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The design of a new versatile control system that will underlie future releases of the automated model-building package ARP/wARP is presented. A sophisticated expert system is under development that will transform ARP/wARP from a very useful model-building aid to a truly automated package capable of delivering complete, well refined and validated m...
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... are iterated millions of times and are suboptimal for the speci®c case. We chose to use what we refer to as a `real-space residual map', a method that has been proposed and implemented for real-space re®nement in O (Jones & Liljas, 1984). A copy of the electron-density map is made in computer memory and all existing atom density is then subtracted from this map (which is in effect equivalent to `masking out' neighbouring atoms in real-space re®nement in O ), resulting in a `real-space residual map' (Fig. 4). Every time that a new side chain is placed, its density values are also subtracted. Since the target for side-chain ®tting is the real-space correlation with the electron-density map, using the correlation with the `real-space residual map' eliminates the need for geometrical antibumping restraints. However, it is important not to perform the side-chain placement sequentially but to ®rst place well ordered side chains with clear positions and proceed later with side chains that are less well ordered. It has to be noted that the `real-space residual map' is only useful in the context of the re®nement scheme we propose, where side chains are re®ned one at a time in real space. 2.4.2. Rotamer fitting . For rotamer placement we use the `Penultimate Rotamer Library' (Lovell et al. , 2000). All conformations are pre-calculated once. The best ®tting rotamers are chosen ®rst for all small hydrophobic and polar residues, then for aromatics and ®nally from shorter towards longer polar charged residues and Met. This way, we ensure that density is occupied ®rst by residues that are usually well ordered and we only position residues that are more likely to be disordered at the end. The order in which different side chains are placed was initially based on `common crystallographic sense'. We are currently revising that order in the context of the results obtained from the EDS server (Kleywegt et al. , 2004) and we aim to derive a statistically valid `order' for different resolutions. For example, an examination of real- space ®t of residues reveals that while between 2.8 and 3.0 A Ê the order should be Cys-Met-Pro-Trp-Thr-Ser-Val-Phe-Tyr- His-Ile-Asp-Leu-Asn-Gln-Arg-Glu-Lys, between 1.2 and 1.4 A Ê it should be Cys-Tyr-Trp-Phe-Thr-Val-His-Ile-Ley-Ser- ...
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Whilst some software measurement research has been unquestionably successful, other research has struggled to enable expected advances in project and process management. Contributing to this lack of advancement has been the incidence of inappropriate or non-optimal application of various model-building procedures. This obviously raises questions ov...
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... The positions of the iron or copper ions were determined based on anomalous difference Fourier maps and geometric parameters calculated using the program FFT (fast Fourier transform) from the CCP4 suite (Collaborative Computational Project, Number 4, 1994). Water molecules were positioned in self-defined residual densities by ARP/wARP (Cohen et al., 2004). The final models were inspected manually and checked with the programs COOT (Emsley and Cowtan, 2004) and PROCHECK (Laskowski et al., 1993). ...
In addition to its role as an iron storage protein, ferritin can function as a major detoxification component in the innate immune defense, and Cu2+ ions can also play crucial antibacterial roles in the blood clam, Tegillarca granosa. However, the mechanism of interaction between iron and copper in recombinant Tegillarca granosa ferritin (TgFer) remains to be investigated. In this study, we investigated the crystal structure of TgFer and examined the effects of Fe2+ and Cu2+ ions on the TgFer structure and catalytic activity. The crystal structure revealed that TgFer presented a typically 4–3–2 symmetry in a cage-like, spherical shell composed of 24 identical subunits, featuring highly conserved organization in both the ferroxidase center and the 3-fold channel. Structural and biochemical analyses indicated that the 4-fold channel of TgFer could be serviced as potential binding sites of metal ions. Cu2+ ions appear to bind preferentially with the 3-fold channel as well as ferroxidase site over Fe2+ ions, possibly inhibiting the ferroxidase activity of TgFer. Our results present a structural and functional characterization of TgFer, providing mechanistic insight into the interactions between TgFer and both Fe2+ and Cu2+ ions.
... The synthetic dsDNA substrates were prepared by annealing oligonucleotides DNA1 and DNA2 by incubating at 90ºC for 4 min followed by 10 min at 37ºC in a thermocycler and a slow cool-down to room temperature on benchtop (Table S2) The data were integrated, scaled and merged with the HKL3000 program suite (68).The heavy atom Se sites were located using the program SHELXD (69) and they were used for phasing with the program MLPHARE (70). After density modification (70), a partial model was built in three J o u r n a l P r e -p r o o f cycles of Arp/Warpmodel building (71). All of the above programs are integrated within the HKL3000 program suite (68). ...
The cariogenic pathogen Streptococcus mutans contains two CRISPR systems (Type I-C and Type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNA transcripts (pre-crRNA) containing repeats and spacers to mature crRNA guides. In this study, we determined the crystal structure of SmuCas5c at 1.72 Å resolution, which revealed the presence of an N-terminal modified RNA-Recognition Motif (RRM) and a C-terminal twisted β-sheet domains with four bound sulphate molecules. Analysis of surface charge and residue conservation of the SmuCas5c structure suggested the location of an RNA-binding site in a shallow groove formed by the RRM domain with several conserved positively charged residues (Arg39, Lys52, Arg109, Arg127, Arg134). Purified SmuCas5c exhibited metal-independent ribonuclease activity against single-stranded pre-CRISPR RNAs containing a stem-loop structure with a seven-nucleotide stem and a pentaloop. We found SmuCas5c cleaves substrate RNA within the repeat sequence at a single cleavage site located at the 3´-base of the stem but shows significant tolerance to substrate sequence variations downstream of the cleavage site. Structure-based mutational analysis revealed that the conserved residues Tyr50, Lys120, and His121 comprise the SmuCas5c catalytic residues. In addition, site-directed mutagenesis of positively charged residues Lys52, Arg109, and Arg134 located near the catalytic triad had strong negative effects on the RNase activity of this protein, suggesting that these residues are involved in RNA binding. Taken together, our results reveal functional diversity of Cas5c ribonucleases and provide further insight into the molecular mechanisms of substrate selectivity and activity of these enzymes.
... However, it reveals very little information on the identity and conformation of the side chains at the positions along the backbone (Figure 1). This is problematic for sequence assignment because current assignment tools rely on correlations between the local electron density and the side-chain geometries to infer the likely residue type at a backbone position (Zou and Jones, 1996;Holton et al., 2000;Terwilliger, 2003;Cohen et al., 2004;Cowtan, 2008). Not surprisingly, all these tools show a very sharp drop in performance for resolutions worse than 3 Å . ...
... We first demonstrate how the correct residue type at a single position along the backbone can be inferred. Similar to existing tools, we mutate each position into the 20 amino acids, score the models, and then predict the residue type to be the bestscoring mutation (Holton et al., 2000;Terwilliger, 2003;Cohen et al., 2004;Cowtan, 2008). What we do differently is the use of reciprocal-space measures to score the mutations rather than relying on real space correlations to the electron density. ...
... Our goal is to find the optimal alignment of the sequence to these preferences. Applications of sequence assignment to find such an alignment (Zou and Jones, 1996;Holton et al., 2000;Terwilliger, 2003;Cohen et al., 2004;Cowtan, 2008) use summation of the positional preferences, and we follow this same procedure. We also note that this task is very similar to the threading done in comparative modeling, which is well-studied in protein structure predication (Dunbrack, 2006). ...
At resolutions worse than 3.5 Å, the electron density is weak or nonexistent at the locations of the side chains. Consequently, the assignment of the protein sequences to their correct positions along the backbone is a difficult problem. In this work, we propose a fully automated computational approach to assign sequence at low resolution. It is based on our surprising observation that standard reciprocal-space indicators, such as the initial unrefined R value, are sensitive enough to detect an erroneous sequence assignment of even a single backbone position. Our approach correctly determines the amino acid type for 15%, 13%, and 9% of the backbone positions in crystallographic datasets with resolutions of 4.0 Å, 4.5 Å, and 5.0 Å, respectively. We implement these findings in an application for threading a sequence onto a backbone structure. For the three resolution ranges, the application threads 83%, 81%, and 64% of the sequences exactly as in the deposited PDB structures.
... [44][45][46] The solution from molecular replacement was subjected to automated model building using the ARP-WARP online server. [47][48][49] The model obtained after ARP-WARP was further built and refined with COOT 50 and REFMAC, [51][52][53] respectively. The anisotropic motion within the molecule was refined with six TLS groups per subunit. ...
Nucleoside hydrolases (NHs) catalyze the hydrolysis of the N-glycoside bond in ribonucleosides and are found in all three domains of life. Although in parasitic protozoa a role in purine salvage has been well established, their precise function in bacteria and higher eukaryotes is still largely unknown. NHs have been classified into three homology groups based on the conservation of active site residues. While many structures are available of representatives of group I and II, structural information for group III NHs is lacking. Here, we report the first crystal structure of a purine-specific nucleoside hydrolase belonging to homology group III from the nematode Caenorhabditis elegans (CeNH) to 1.65Å resolution. In contrast to dimeric purine-specific NHs from group II, CeNH is a homotetramer. A cysteine residue that characterizes group III NHs (Cys253) structurally aligns with the catalytic histidine and tryptophan residues of group I and group II enzymes, respectively. Moreover, a second cysteine (Cys42) points into the active site of CeNH. Substrate docking shows that both cysteine residues are appropriately positioned to interact with the purine ring. Site-directed mutagenesis and kinetic analysis proposes a catalytic role for both cysteines residues, with Cys253 playing the most prominent role in leaving group activation. This article is protected by copyright. All rights reserved.
... 41 Following density modification, 42 a partial model was built by three cycles of ARP/wARP. 43 The structures were completed with alternating rounds of manual model building with COOT 44 and refinement with PHENIX 1.8.2_1309. 45 Data collection and refinement statistics were summarized in Table I. ...
CalE6 from Micromonospora echinospora is a (pyridoxal 5′ phosphate) PLP-dependent methionine γ-lyase involved in the biosynthesis of calicheamicins. We report the crystal structure of a CalE6 2-(N-morpholino)ethanesulfonic acid complex showing ligand-induced rotation of Tyr100, which stacks with PLP, resembling the corresponding tyrosine rotation of true catalytic intermediates of CalE6 homologs. Elastic network modeling and crystallographic ensemble refinement reveal mobility of the N-terminal loop, which involves both tetrameric assembly and PLP binding. Modeling and comparative structuralanalysis of PLP-dependent enzymes involved in Cys/Met metabolism shine light on the functional implications of the intrinsic dynamic properties of CalE6 in catalysis and holoenzyme maturation.
... The electron density map was improved by solvent flattening using the programs SOLOMON and DM (Abrahams and Leslie 1996;Cowtan 1994). A structure model was constructed with the program ARP/wARP (Cohen et al. 2004). A finalized set of atomic coordinates was obtained after iterative rounds of model building with the program XtalView (McRee 1999) and refinement with CNS (Brunger et al. 1998) by simulated annealing, positional minimization, water molecule identification, and individual isotropic B-value refinement. ...
TTHA0829 from Thermus thermophilus HB8 has a molecular mass of 22,754 Da and is composed of 210 amino acid residues. The expression of TTHA0829 is remarkably elevated in the latter half of logarithmic growth phase. TTHA0829 can form either a tetrameric or dimeric structure, and main-chain folding provides an N-terminal cystathionine-β-synthase (CBS) domain and a C-terminal aspartate-kinase chorismate-mutase tyrA (ACT) domain. Both CBS and ACT are regulatory domains to which a small ligand molecule can bind. The CBS domain is found in proteins from organisms belonging to all kingdoms and is observed frequently as two or four tandem copies. This domain is considered as a small intracellular module with a regulatory function and is typically found adjacent to the active (or functional) site of several enzymes and integral membrane proteins. The ACT domain comprises four β-strands and two α-helices in a βαββαβ motif typical of intracellular small molecule binding domains that help control metabolism, solute transport and signal transduction. We discuss the possible role of TTHA0829 based on its structure and expression pattern. The results imply that TTHA0829 acts as a cell-stress sensor or a metabolite acceptor.
... Initial phases were obtained after density modification using RESOLVE in the phenix.autosol pipeline and the resulting maps used to auto-build secondary structure elements using ARP/wARP from the CCP4 package 34 . The resulting partial model was then used as a search model for molecular replacement against the native data using Phaser and run through 25 cycles of backbone auto-tracing using the native data set in SHELXE 35 . ...
Phosphorus is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use phosphonate compounds, which require specialized enzymatic machinery to break the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolizes phosphonates remain unknown. Here we determine the crystal structure of the 240-kilodalton Escherichia coli C-P lyase core complex (PhnG-PhnH-PhnI-PhnJ; PhnGHIJ), and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that probably couple phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy, and show that it binds to a conserved insertion domain of PhnJ. Our results provide a structural basis for understanding microbial phosphonate breakdown.
... The phase was improved using the programs solomon and DM in CCP4 suite (26). The initial model of the Se-Met-labelled PhoExo I was built using the program ARP/wARP (29). The model was manually rebuilt and refined using the programs COOT and Phenix.refine, ...
Nucleases play important roles in nucleic acid processes, such as replication, repair and recombination. Recently, we identified a novel single-strand specific 3'-5' exonuclease, PfuExo I, from the hyperthermophilic archaeon Pyrococcus furiosus, which may be involved in the Thermococcales-specific DNA repair system. PfuExo I forms a trimer and cleaves single-stranded DNA at every two nucleotides. Here, we report the structural basis for the cleavage mechanism of this novel exonuclease family. A structural analysis of PhoExo I, the homologous enzyme from P. horikoshii OT3, showed that PhoExo I utilizes an RNase H-like active site and possesses a 3'-OH recognition site ∼9 Å away from the active site, which enables cleavage at every two nucleotides. Analyses of the heterotrimeric and monomeric PhoExo I activities showed that trimerization is indispensable for its processive cleavage mechanism, but only one active site of the trimer is required.
© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
... 30 After density modification, 30 partial models were built in three cycles of Arp/Warp model building. 31 All of the above programs are integrated within the program suite HKL3000. 28 The models were then completed manually using the program COOT. ...
The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (∼10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for two alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown. This article is protected by copyright. All rights reserved.
© 2015 The Protein Society.
... All of the nine heavy atoms requested were found with SHELXD [37]. Ninety-six per cent of the model was built with ARP/WARP [38]. Several rounds of manual model building with COOT [21] and refinement cycles with PHENIX [39] were performed until R/R free of 0.212/0.272 ...
The growing emergence of antibiotic‐resistant bacteria has led to the exploration of naturally occurring defense peptides as antimicrobials. In this study, we found that laterosporulin ( LS ), a class II d bacteriocin, effectively kills active and nonmultiplying cells of both Gram‐positive and Gram‐negative bacteria. Fluorescence and electron microscopy suggest that growth inhibition occurs because of increased membrane permeability. The crystal structure of LS at 2.0 Å resolution reveals an all‐β conformation of this peptide, with four β‐strands forming a twisted β‐sheet. All six intrinsic cysteines are intramolecularly disulfide‐bonded, with two disulfides constraining the N terminus of the peptide and the third disulfide crosslinking the extreme C terminus, resulting in the formation of a closed structure. The significance of disulfides in maintaining the in‐solution peptide structure was confirmed by CD and fluorescence analyses. Despite a low overall sequence similarity, LS has disulfide connectivity [ C I – C V , C II – C IV , and C III – C VI ] like that of β‐defensins and a striking architectural similarity with α‐defensins. Therefore LS presents a missing link between bacteriocins and mammalian defensins, and is also a potential antimicrobial lead, in particular against nonmultiplying bacteria.
Database
The atomic coordinates and the structure factors have been deposited in the Protein Data Bank under accession number 4OZK
