Search

The bacterial genus Mycobacterium includes important pathogens, most notably M. tuberculosis, which infects one‐quarter of the entire human population, resulting in around 1.4 million deaths from tuberculosis each year. Mycobacteria, and the closely related corynebacteria, synthesize a class of abundant glycolipids, the phosphatidyl‐myo‐inositol mannosides (PIMs). PIMs serve as membrane anchors for hyperglycosylated species, lipomannan (LM) and lipoarabinomannan (LAM), which are surface‐exposed and modulate the host immune response. Previously, in studies using the model species Corynebacterium glutamicum, NCgl2760 was identified as a novel membrane protein that is required for the synthesis of full‐length LM and LAM. Here, the first crystal structure of its ortholog in Mycobacterium smegmatis, MSMEG_0317, is reported at 1.8 Å resolution. The structure revealed an elongated β‐barrel fold enclosing two distinct cavities and one α‐helix extending away from the β‐barrel core, resembling a 'cone with a flake' arrangement. Through xenon derivatization and structural comparison with AlphaFold2‐derived predictions of the M. tuberculosis homolog Rv0227c, structural elements were identified that may undergo conformational changes to switch from 'closed' to 'open' conformations, allowing cavity access. An AlphaFold2‐derived NCgl2760 model predicted a smaller β‐barrel core with an enclosed central cavity, suggesting that all three proteins, which were collectively termed LmcA, may have a common mechanism of ligand binding through these cavities. These findings provide new structural insights into the biosynthetic pathway for a family of surface lipoglycans with important roles in mycobacterial pathogenesis. [ABSTRACT FROM AUTHOR]

Lysine biosynthesis in plants occurs via the diaminopimelate pathway. The first committed and rate‐limiting step of this pathway is catalysed by dihydrodipicolinate synthase (DHDPS), which is allosterically regulated by the end product, l‐lysine (lysine). Given that lysine is a common nutritionally limiting amino acid in cereal crops, there has been much interest in probing the regulation of DHDPS. Interestingly, knockouts in Arabidopsis thaliana of each isoform (AtDHDPS1 and AtDHDPS2) result in different phenotypes, despite the enzymes sharing > 85% protein sequence identity. Accordingly, in this study, we compared the catalytic activity, lysine‐mediated inhibition and structures of both A. thaliana DHDPS isoforms. We found that although the recombinantly produced enzymes have similar kinetic properties, AtDHDPS1 is 10‐fold more sensitive to lysine. We subsequently used X‐ray crystallography to probe for structural differences between the apo‐ and lysine‐bound isoforms that could account for the differential allosteric inhibition. Despite no significant changes in the overall structures of the active or allosteric sites, we noted differences in the rotamer conformation of a key allosteric site residue (Trp116) and proposed that this could result in differences in lysine dissociation. Microscale thermophoresis studies supported our hypothesis, with AtDHDPS1 having a ~ 6‐fold tighter lysine dissociation constant compared to AtDHDPS2, which agrees with the lower half minimal inhibitory concentration for lysine observed. Thus, we highlight that subtle differences in protein structures, which could not have been predicted from the primary sequences, can have profound effects on the allostery of a key enzyme involved in lysine biosynthesis in plants. Databases: Structures described are available in the Protein Data Bank under the accession numbers 6VVH and 6VVI. [ABSTRACT FROM AUTHOR]

The rise of antibiotic resistance combined with the lack of new products entering the market has led to bacterial infections becoming one of the biggest threats to global health. Therefore, there is an urgent need to identify novel antibiotic targets, such as dihydrodipicolinate synthase (DHDPS), an enzyme involved in the production of essential metabolites in cell wall and protein synthesis. Here, we utilised a 7‐residue sequence motif to identify mis‐annotation of multiple DHDPS genes in the high‐priority Gram‐negative bacteria Acinetobacter baumannii and Klebsiella pneumoniae. We subsequently confirmed these mis‐annotations using a combination of enzyme kinetics and X‐ray crystallography. Thus, this study highlights the need to ensure genes encoding promising drug targets, like DHDPS, are annotated correctly, especially for clinically important pathogens. PDB ID: 6UE0. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is one of the leading causes of nosocomial infections, accounting for 10% of all hospital‐acquired infections. Current antibiotics against P. aeruginosa are becoming increasingly ineffective due to the exponential rise in drug resistance. Thus, there is an urgent need to validate and characterize novel drug targets to guide the development of new classes of antibiotics against this pathogen. One such target is the diaminopimelate (DAP) pathway, which is responsible for the biosynthesis of bacterial cell wall and protein building blocks, namely meso‐DAP and lysine. The rate‐limiting step of this pathway is catalysed by the enzyme dihydrodipicolinate synthase (DHDPS), typically encoded for in bacteria by a single dapA gene. Here, we show that P. aeruginosa encodes two functional DHDPS enzymes, PaDHDPS1 and PaDHDPS2. Although these isoforms have similar catalytic activities (kcat = 29 s−1 and 44 s−1 for PaDHDPS1 and PaDHDPS2, respectively), they are differentially allosterically regulated by lysine, with only PaDHDPS2 showing inhibition by the end product of the DAP pathway (IC50 = 130 μm). The differences in allostery are attributed to a single amino acid difference in the allosteric binding pocket at position 56. This is the first example of a bacterium that contains multiple bona fide DHDPS enzymes, which differ in allosteric regulation. We speculate that the presence of the two isoforms allows an increase in the metabolic flux through the DAP pathway when required in this clinically important pathogen. Databases: PDB ID: 6P90. [ABSTRACT FROM AUTHOR]

Two commonly encountered bottlenecks in the structure determination of a protein by X‐ray crystallography are screening for conditions that give high‐quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5‐amino‐2,4,6‐triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high‐quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg‐white lysozyme and the N‐terminal domain of the Orf11 protein from Staphylococcus phage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion‐associated lysin or endolysin. [ABSTRACT FROM AUTHOR]

MX2 is an in‐vacuum undulator‐based crystallography beamline at the 3 GeV Australian Synchrotron. The beamline delivers hard X‐rays in the energy range 4.8–21 keV to a focal spot of 22 × 12 µm FWHM (H × V). At 13 keV the flux at the sample is 3.4 × 1012 photons s−1. The beamline endstation allows robotic handling of cryogenic samples via an updated SSRL SAM robot. This beamline is ideal for weakly diffracting hard‐to‐crystallize proteins, virus particles, protein assemblies and nucleic acids as well as smaller molecules such as inorganic catalysts and organic drug molecules. The beamline is now mature and has enjoyed a full user program for the last nine years. This paper describes the beamline status, plans for its future and some recent scientific highlights. [ABSTRACT FROM AUTHOR]

Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first committed step of the histidine biosynthesis in plants and microorganisms. Here, we present the functional and structural characterization of the ATP-PRT from the pathogenic ε-proteobacteria Campylobacter jejuni ( CjeATP-PRT). This enzyme is a member of the long form (HisGL) ATP-PRT and is allosterically inhibited by histidine, which binds to a remote regulatory domain, and competitively inhibited by AMP. In the crystalline form, CjeATP-PRT was found to adopt two distinctly different hexameric conformations, with an open homohexameric structure observed in the presence of substrate ATP, and a more compact closed form present when inhibitor histidine is bound. CjeATP-PRT was observed to adopt only a hexameric quaternary structure in solution, contradicting previous hypotheses favoring an allosteric mechanism driven by an oligomer equilibrium. Instead, this study supports the conclusion that the ATP-PRT long form hexamer is the active species; the tightening of this structure in response to remote histidine binding results in an inhibited enzyme. [ABSTRACT FROM AUTHOR]

Proteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63-residue member of this family, snakin-1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Å structure of snakin-1, determined by a novel combination of racemic protein crystallization and radiation-damage-induced phasing (RIP), is reported. Racemic crystals of snakin-1 and quasiracemic crystals incorporating an unnatural 4-iodophenylalanine residue were prepared from chemically synthesized d- and l-proteins. Breakage of the C-I bonds in the quasi-racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces. [ABSTRACT FROM AUTHOR]

Proteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63-residue member of this family, snakin-1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Å structure of snakin-1, determined by a novel combination of racemic protein crystallization and radiation-damage-induced phasing (RIP), is reported. Racemic crystals of snakin-1 and quasiracemic crystals incorporating an unnatural 4-iodophenylalanine residue were prepared from chemically synthesized D- and L-proteins. Breakage of the C-I bonds in the quasi-racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces. [ABSTRACT FROM AUTHOR]

Non-POU domain-containing octamer-binding protein (NONO, a.k.a. p54nrb) is a central player in nuclear gene regulation with rapidly emerging medical significance. NONO is a member of the highly conserved Drosophila behaviour/human splicing (DBHS) protein family, a dynamic family of obligatory dimeric nuclear regulatory mediators. However, work with the NONO homodimer has been limited by rapid irreversible sample aggregation. Here, it is reported that l-proline stabilizes purified NONO homodimers, enabling good-quality solution small-angle X-ray structure determination and crystallization. NONO crystallized in the apparent space group P21 with a unique axis ( b) of 408.9 Å and with evidence of twinning, as indicated by the cumulative intensity distribution L statistic, suggesting the possibility of space group P1. Structure solution by molecular replacement shows a superhelical arrangement of six NONO homodimers (or 12 in P1) oriented parallel to the long axis, resulting in extensive noncrystallographic symmetry. Further analysis revealed that the crystal was not twinned, but the collected data suffered from highly overlapping reflections that obscured the L-test. Optimized data collection on a new crystal using higher energy X-rays, a smaller beam width and an increased sample-to-detector distance produced non-overlapping reflections to 2.6 Å resolution. The steps taken to analyse and overcome this series of practical difficulties and to produce a biologically informative structure are discussed. [ABSTRACT FROM AUTHOR]

The B and C proteins from the ABC toxin complex of Yersinia entomophaga form a large heterodimer that cleaves and encapsulates the C-terminal toxin domain of the C protein. Determining the structure of the complex formed by B and the N-terminal region of C was challenging owing to its large size, the non-isomorphism of different crystals and their sensitivity to radiation damage. A native data set was collected to 2.5 Å resolution and a non-isomorphous Ta6Br12-derivative data set was collected that showed strong anomalous signal at low resolution. The tantalum-cluster sites could be found, but the anomalous signal did not extend to a high enough resolution to allow model building. Selenomethionine (SeMet)-derivatized protein crystals were produced, but the high number (60) of SeMet sites and the sensitivity of the crystals to radiation damage made phasing using the SAD or MAD methods difficult. Multiple SeMet data sets were combined to provide 30-fold multiplicity, and the low-resolution phase information from the Ta6Br12 data set was transferred to this combined data set by cross-crystal averaging. This allowed the Se atoms to be located in an anomalous difference Fourier map; they were then used in Auto-Rickshaw for multiple rounds of autobuilding and MRSAD. [ABSTRACT FROM AUTHOR]

Using the UHV experimental endstation on the soft X-ray beamline at the Australian Synchrotron, lysozyme and proteinase K crystals have been exposed to a vacuum of 10−5 mbar, prior to flash-cooling in a bath of liquid nitrogen. Subsequent data collection on the MX2 beamline at the Australian Synchrotron demonstrated that, for lysozyme and proteinase K, it is possible to subject these mounted crystals to a vacuum pressure of 10−5 mbar without destroying the crystal lattice. Despite the lower data quality of the vacuum-pumped crystals compared with control crystals, it is demonstrated that the protein crystals can survive in a vacuum under suitable conditions. [ABSTRACT FROM AUTHOR]

The Zn inactive class of glyoxalase I (Glo1) metalloenzymes are typically homodimeric with two metal-dependent active sites. While the two active sites share identical amino acid composition, this class of enzyme is optimally active with only one metal per homodimer. We have determined the X-ray crystal structure of GloA2, a Zn inactive Glo1 enzyme from Pseudomonas aeruginosa. The presented structures exhibit an unprecedented metal-binding arrangement consistent with half-of-sites activity: one active site contains a single activating Ni2+ ion, whereas the other contains two inactivating Zn2+ ions. Enzymological experiments prompted by the binuclear Zn2+ site identified a novel catalytic property of GloA2. The enzyme can function as a Zn2+/Co2+-dependent hydrolase, in addition to its previously determined glyoxalase I activity. The presented findings demonstrate that GloA2 can accommodate two distinct metal-binding arrangements simultaneously, each of which catalyzes a different reaction. [ABSTRACT FROM AUTHOR]

MX1 is a bending-magnet crystallography beamline at the 3 GeV Australian Synchrotron. The beamline delivers hard X-rays in the energy range from 8 to 18 keV to a focal spot at the sample position of 120 µm FWHM. The beamline endstation and ancillary equipment facilitate local and remote access for both chemical and biological macromolecular crystallography. Here, the design of the beamline and endstation are discussed. The beamline has enjoyed a full user program for the last seven years and scientific highlights from the user program are also presented. [ABSTRACT FROM AUTHOR]

The crystal structure of banyan peroxidase purified from the latex of Ficus benghalensis has been solved at 1.67 Å resolution by single-wavelength anomalous diffraction phasing. The refined structure includes 306 amino acid residues, a heme and two calcium ions. The protein belongs to class III peroxidases and is the first one from plant latex. Extensive glycosylation was observed with N-linked glycans attached to seven asparagine residues. The enzyme is stable with respect to a wide pH range, temperature, chemical denaturants and organic solvents, probably as a result of its high glycosylation. An unexpected post-translational modification of Asp290 was identified as succinimide moiety. Kinetic parameters of banyan peroxidase have been determined using various hydrogen donor substrates and hydrogen peroxide. [ABSTRACT FROM AUTHOR]

Expression of the aromatic hydroxylase TetX under aerobic conditions confers bacterial resistance against tetracycline antibiotics. Hydroxylation inactivates and degrades tetracyclines, preventing inhibition of the prokaryotic ribosome. X-ray crystal structure analyses of TetX in complex with the second-generation and third-generation tetracyclines minocycline and tigecycline at 2.18 and 2.30 Å resolution, respectively, explain why both clinically potent antibiotics are suitable substrates. Both tetracyclines bind in a large tunnel-shaped active site in close contact to the cofactor FAD, pre-oriented for regioselective hydroxylation to 11a-hydroxytetracyclines. The characteristic bulky 9- tert-butylglycylamido substituent of tigecycline is solvent-exposed and does not interfere with TetX binding. In the TetX-minocycline complex a second binding site for a minocycline dimer is observed close to the active-site entrance. The pocket is formed by the crystal packing arrangement on the surface of two neighbouring TetX monomers. Crystal structure analysis at 2.73 Å resolution of xenon-pressurized TetX identified two adjacent Xe-binding sites. These putative dioxygen-binding cavities are located in the substrate-binding domain next to the active site. Molecular-dynamics simulations were performed in order to characterize dioxygen-diffusion pathways to FADH2 at the active site. [ABSTRACT FROM AUTHOR]

The cyanuric acid hydrolase, AtzD, is the founding member of a newly identified family of ring-opening amidases. We report the first X-ray structure for this family, which is a novel fold (termed the 'Toblerone' fold) that likely evolved via the concatenation of monomers of the trimeric YjgF superfamily and the acquisition of a metal binding site. Structures of AtzD with bound substrate (cyanuric acid) and inhibitors (phosphate, barbituric acid and melamine), along with mutagenesis studies, allowed the identification of the active site. The AtzD monomer, active site and substrate all possess threefold rotational symmetry, to the extent that the active site possesses three potential Ser- Lys catalytic dyads. A single catalytic dyad ( Ser85- Lys42) is hypothesized, based on biochemical evidence and crystallographic data. A plausible catalytic mechanism based on these observations is also presented. A comparison with a homology model of the related barbiturase, Bar, was used to infer the active-site residues responsible for substrate specificity, and the phylogeny of the 68 AtzD-like enzymes in the database were analysed in light of this structure-function relationship. [ABSTRACT FROM AUTHOR]

F18-positive enterotoxigenic and Shiga toxin-producing Escherichia coli are responsible for post-weaning diarrhoea and oedema disease in pigs and lead to severe production losses in the farming industry. F18 fimbriae attach to the small intestine of young piglets by latching onto glycosphingolipids with A/H blood group determinants on type 1 core. We demonstrate the N-terminal domain of the F18 fimbrial subunit FedF to be responsible for ABH-mediated attachment and present its X-ray structure in ligand-free form and bound to A and B type 1 hexaoses. The FedF lectin domain comprises a 10-stranded immunoglobulin-like β-sandwich. Three linear motives, Q47-N50, H88-S90 and R117-T119, form a shallow glycan binding pocket near the tip of the domain that is selective for type 1 core glycans in extended conformation. In addition to the glycan binding pocket, a polybasic loop on the membrane proximal surface of FedF lectin domain is shown to be required for binding to piglet enterocytes. Although dispensable for ABH glycan recognition, the polybasic surface adds binding affinity in the context of the host cell membrane, a mechanism that is proposed to direct ABH-glycan binding to cell-bound glycosphingolipids and could allow bacteria to avoid clearance by secreted glycoproteins. [ABSTRACT FROM AUTHOR]

Three crystal structures of a lipoprotein (Bmlp7) of unknown function, a member of the 30 kDa lipoprotein family from mulberry silkworm ( Bombyx mori L.) haemolymph, have been determined. The 1.33 Å resolution structure is an excellent example of how a precise crystallographic study can contribute to protein identification. The correct sequence of this haemolymph-isolated protein was assigned thanks to superb-quality electron-density maps. Two unexpected cadmium cations were found in this crystal structure [Bmlp7-I(Cd)] and their presence may be connected to a detoxification mechanism in this insect. For a comparison of the metal-binding sites, the crystal structure of a platinum complex (Bmlp7-Pt) was also solved at 1.94 Å resolution. The third (2.50 Å resolution) structure, of the native protein harvested in a different season (Bmlp7-II), corresponds to a different polymorph with an altered pattern of intermolecular interactions and with a total absence of cadmium ions and highlights the possible involvement of Bmlp7 in the response to environmental pollution. The N-terminal domain of Bmlp7 has a fold resembling a clockwise spiral created by six helices and can be classified as a VHS domain. The C-terminal domain is folded as a β-trefoil. The biological function of Bmlp7 is unknown, but its structural homology to sugar-binding proteins suggests that, in analogy to other 30 kDa haemolymph lipoproteins, it could play a role as an anti-apoptotic factor or function in the immune response of the insect to fungal infections. [ABSTRACT FROM AUTHOR]

Formylglycinamide ribonucleotide (FGAR) amidotransferase (FGAR-AT) takes part in purine biosynthesis and is a multidomain enzyme with multiple spatially separated active sites. FGAR-AT contains a glutaminase domain that is responsible for the generation of ammonia from glutamine. Ammonia is then transferred via a channel to a second active site located in the synthetase domain and utilized to convert FGAR to formylglycinamidine ribonucleotide (FGAM) in an adenosine triphosphate (ATP) dependent reaction. In some ammonia-channelling enzymes ligand binding triggers interdomain signalling between the two diverse active centres and also assists in formation of the ammonia channel. Previously, the structure of FGAR-AT from Salmonella typhimurium containing a glutamyl thioester intermediate covalently bound in the glutaminase active site was determined. In this work, the roles played by various ligands of FGAR-AT in inducing catalytic coupling are investigated. Structures of FGAR-AT from S. typhimurium were determined in two different states: the unliganded form and the binary complex with an ATP analogue in the presence of the glutamyl thioester intermediate. The structures were compared in order to decipher the roles of these two states in interdomain communication. Using a process of elimination, the results indicated that binding of FGAR is most likely to be the major mechanism by which catalytic coupling occurs. This is because conformational changes do not occur either upon formation of the glutamyl thioester intermediate or upon subsequent ATP complexation. A model of the FGAR-bound form of the enzyme suggested that the loop in the synthetase domain may be responsible for initiating catalytic coupling via its interaction with the N-terminal domain. [ABSTRACT FROM AUTHOR]

Ubiquitin-conjugating enzymes (E2s) coordinate distinct types of ubiquitination via specific E3 ligases, to a large number of protein substrates. While many E2 enzymes need only the presence of an E3 ligase for substrate ubiquitination, a number of E2s require additional, non-canonical binding partners to specify their function. Here, we have determined the crystal structure and function of an E2/co-activator assembly, the Pex4p:Pex22p complex. The peroxisome-associated E2 enzyme Pex4p binds the peroxisomal membrane protein Pex22p through a binding site that does not overlap with any other known interaction interface in E2 enzymes. Pex22p association enhances Pex4p's ability to transfer ubiquitin to a substrate in vitro, and Pex22p binding-deficient forms of Pex4p are unable to ubiquitinate the peroxisomal import receptor Pex5p in vivo. Our data demonstrate that the Pex4p:Pex22p assembly, and not Pex4p alone, functions as the E2 enzyme required for Pex5p ubiquitination, establishing a novel mechanism of E2 enzyme regulation. [ABSTRACT FROM AUTHOR]

The article demonstrates the beneficial use of the combined anomalous differences and structure-factor amplitudes in molecular replacement (MR) in analyzing macromolecular structures. The study indicates that anomalous intensity differences can be utilized to enhance the performance of molecular replacement to study macromolecular structures. It demonstrates that this combined method is a promising approach to macromolecular structure studies.

The ABC transporter haemolysin B (HlyB) from Escherichia coli is part of a type I secretion system that translocates a 110 kDa toxin in one step across both membranes of this Gram-negative bacterium in an ATP-dependent manner. Sequence analysis indicates that HlyB contains a C39 peptidase-like domain at its N-terminus. C39 domains are thiol-dependent peptidases that cleave their substrates after a GG motif. Interestingly, the catalytically invariant cysteine is replaced by a tyrosine in the C39-like domain of HlyB. Here, the overexpression, purification and crystallization of the isolated C39-like domain are described as a first step towards obtaining structural insights into this domain and eventually answering the question concerning the function of a degenerated C39 domain in the ABC transporter HlyB. [ABSTRACT FROM AUTHOR]

The article discusses a research study on the phasing of selenomethionine-containing proteins with single isomorphous replacement through the use of inhouse ultraviolet (UV)-induced radiation damage. Experimental procedure include the use of three selenium-labelled proteins, simulation of protein crystal penetration depths and measurement UV absorption spectrum with the data collected on European Synchrotron Radiation Facility (ESRF). Conclusions indicated that UV can bring changes to protein crystals more than X-ray.

The article discusses the addition to the tutorial for teaching macromolecular crystallography. The updates include experiments on structure determination by single isomorphous replacement including anomalous scattering (SIRAS) using a gold derivative of tetragonal HEWL and a structure determination by ultraviolet radiation damage induced phasing on the protein thaumatin. The update is said to provide a complete description of crystallographic experiment covering all aspects from crystallization to structure determination and interpretation.

A combination of molecular replacement and single-wavelength anomalous diffraction phasing has been incorporated into the automated structure-determination platform Auto-Rickshaw. The complete MRSAD procedure includes molecular replacement, model refinement, experimental phasing, phase improvement and automated model building. The improvement over the standard SAD or MR approaches is illustrated by ten test cases taken from the JCSG diffraction data-set database. Poor MR or SAD phases with phase errors larger than 70° can be improved using the described procedure and a large fraction of the model can be determined in a purely automatic manner from X-ray data extending to better than 2.6 Å resolution. [ABSTRACT FROM AUTHOR]

Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26-31 kb) encodes 15-16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication-transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (~100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P43. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein-protein interactions. [ABSTRACT FROM AUTHOR]

Many Gram-negative bacteria use the chaperone-usher pathway to express adhesive surface structures, such as fimbriae, in order to mediate attachment to host cells. Periplasmic chaperones are required to shuttle fimbrial subunits or pilins through the periplasmic space in an assembly-competent form. The chaperones cap the hydrophobic surface of the pilins through a donor-strand complementation mechanism. Face is the periplasmic chaperone required for the assembly of the F4 fimbriae of enterotoxigenic Escherichia coli. The FacE crystal structure shows a dimer formed by interaction between the pilin-binding interfaces of the two monomers. Dimerization and tetramerization have been observed previously in crystal structures of fimbrial chaperones and have been suggested to serve as a self-capping mechanism that protects the pilin-interactive surfaces in solution in the absence of the pilins. However, thermodynamic and biochemical data show that Face occurs as a stable monomer in solution. Other lines of evidence indicate that self-capping of the pilin-interactive interfaces is not a mechanism that is conservedly applied by all periplasmic chaperones, but is rather a case-specific solution to cap aggregation-prone surfaces. [ABSTRACT FROM AUTHOR]

Five experiments have been designed to be used for teaching macromolecular crystallography. The three proteins used in this tutorial are all commercially available; they can be easily and reproducibly crystallized and mounted for diffraction data collection. For each of the five experiments the raw images and the processed data of a sample diffraction data set as well as the refined coordinates and phases are provided for teaching the steps of data processing and structure determination. [ABSTRACT FROM AUTHOR]

A cDNA from the plant Thalictrum flavum encoding pavine N-methyltransferase, an enzyme belonging to a novel class of S-adenosylmethionine-dependent N-methyltransferases specific for benzylisoquinoline alkaloids, has been heterologously expressed in Escherichia coli. The enzyme was purified using affinity and gel-filtration chromatography and was crystallized in space group P21. The structure was solved at 2.0 Å resolution using a xenon derivative and the single isomorphous replacement with anomalous scattering method. [ABSTRACT FROM AUTHOR]

Copyright of Protein Science: A Publication of the Protein Society is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Aspartate semialdehyde dehydrogenase fromMycobacterium tuberculosis (Asd, ASADH, Rv3708c), which is the second enzyme in the lysine/homoserinebiosynthetic pathways, has been expressed heterologously in Escherichia coli. The enzyme was purified using affinity and gel-filtration chromatographic techniques and crystallized in two different crystal forms. Preliminary diffraction data analysis suggested the presence of up to four monomers in the asymmetric unit of the orthorhombic crystal form A and of one or two monomers in the cubic crystal form B. [ABSTRACT FROM AUTHOR]

EcoR124I is a multicomplex enzyme belonging to the type I restriction-modification system from Escherichia coli. Although EcoR124I has been extensively characterized biochemically, there is no direct structural information available about particular subunits. HsdR is a motor subunit that is responsible for ATP hydrolysis, DNA translocation and cleavage of the DNA substrate recognized by the complex. Recombinant HsdR subunit was crystallized using the sitting-drop vapour-diffusion method. Crystals belong to the primitive monoclinic space group, with unit-cell parameters a = 85.75, b = 124.71, c = 128.37 Å, β = 108.14°. Native data were collected to 2.6 Å resolution at the X12 beamline of EMBL Hamburg. [ABSTRACT FROM AUTHOR]

Crystal structure determination of catabolite control protein A (CcpA) at 2.6 Å resolution reveals for the first time the structure of a full-length apo-form LacI-GalR family repressor protein. In the crystal structures of these transcription regulators, the three-helix bundle of the DNA-binding domain has only been observed in cognate DNA complexes; it has not been observed in other crystal structures owing to its mobility. In the crystal packing of apo-CcpA, the protein–protein contacts between the N-terminal three-helix bundle and the core domain consisted of interactions between the homodimers that were similar to those between the corepressor protein HPr and the CcpA N-subdomain in the ternary DNA complex. In contrast to the DNA complex, the apo-CcpA structure reveals large subdomain movements in the core, resulting in a complete loss of contacts between the N-subdomains of the homodimer. [ABSTRACT FROM AUTHOR]

23 different crystal forms of 19 different biological macromolecules were examined with respect to their anomalously scattering substructures using diffraction data collected at a wavelength of 2.0 Å (6.2 keV). In more than 90% of the cases the substructure was found to contain more than just the protein S atoms. The data presented suggest that chloride, sulfate, phosphate or metal ions from the buffer or even from the purification protocol are frequently bound to the protein molecule and that these ions are often overlooked, especially if they are not bound at full occupancy. Thus, in order to fully describe the macromolecule under study, it seems desirable that any structure determination be complemented with a long-wavelength data set. [ABSTRACT FROM AUTHOR]

Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 m M sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C2221 and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å. [ABSTRACT FROM AUTHOR]

The 22 kDa SoxYZ protein complex from the green sulfur bacterium Chlorobium limicola f. thiosulfatophilum is a central player in the sulfur-oxidizing (Sox) enzyme system of the organism by activating thiosulfate for oxidation by SoxXA and SoxB. It has been proposed that SoxYZ exists as a heterodimer or heterotetramer, but the properties and role of the individual components of the complex thus far remain unknown. Here, the heterologous expression, purification, and the crystallization of stable tetrameric SoxY are reported. Crystals of SoxY diffract to 2.15 Å resolution and belong to space group C2221, with unit-cell parameters a = 41.22, b = 120.11, c = 95.30 Å. MIRAS data from Pt2+- and Hg2+-derivatized SoxY crystals resulted in an interpretable electron-density map at 3 Å resolution after density modification. [ABSTRACT FROM AUTHOR]

Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 Å, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 Å. [ABSTRACT FROM AUTHOR]

Complete and highly redundant data sets were collected at different wavelengths between 0.80 and 2.65 Å for a total of ten different protein and DNA model systems. The magnitude of the anomalous signal-to-noise ratio as assessed by the quotient Ranom/Rr.i.m. was found to be influenced by the data-collection wavelength and the nature of the anomalously scattering substructure. By utilizing simple empirical correlations, for instance between the estimated ΔF/F and the expected Ranom or the data-collection wavelength and the expected Rr.i.m., the wavelength at which the highest anomalous signal-to-noise ratio can be expected could be estimated even before the experiment. Almost independent of the nature of the anomalously scattering substructure and provided that no elemental X-ray absorption edge is nearby, this optimal wavelength is 2.1 Å. [ABSTRACT FROM AUTHOR]

Dihydrodipicolinate reductase from Mycobacterium tuberculosis (DapB, DHDPR, Rv2773c) has been cloned and heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized in three different crystal forms. Preliminary diffraction data analysis suggests the presence of two tetramers in the asymmetric unit of one crystal form and half a tetramer in the other two crystal forms. [ABSTRACT FROM AUTHOR]

Two series of complete and highly redundant data sets were collected at wavelengths of 1.00 and 2.00 A. on a cadmium derivative of porcine pancreatic elastase (PPE). Radiation damage to the sample was evaluated qualitatively by inspecting consecutive difference electron density maps during the course of the experiment. The nature of the radiation damage was found to be identical at both wavelengths and was localized primarily at the four disulfide bridges of PPE, the cadmium site and the two methionine residues. For a quantitative examination of the radiation damage, the decrease in the peak height of the cadmium ion in various electron density maps was exploited. Again, no significant difference in radiation damage between the two wavelengths was observed. This can be rationalized by considering the wavelength dependencies of the number of diffracted photons versus the number of absorbed photons and the energy deposited in the crystal by the latter. [ABSTRACT FROM AUTHOR]

The EMBL-Hamburg Automated Crystal Structure Determination Platform is a sys-tem that combines a number of existing macromolecular crystallographic computer programs and several decision-makers into a software pipeline for automated and efficient crystal structure determination. The pipeline can be invoked as soon as X-ray data from derivatized protein crystals have been collected and processed. It is controlled by a web-based graphical user interface for data and parameter input, and for monitoring the progress of structure determination. A large number of possible structure-solution paths are encoded in the system and the optimal path is selected by the decision-makers as the structure solution evolves. The processes have been optimized for speed so that the pipeline can be used effectively for validating the X-ray experiment at a synchrotron beamline. [ABSTRACT FROM AUTHOR]

Complete and highly redundant data sets were collected at nine different wavelengths between 0.80 and 2.65 Å on a xenon derivative of porcine pancreatic elastase in both air and helium atmospheres. The magnitude of the anomalous signal, as assessed by the xenon-peak height in the anomalous difference Patterson synthesis, is affected by the wavelength of data collection as well as by the scaling model used. For data collected at wavelengths longer than 1.7 Å, the use of a three-dimensional scaling protocol is essential in order to obtain the highest possible anomalous signal. Based on the scaling protocols currently available, the optimal wavelength range for data collection appears to be between 2.1 and 2.4 Å. Beyond that, any further increase in signal will be compensated for or even superseded by a concomitant increase in noise, which cannot be fully corrected for. Data collection in a helium atmosphere yields higher I/σ(I) values, but not significantly better anomalous differences, than data collection in air. [ABSTRACT FROM AUTHOR]

Investigates the crystallization of porcine pancreatic elastase (PPE) at acidic hydrogen-ion concentration. Detection of calcium ion in the metal-binding site of PPE; Coordination distance of the metal ions; Structure of PPE.

Investigates the xenon (Xe) derivatization of halide-soaked protein crystals. Use of Xe sites in determining the bromide substructure; Refinement of the bromide-containing structures; Crystallization of porcine pancreatic elastase.

Assesses the phasing possibilities using different wavelengths with a xenon derivative. Examination of various phasing procedures for xenon-derivatized porcine pancreatic elastase; Importance of highly redundant data in measuring precise anomalous differences; Evaluation of structure refinement and automated model building.

1,3-Propanediol dehydrogenase is an enzyme that catalyzes the oxidation of 1,3-propanediol to 3-hydroxypropanal with the simultaneous reduction of NADP+ to NADPH. SeMet-labelled 1,3-propanediol dehydrogenase protein from the hyperthermophilic bacterium Aquifex aeolicus VF5 was overexpressed in Escherichia coli and purified to homogeneity. Crystals of this protein were grown from an acidic buffer with ammonium sulfate as the precipitant. Single-wavelength data were collected at the selenium peak to a resolution of 2.4 Å. The crystal belonged to space group P32, with unit-cell parameters a = b = 142.19, c = 123.34 Å. The structure contained two dimers in the asymmetric unit and was solved by the MR-SAD approach. [ABSTRACT FROM AUTHOR]