amino-acid composition of the ice-binding site, which correlates with differences in affinity and surface complementarity to the ice crystal. In conclusion, this study provides a molecular basis for understanding the antifreeze mechanism of FfIBP and provides new insights into the reasons for the higher TH activity of FfIBP compared with LeIBP.

Structural basis for the broad specificity of a new family of amino-acid racemases

Abstract: Broad-spectrum amino-acid racemases (Bsrs) enable bacteria to generate noncanonical d-amino acids, the roles of which in microbial physiology, including the modulation of cell-wall structure and the dissolution of biofilms, are just beginning to be appreciated. Here, extensive crystallographic, mutational, biochemical and bioinformatic studies were used to define the molecular features of the racemase BsrV that enable this enzyme to accommodate more diverse substrates than the related PLP-dependent alanine racemases. Conserved residues were identified that distinguish BsrV and a newly defined family of broad-spectrum racemases from alanine racemases, and these residues were found to be key mediators of the multispecificity of BrsV. Finally, the structural analysis of an additional Bsr that was identified in the bioinformatic analysis confirmed that the distinguishing features of BrsV are conserved among Bsr family members.

ACHESYM: an algorithm and server for standardized placement of macromolecular models in the unit cell.

Abstract: Despite the existence of numerous useful conventions in structural crystallography, for example for the choice of the asymmetric part of the unit cell or of reciprocal space, surprisingly no standards are in use for the placement of the molecular model in the unit cell, often leading to inconsistencies or confusion. A conceptual solution for this problem has been proposed for macromolecular crystal structures based on the idea of the anti-Cheshire unit cell. Here, a program and server (called ACHESYM; http://achesym.ibch.poznan.pl) are presented for the practical implementation of this concept. In addition, the first task of ACHESYM is to find an optimal (compact) macromolecular assembly if more than one polymer chain exists. ACHESYM processes PDB (atomic parameters and TLS matrices) and mmCIF (diffraction data) input files to produce a new coordinate set and to reindex the reflections and modify their phases, if necessary.

Structural basis for catalysis and ubiquitin recognition by the Severe acute respiratory syndrome coronavirus papain-like protease

Abstract: Papain-like protease (PLpro) is one of two cysteine proteases involved in the proteolytic processing of the polyproteins of Severe acute respiratory syndrome coronavirus (SARS-CoV). PLpro also shows significant in vitro deubiquitinating and de-ISGylating activities, although the detailed mechanism is still unclear. Here, the crystal structure of SARS-CoV PLpro C112S mutant in complex with ubiquitin (Ub) is reported at 1.4 A resolution. The Ub core makes mostly hydrophilic interactions with PLpro, while the Leu-Arg-Gly-Gly C-terminus of Ub is located in the catalytic cleft of PLpro, mimicking the P4–P1 residues and providing the first atomic insights into its catalysis. One of the O atoms of the C-­terminal Gly residue of Ub is located in the oxyanion hole consisting of the main-chain amides of residues 112 and 113. Mutations of residues in the PLpro–Ub interface lead to reduced catalytic activity, confirming their importance for Ub binding and/or catalysis. The structure also revealed an N-­cyclohexyl-2-aminethanesulfonic acid molecule near the catalytic triad, and kinetic studies suggest that this binding site is also used by other PLpro inhibitors. Overall, the structure provides a foundation for understanding the molecular basis of coronaviral PLpro catalysis.

The crystal structure of wild-type human brain neuroglobin reveals flexibility of the disulfide bond that regulates oxygen affinity.

Abstract: Neuroglobin plays an important function in the supply of oxygen in nervous tissues. In human neuroglobin, a cysteine at position 46 in the loop connecting the C and D helices of the globin fold is presumed to form an intramolecular disulfide bond with Cys55. Rupture of this disulfide bridge stabilizes bi-­histidyl haem hexacoordination, causing an overall decrease in the affinity for oxygen. Here, the first X-ray structure of wild-type human neuroglobin is reported at 1.74 A resolution. This structure provides a direct observation of two distinct conformations of the CD region containing the intramolecular disulfide link and highlights internal cavities that could be involved in ligand migration and/or are necessary to enable the conformational transition between the low and high oxygen-affinity states following S—S bond formation.

Structural insights into the molecular mechanism of Escherichia coli SdiA, a quorum-sensing receptor

Abstract: Escherichia coli SdiA is a quorum-sensing (QS) receptor that responds to autoinducers produced by other bacterial species to control cell division and virulence. Crystal structures reveal that E. coli SdiA, which is composed of an N-­terminal ligand-binding domain and a C-terminal DNA-binding domain (DBD), forms a symmetrical dimer. Although each domain shows structural similarity to other QS receptors, SdiA differs from them in the relative orientation of the two domains, suggesting that its ligand-binding and DNA-binding functions are independent. Consistently, in DNA gel-shift assays the binding affinity of SdiA for the ftsQP2 promoter appeared to be insensitive to the presence of autoinducers. These results suggest that autoinducers increase the functionality of SdiA by enhancing the protein stability rather than by directly affecting the DNA-binding affinity. Structural analyses of the ligand-binding pocket showed that SdiA cannot accommodate ligands with long acyl chains, which was corroborated by isothermal titration calorimetry and thermal stability analyses. The formation of an intersubunit disulfide bond that might be relevant to modulation of the DNA-binding activity was predicted from the proximal position of two Cys residues in the DBDs of dimeric SdiA. It was confirmed that the binding affinity of SdiA for the uvrY promoter was reduced under oxidizing conditions, which suggested the possibility of regulation of SdiA by multiple independent signals such as quorum-sensing inducers and the oxidation state of the cell.

Structure, mechanism and ensemble formation of the alkylhydroperoxide reductase subunits AhpC and AhpF from Escherichia coli

Abstract: Hydroperoxides are reactive oxygen species (ROS) that are toxic to all cells and must be converted into the corresponding alcohols to alleviate oxidative stress. In Escherichia coli, the enzyme primarily responsible for this reaction is alkylhydroperoxide reductase (AhpR). Here, the crystal structures of both of the subunits of EcAhpR, EcAhpF (57 kDa) and EcAhpC (21 kDa), have been solved. The EcAhpF structures (2.0 and 2.65 A resolution) reveal an open and elongated conformation, while that of EcAhpC (3.3 A resolution) forms a decameric ring. Solution X-ray scattering analysis of EcAhpF unravels the flexibility of its N-terminal domain, and its binding to EcAhpC was demonstrated by isothermal titration calorimetry. These studies suggest a novel overall mechanistic model of AhpR as a hydroperoxide scavenger, in which the dimeric, extended AhpF prefers complex formation with the AhpC ring to accelerate the catalytic activity and thus to increase the chance of rescuing the cell from ROS.

The structure of human interleukin-11 reveals receptor-binding site features and structural differences from interleukin-6.

Abstract: Interleukin (IL)-11 is a multifunctional member of the IL-6 family of cytokines. Recombinant human IL-11 is administered as a standard clinical treatment for chemotherapy-induced thrombocytopaenia. Recently, a new role for IL-11 signalling as a potent driver of gastrointestinal cancers has been identified, and it has been demonstrated to be a novel therapeutic target for these diseases. Here, the crystal structure of human IL-11 is reported and the structural resolution of residues previously identified as important for IL-11 activity is presented. While IL-11 is thought to signal via a complex analogous to that of IL-6, comparisons show important differences between the two cytokines and it is suggested that IL-11 engages GP130 differently to IL-6. In addition to providing a structural platform for further study of IL-11, these data offer insight into the binding interactions of IL-11 with each of its receptors and the structural mechanisms underlying agonist and antagonist variants of the protein.

xMDFF: molecular dynamics flexible fitting of low‐resolution X‐ray structures

Abstract: X-ray crystallography remains the most dominant method for solving atomic structures. However, for relatively large systems, the availability of only medium-to-low-resolution diffraction data often limits the determination of all-atom details. A new molecular dynamics flexible fitting (MDFF)-based approach, xMDFF, for determining structures from such low-resolution crystallographic data is reported. xMDFF employs a real-space refinement scheme that flexibly fits atomic models into an iteratively updating electron-density map. It addresses significant large-scale deformations of the initial model to fit the low-resolution density, as tested with synthetic low-resolution maps of d-ribose-binding protein. xMDFF has been successfully applied to re-refine six low-resolution protein structures of varying sizes that had already been submitted to the Protein Data Bank. Finally, via systematic refinement of a series of data from 3.6 to 7 A resolution, xMDFF refinements together with electro­physiology experiments were used to validate the first all-atom structure of the voltage-sensing protein Ci-VSP.

The first structure of a bacterial diterpene cyclase: CotB2.

Abstract: Sesquiterpenes and diterpenes are a diverse class of secondary metabolites that are predominantly derived from plants and some prokaryotes. The properties of these natural products encompass antitumor, antibiotic and even insecticidal activities. Therefore, they are interesting commercial targets for the chemical and pharmaceutical industries. Owing to their structural complexity, these compounds are more efficiently accessed by metabolic engineering of microbial systems than by chemical synthesis. This work presents the first crystal structure of a bacterial diterpene cyclase, CotB2 from the soil bacterium Streptomyces melanosporofaciens, at 1.64 A resolution. CotB2 is a diterpene cyclase that catalyzes the cyclization of the linear geranylgeranyl diphosphate to the tricyclic cyclooctat-9-en-7-ol. The subsequent oxidation of cyclooctat-9-en-7-ol by two cytochrome P450 monooxygenases leads to bioactive cyclo­octatin. Plasticity residues that decorate the active site of CotB2 have been mutated, resulting in alternative monocyclic, dicyclic and tricyclic compounds that show bioactivity. These new compounds shed new light on diterpene cyclase reaction mechanisms. Furthermore, the product of mutant CotB2W288G produced the new antibiotic compound (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene, which acts specifically against multidrug-resistant Staphylococcus aureus. This opens a sustainable route for the industrial-scale production of this bioactive compound.

The structure of a tetrameric α-carbonic anhydrase from Thermovibrio ammonificans reveals a core formed around intermolecular disulfides that contribute to its thermostability

Abstract: Carbonic anhydrase enzymes catalyse the reversible hydration of carbon dioxide to bicarbonate. A thermophilic Thermovibrio ammonificans α-carbonic anhydrase (TaCA) has been expressed in Escherichia coli and structurally and biochemically characterized. The crystal structure of TaCA has been determined in its native form and in two complexes with bound inhibitors. The tetrameric enzyme is stabilized by a unique core in the centre of the molecule formed by two intersubunit disulfides and a single lysine residue from each monomer that is involved in intersubunit ionic interactions. The structure of this core protects the intersubunit disulfides from reduction, whereas the conserved intrasubunit disulfides are not formed in the reducing environment of the E. coli host cytosol. When oxidized to mimic the environment of the periplasmic space, TaCA has increased thermostability, retaining 90% activity after incubation at 70°C for 1 h, making it a good candidate for industrial carbon-dioxide capture. The reduction of all TaCA cysteines resulted in dissociation of the tetrameric molecule into monomers with lower activity and reduced thermostability. Unlike other characterized α-carbonic anhydrases, TaCA does not display esterase activity towards p-nitrophenyl acetate, which appears to result from the increased rigidity of its protein scaffold.

Improving the chances of successful protein structure determination with a random forest classifier

Abstract: Obtaining diffraction quality crystals remains one of the major bottlenecks in structural biology. The ability to predict the chances of crystallization from the amino-acid sequence of the protein can, at least partly, address this problem by allowing a crystallographer to select homologs that are more likely to succeed and/or to modify the sequence of the target to avoid features that are detrimental to successful crystallization. In 2007, the now widely used XtalPred algorithm [Slabinski et al. (2007), Protein Sci. 16, 2472-2482] was developed. XtalPred classifies proteins into five `crystallization classes' based on a simple statistical analysis of the physicochemical features of a protein. Here, towards the same goal, advanced machine-learning methods are applied and, in addition, the predictive potential of additional protein features such as predicted surface ruggedness, hydrophobicity, side-chain entropy of surface residues and amino-acid composition of the predicted protein surface are tested. The new XtalPred-RF (random forest) achieves significant improvement of the prediction of crystallization success over the original XtalPred. To illustrate this, XtalPred-RF was tested by revisiting target selection from 271 Pfam families targeted by the Joint Center for Structural Genomics (JCSG) in PSI-2, and it was estimated that the number of targets entered into the protein-production and crystallization pipeline could have been reduced by 30% without lowering the number of families for which the first structures were solved. The prediction improvement depends on the subset of targets used as a testing set and reaches 100% (i.e. twofold) for the top class of predicted targets.

Structural basis of the heterodimerization of the MST and RASSF SARAH domains in the Hippo signalling pathway.

Abstract: Despite recent progress in research on the Hippo signalling pathway, the structural information available in this area is extremely limited. Intriguingly, the homodimeric and heterodimeric interactions of mammalian sterile 20-like (MST) kinases through the so-called `SARAH' (SAV/RASSF/HPO) domains play a critical role in cellular homeostasis, dictating the fate of the cell regarding cell proliferation or apoptosis. To understand the mechanism of the heterodimerization of SARAH domains, the three-dimensional structures of an MST1–RASSF5 SARAH heterodimer and an MST2 SARAH homodimer were determined by X-ray crystallography and were analysed together with that previously determined for the MST1 SARAH homodimer. While the structure of the MST2 homodimer resembled that of the MST1 homodimer, the MST1–RASSF5 heterodimer showed distinct structural features. Firstly, the six N-terminal residues (Asp432–Lys437), which correspond to the short N-terminal 310-helix h1 kinked from the h2 helix in the MST1 homodimer, were disordered. Furthermore, the MST1 SARAH domain in the MST1–RASSF5 complex showed a longer helical structure (Ser438–Lys480) than that in the MST1 homodimer (Val441–Lys480). Moreover, extensive polar and nonpolar contacts in the MST1–RASSF5 SARAH domain were identified which strengthen the interactions in the heterodimer in comparison to the interactions in the homodimer. Denaturation experiments performed using urea also indicated that the MST–RASSF heterodimers are substantially more stable than the MST homodimers. These findings provide structural insights into the role of the MST1–RASSF5 SARAH domain in apoptosis signalling.

Structural characteristics of an insect group I chitinase, an enzyme indispensable to moulting

Abstract: Insects possess a greater number of chitinases than any other organisms. This work is the first report of unliganded and oligosaccharide-complexed crystal structures of the insect chitinase OfChtI from Ostrinia furnacalis, which is essential to moulting. The obtained crystal structures were solved at resolutions between 1.7 and 2.2 A. A structural comparison with other chitinases revealed that OfChtI contains a long substrate-binding cleft similar to the bacterial chitinase SmChiB from Serratia marcescens. However, unlike the exo-acting SmChiB, which has a blocked and tunnel-like cleft, OfChtI possesses an open and groove-like cleft. The complexed structure of the catalytic domain of OfChtI (OfChtI-CAD) with (GlcNAc)2/3 indicates that the reducing sugar at subsite −1 is in an energetically unfavoured `boat' conformation, a state that possibly exists just before the completion of catalysis. Because OfChtI is known to act from nonreducing ends, (GlcNAc)3 would be a hydrolysis product of (GlcNAc)6, suggesting that OfChtI possesses an endo enzymatic activity. Furthermore, a hydrophobic plane composed of four surface-exposed aromatic residues is adjacent to the entrance to the substrate-binding cleft. Mutations of these residues greatly impair the chitin-binding activity, indicating that this hydrophobic plane endows OfChtI-CAD with the ability to anchor chitin. This work reveals the unique structural characteristics of an insect chitinase.

Structure of the epimerization domain of tyrocidine synthetase A

Abstract: Tyrocidine, a macrocyclic decapeptide from Bacillus brevis, is nonribosomally assembled by a set of multimodular peptide synthetases, which condense two D-amino acids and eight L-amino acids to produce this membrane-disturbing antibiotic. D-Phenylalanine, the first amino acid incorporated into tyrocidine, is catalytically derived from enzyme-bound L-Phe by the C-terminal epimerization (E) domain of tyrocidine synthetase A (TycA). The 1.5 A resolution structure of the cofactor-independent TycA E domain reveals an intimate relationship to the condensation (C) domains of peptide synthetases. In contrast to the latter, the TycA E domain uses an enlarged bridge region to plug the active-site canyon from the acceptor side, whereas at the donor side a latch-like floor loop is suitably extended to accommodate the αIII helix of the preceding peptide-carrier domain. Additionally, E domains exclusively harbour a conserved glutamate residue, Glu882, that is opposite the active-site residue His743. This active-site topology implies Glu882 as a candidate acid-base catalyst, whereas His743 stabilizes in the protonated state a transient enolate intermediate of the L↔D isomerization.

The structure of the C-terminal domain of the Zaire ebolavirus nucleoprotein.

Abstract: Ebolavirus (EBOV) causes severe hemorrhagic fever with a mortality rate of up to 90%. EBOV is a member of the order Mononegavirales and, like other viruses in this taxonomic group, contains a negative-sense single-stranded (ss) RNA. The EBOV ssRNA encodes seven distinct proteins. One of them, the nucleoprotein (NP), is the most abundant viral protein in the infected cell and within the viral nucleocapsid. Like other EBOV proteins, NP is multifunctional. It is tightly associated with the viral genome and is essential for viral transcription, RNA replication, genome packaging and nucleocapsid assembly prior to membrane encapsulation. NP is unusual among the Mononegavirales in that it contains two distinct regions, or putative domains, the C-terminal of which shows no homology to any known proteins and is purported to be a hub for protein–protein interactions within the nucleocapsid. The atomic structure of NP remains unknown. Here, the boundaries of the N- and C-terminal domains of NP from Zaire EBOV are defined, it is shown that they can be expressed as highly stable recombinant proteins in Escherichia coli, and the atomic structure of the C-terminal domain (residues 641–739) derived from analysis of two distinct crystal forms at 1.98 and 1.75 A resolution is described. The structure reveals a novel tertiary fold that is distantly reminiscent of the β-grasp architecture.