Analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria.

Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock, considering that vancomycin is the last resort for controlling methicillin-resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, the analyses, which are typically not as straightforward as those of small proteins around 10 kDa, were successfully conducted. We assigned the main chain using an amino acid-selective unlabeling method. Because we found that the zinc ion-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by replacing an amino acid at the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.

Synthesis of 3,3'-dihydroxy-2,2'-diindan-1,1'-dione derivatives for tautomeric organic semiconductors exhibiting intramolecular double proton transfer.

To investigate potential applications of the 3,3'-dihydroxy-2,2'-biindan-1,1'-dione (BIT) structure as an organic semiconductor with intramolecular hydrogen bonds, a new synthetic route under mild conditions is developed based on the addition reaction of 1,3-dione to ninhydrin and the subsequent hydrogenation of the hydroxyl group. This route affords several new BIT derivatives, including asymmetrically substituted structures that are difficult to access by conventional high-temperature synthesis. The BIT derivatives exhibit rapid tautomerization by intramolecular double proton transfer in solution. The tautomerizations are also observed in the solid state by variable temperature measurements of X-ray diffractometry and magic angle spinning 13C solid-state NMR. Possible interplay between the double proton transfer and the charge transport is suggested by quantum chemical calculations. The monoalkylated BIT derivative with a lamellar packing structure suitable for lateral charge transport in films shows a hole mobility of up to 0.012 cm2 V-1 s-1 with a weak temperature dependence in an organic field effect transistor.

Nuclear Magnetic Resonance Detection of Hydrogen Bond Network in a Proton Pump Rhodopsin RxR and Its Alteration during the Cyclic Photoreaction.

Hydrogen bond formation and deformation are crucial for the structural construction and functional expression of biomolecules. However, direct observation of exchangeable hydrogens, especially for oxygen-bound hydrogens, relevant to hydrogen bonds is challenging for current structural analysis approaches. Using solution-state NMR spectroscopy, this study detected the functionally important exchangeable hydrogens (i.e., Y49-ηOH and Y178-ηOH) involved in the pentagonal hydrogen bond network in the active site of R. xylanophilus rhodopsin (RxR), which functions as a light-driven proton pump. Moreover, utilization of the original light-irradiation NMR approach allowed us to detect and characterize the late photointermediate state (i.e., O-state) of RxR and revealed that hydrogen bonds relevant to Y49 and Y178 are still maintained during the photointermediate state. In contrast, the hydrogen bond between W75-εNH and D205-γCOO- is strengthened and stabilizes the O-state.

Biophysical analysis of Gaussia luciferase bioluminescence mechanisms using a non-oxidizable coelenterazine.

Gaussia luciferase (GLuc 18.2kDa; 168 residues) is a marine copepod luciferase that emits a bright blue light when oxidizing coelenterazine (CTZ). It is a helical protein where two homologous sequential repeats form two anti-parallel bundles, each made of four helices. We previously identified a hydrophobic cavity as a prime candidate for the catalytic site, but GLuc's fast bioluminescence reaction hampered a detailed analysis. Here, we used azacoelenterazine (Aza-CTZ), a non-oxidizable coelenterazine (CTZ) analog, as a probe to investigate its binding mode to GLuc. While analysing GLuc's activity, we unexpectedly found that salt and monovalent anions are absolutely required for Gluc's bioluminescence, which retrospectively appears reasonable for a sea-dwelling organism. The NMR-based investigation, using chemical shift perturbations monitored by 15N-1H HSQC, suggested that Aza-CTZ (and thus unoxidized CTZ) binds to residues in or near the hydrophobic cavity. These NMR data are in line with a recent structural prediction of GLuc, hypothesizing that large structural changes occur in regions remote from the hydrophobic cavity upon the addition of CTZ. Interestingly, these results point toward a unique mode of catalysis to achieve CTZ oxidative decarboxylation.

Characterization of retinal chromophore and protonated Schiff base in Thermoplasmatales archaeon heliorhodopsin using solid-state NMR spectroscopy.

Heliorhodopsin (HeR) is a seven-helical transmembrane protein with a retinal chromophore that corresponds to a new rhodopsin family. HeR from the archaebacterium Thermoplasmatales archaeon (TaHeR) exhibits unique features, such as the inverted protein orientation in the membrane compared to other rhodopsins and a long photocycle. Here, we used solid-state nuclear magnetic resonance (NMR) spectroscopy to investigate the 13C and 15N NMR signals of the retinal chromophore and protonated Schiff base (RPSB) in TaHeR embedded in POPE/POPG membrane. Although the 14- and 20-13C retinal signals indicated 13-trans/15-anti (all-trans) configurations, the 20-13C chemical shift value was different from that of other microbial rhodopsins, indicating weakly steric hinderance between Phe203 and the C20 methyl group. 15N RPSB/λmax plot deviated from the linear correlation based on retinylidene-halide model compounds. Furthermore, 15N chemical shift anisotropy (CSA) suggested that Ser112 and Ser234 polar residues distinguish the electronic environment tendencies of RPSB from those of other microbial rhodopsins. Our NMR results revealed that the retinal chromophore and the RPSB in TaHeR exhibit unique electronic environments.

Reflecting on mutational and biophysical analysis of Gaussia princeps Luciferase from a structural perspective: a unique bioluminescent enzyme.

Gaussia princeps luciferase (GLuc 18.2 kDa; 168 residues) is a marine copepod luciferase that emits a bright blue light when oxidizing coelenterazine (CTZ). GLuc is a small luciferase, attracting much attention as a potential reporter protein. However, compared to firefly and Renilla luciferases, which have been thoroughly characterized and are used in a wide range of applications, structural and biophysical studies of GLuc have been slow to appear. Here, we review the biophysical and mutational studies of GLuc's bioluminescence from a structural viewpoint, particularly in view of its recent NMR solution structure, where two homologous sequential repeats form two anti-parallel bundles, each made of four helices, grabbing a short N-terminal helix. Additionally, a long loop classified as an intrinsically disordered region separates the two bundles forming one side of a hydrophobic pocket that is most likely the binding/catalytic site. We compare the NMR-determined structure with a recent AlphaFold2 prediction. Overall, the AlphaFold2 structure was in line with the solution structure; however, it surprisingly revealed a possible, alternative conformation, where the N-terminal helix is replaced by a newly formed α helix in the C-terminal tail that is unfolded in the NMR structure. In addition, we discuss the results of previous mutational analysis focusing on a putative catalytic core identified by chemical shift perturbation analysis and molecular dynamics simulations performed using both the NMR and the AlphaFold2 structures. In particular, we discuss the role of the possible conformational change and the hydrophobic pocket in GLuc's activity. Overall, the discussion points toward GLuc's unexpected and unusual characteristics that appear to be much more flexible than traditional enzymes, resulting in a unique mode of catalysis to achieve CTZ oxidative decarboxylation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-022-01025-6.

Assessment of prediction methods for protein structures determined by NMR in CASP14: Impact of AlphaFold2.

NMR studies can provide unique information about protein conformations in solution. In CASP14, three reference structures provided by solution NMR methods were available (T1027, T1029, and T1055), as well as a fourth data set of NMR-derived contacts for an integral membrane protein (T1088). For the three targets with NMR-based structures, the best prediction results ranged from very good (GDT_TS = 0.90, for T1055) to poor (GDT_TS = 0.47, for T1029). We explored the basis of these results by comparing all CASP14 prediction models against experimental NMR data. For T1027, NMR data reveal extensive internal dynamics, presenting a unique challenge for protein structure prediction methods. The analysis of T1029 motivated exploration of a novel method of "inverse structure determination," in which an AlphaFold2 model was used to guide NMR data analysis. NMR data provided to CASP predictor groups for target T1088, a 238-residue integral membrane porin, was also used to assess several NMR-assisted prediction methods. Most groups involved in this exercise generated similar beta-barrel models, with good agreement with the experimental data. However, as was also observed in CASP13, some pure prediction groups that did not use any NMR data generated models for T1088 that better fit the NMR data than the models generated using these experimental data. These results demonstrate the remarkable power of modern methods to predict structures of proteins with accuracies rivaling solution NMR structures, and that it is now possible to reliably use prediction models to guide and complement experimental NMR data analysis.

Structure Solution of Nano-Crystalline Small Molecules Using MicroED and Solid-State NMR Dipolar-Based Experiments.

Three-dimensional electron diffraction crystallography (microED) can solve structures of sub-micrometer crystals, which are too small for single crystal X-ray crystallography. However, R factors for the microED-based structures are generally high because of dynamic scattering. That means R factor may not be reliable provided that kinetic analysis is used. Consequently, there remains ambiguity to locate hydrogens and to assign nuclei with close atomic numbers, like carbon, nitrogen, and oxygen. Herein, we employed microED and ssNMR dipolar-based experiments together with spin dynamics numerical simulations. The NMR dipolar-based experiments were 1H-14N phase-modulated rotational-echo saturation-pulse double-resonance (PM-S-RESPDOR) and 1H-1H selective recoupling of proton (SERP) experiments. The former examined the dephasing effect of a specific 1H resonance under multiple 1H-14N dipolar couplings. The latter examined the selective polarization transfer between a 1H-1H pair. The structure was solved by microED and then validated by evaluating the agreement between experimental and calculated dipolar-based NMR results. As the measurements were performed on 1H and 14N, the method can be employed for natural abundance samples. Furthermore, the whole validation procedure was conducted at 293 K unlike widely used chemical shift calculation at 0 K using the GIPAW method. This combined method was demonstrated on monoclinic l-histidine.

Sensitivity-Enhanced Solid-State NMR Detection of Structural Differences and Unique Polymorphs in Pico- to Nanomolar Amounts of Brain-Derived and Synthetic 42-Residue Amyloid-ß Fibrils.

Amyloid-ß (Aß) fibrils in neuritic plaques are a hallmark of Alzheimer's disease (AD). Since the 42-residue Aß (Aß42) fibril is the most pathogenic among different Aß species, its structural characterization is crucial to our understanding of AD. While several polymorphs have been reported for Aß40, previous studies of Aß42 fibrils prepared at neutral pH detected essentially only one structure, with an S-shaped ß-sheet arrangement (e.g., Xiao et al. Nat. Struct. Mol. Biol. 2015, 22, 499). Herein, we demonstrate the feasibility of characterizing the structure of trace amounts of brain-derived and synthetic amyloid fibrils by sensitivity-enhanced 1H-detected solid-state NMR (SSNMR) under ultrafast magic angle spinning. By taking advantage of the high sensitivity of this technique, we first demonstrate its applicability for the high-throughput screening of trace amounts of selectively 13C- and 15N-labeled Aß42 fibril prepared with ∼0.01% patient-derived amyloid (ca. 4 pmol) as a seed. The comparison of 2D 13C/1H SSNMR data revealed marked structural differences between AD-derived Aß42 (∼40 nmol or ∼200 µg) and synthetic fibrils in less than 10 min, confirming the feasibility of assessing the fibril structure from ∼1 pmol of brain amyloid seed in ∼2.5 h. We also present the first structural characterization of synthetic fully protonated Aß42 fibril by 1H-detected 3D and 4D SSNMR. With procedures assisted by automated assignments, main-chain resonance assignments were completed for trace amounts (∼42 nmol) of a fully protonated amyloid fibril in the 1H-detection approach. The results suggest that this Aß42 fibril exhibits a novel fold or polymorph structure.

Nanodisc reconstitution of flavin mononucleotide binding domain of cytochrome-P450-reductase enables high-resolution NMR probing.

Cytochrome-P450-reductase transfers electrons to cytochrome-P450 through its flavin mononucleotide binding domain (FBD). Despite the importance of membrane-anchoring for FBD function, studies have focused on its soluble domain lacking the transmembrane-domain. Here we demonstrate that the reconstitution of FBD in nanodiscs enables high-resolution NMR measurements and renders a stable conformation.

Evaluation of a booster tuberculosis vaccine containing mycobacterial DNA-binding protein 1 and CpG oligodeoxynucleotide G9.1 using a Guinea pig model that elicits immunity to Bacillus Calmette-Guérin.

Tuberculosis is a major threat to global health and its increased incidence in adolescents as well as onset in the elderly presents a serious problem. One strategy to control tuberculosis involves taking advantage of Bacillus Calmette-Guérin's (BCG) superior effects on childhood tuberculosis. Accordingly, here we aimed to develop a booster vaccine for adults who received the BCG vaccine during early childhood. Therefore, we first devised a system to assess the efficacy of a candidate booster vaccine. Specifically, variant strain BCG-II, a minor component of BCG-Tokyo strain, which elicits weak immunity, was administered to guinea pigs. Vaccine-induced immunity and protection against Mycobacterium tuberculosis (Mtb) infection were evaluated using skin delayed-type hypersensitivity (DTH) and Mtb colony forming unit counts in organs, respectively. Candidate booster vaccine containing the mycobacterial DNA-binding protein 1 (MDP1) as antigen and CpG oligodeoxynucleotide G9.1 as adjuvant increased T-bet expression and IFN-γ production in human peripheral blood mononuclear cells. Intradermal administration of MDP1 or MDP1 and G9.1 to unimmunized guinea pigs produced DTH on MDP1-inoculated skin. Boosting BCG-II-primed guinea pigs with this protocol effectively enhanced DTH against MDP1 and protection against Mtb infection, particularly when combined with G9.1. The candidate vaccine may contribute to efforts to prevent tuberculosis.

Decoherence optimized tilted-angle cross polarization: A novel concept for sensitivity-enhanced solid-state NMR using ultra-fast magic angle spinning.

Ultra-fast magic-angle spinning (UFMAS) at a MAS rate (ωR/2π) of 60 kHz or higher has dramatically improved the resolution and sensitivity of solid-state NMR (SSNMR). However, limited polarization transfer efficiency using cross-polarization (CP) between 1H and rare spins such as 13C still restricts the sensitivity and multi-dimensional applications of SSNMR using UFMAS. We propose a novel approach, which we call decoherence-optimized tilted-angle CP (DOTA CP), to improve CP efficiency with prolonged lifetime of 1H coherence in the spin-locked condition and efficient band-selective polarization transfer by incorporating off-resonance irradiation to 1H spins. 13C CP-MAS at ωR/2π of 70-90 kHz suggested that DOTA CP notably outperformed traditional adiabatic CP, a de-facto-standard CP scheme over the past decade, in sensitivity for the aliphatic-region spectra of 13C-labeled GB1 protein and N-formyl-Met-Leu-Phe samples by up to 1.4- and 1.2-fold, respectively. 1H-detected 2D 1H/13C SSNMR for the GB1 sample indicated the effectiveness of this approach in various multidimensional applications.

Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR.

Gaussia luciferase (GLuc) is a small luciferase (18.2 kDa; 168 residues) and is thus attracting much attention as a reporter protein, but the lack of structural information is hampering further application. Here, we report the first solution structure of a fully active, recombinant GLuc determined by heteronuclear multidimensional NMR. We obtained a natively folded GLuc by bacterial expression and efficient refolding using a Solubility Enhancement Petide (SEP) tag. Almost perfect assignments of GLuc's 1H, 13C and 15N backbone signals were obtained. GLuc structure was determined using CYANA, which automatically identified over 2500 NOEs of which > 570 were long-range. GLuc is an all-alpha-helix protein made of nine helices. The region spanning residues 10-18, 36-81, 96-145 and containing eight out of the nine helices was determined with a Cα-atom RMSD of 1.39 Å ± 0.39 Å. The structure of GLuc is novel and unique. Two homologous sequential repeats form two anti-parallel bundles made by 4 helices and tied together by three disulfide bonds. The N-terminal helix 1 is grabbed by these 4 helices. Further, we found a hydrophobic cavity where several residues responsible for bioluminescence were identified in previous mutational studies, and we thus hypothesize that this is a catalytic cavity, where the hydrophobic coelenterazine binds and the bioluminescence reaction takes place.

Silkworm Pupae Function as Efficient Producers of Recombinant Glycoproteins with Stable-Isotope Labeling.

Baculovirus-infected silkworms are promising bioreactors for producing recombinant glycoproteins, including antibodies. Previously, we developed a method for isotope labeling of glycoproteins for nuclear magnetic resonance (NMR) studies using silkworm larvae reared on an artificial diet containing 15N-labeled yeast crude protein extract. Here, we further develop this method by introducing a technique for the expression of isotope-labeled glycoproteins by silkworm pupae, which has several potential advantages relative to larvae-based techniques in terms of production yield, ease of handling, and storage. Here, we fed fifth instar larvae an artificial diet with an optimized composition containing [methyl-13C]methionine, leading to pupation. Nine-day-old pupae were then injected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid for expression of recombinant human immunoglobulin G (IgG). From the whole-body homogenates of pupae, 0.35 mg/pupa of IgG was harvested, which is a yield that is five times higher than can be obtained from larvae. Recombinant IgG, thus prepared, exhibited mainly three kinds of pauci-mannose-type oligosaccharides and had a 13C-enrichment ratio of approximately 80%. This enabled selective observation of NMR signals originating from the methionyl methyl group of IgG, confirming its conformational integrity. These data demonstrate the utility of silkworm pupae as factories for producing recombinant glycoproteins with amino-acid-selective isotope labeling.

An NMR-based approach reveals the core structure of the functional domain of SINEUP lncRNAs.

Long non-coding RNAs (lncRNAs) are attracting widespread attention for their emerging regulatory, transcriptional, epigenetic, structural and various other functions. Comprehensive transcriptome analysis has revealed that retrotransposon elements (REs) are transcribed and enriched in lncRNA sequences. However, the functions of lncRNAs and the molecular roles of the embedded REs are largely unknown. The secondary and tertiary structures of lncRNAs and their embedded REs are likely to have essential functional roles, but experimental determination and reliable computational prediction of large RNA structures have been extremely challenging. We report here the nuclear magnetic resonance (NMR)-based secondary structure determination of the 167-nt inverted short interspersed nuclear element (SINE) B2, which is embedded in antisense Uchl1 lncRNA and upregulates the translation of sense Uchl1 mRNAs. By using NMR 'fingerprints' as a sensitive probe in the domain survey, we successfully divided the full-length inverted SINE B2 into minimal units made of two discrete structured domains and one dynamic domain without altering their original structures after careful boundary adjustments. This approach allowed us to identify a structured domain in nucleotides 31-119 of the inverted SINE B2. This approach will be applicable to determining the structures of other regulatory lncRNAs.

Understanding hydrogen-bonding structures of molecular crystals via electron and NMR nanocrystallography.

Understanding hydrogen-bonding networks in nanocrystals and microcrystals that are too small for X-ray diffractometry is a challenge. Although electron diffraction (ED) or electron 3D crystallography are applicable to determining the structures of such nanocrystals owing to their strong scattering power, these techniques still lead to ambiguities in the hydrogen atom positions and misassignments of atoms with similar atomic numbers such as carbon, nitrogen, and oxygen. Here, we propose a technique combining ED, solid-state NMR (SSNMR), and first-principles quantum calculations to overcome these limitations. The rotational ED method is first used to determine the positions of the non-hydrogen atoms, and SSNMR is then applied to ascertain the hydrogen atom positions and assign the carbon, nitrogen, and oxygen atoms via the NMR signals for 1H, 13C, 14N, and 15N with the aid of quantum computations. This approach elucidates the hydrogen-bonding networks in L-histidine and cimetidine form B whose structure was previously unknown.

Misfolding of a Single Disulfide Bonded Globular Protein into a Low-Solubility Species Conformationally and Biophysically Distinct from the Native One.

In practice and despite Anfinsen's dogma, the refolding of recombinant multiple SS-bonded proteins is famously difficult because misfolded species with non-native SS-bonds appear upon the oxidization of their cysteine residues. On the other hand, single SS-bond proteins are thought to be simple to refold because their cysteines have only one SS-bond partner. Here, we report that dengue 4 envelope protein domain 3 (DEN4 ED3), a single SS-bonded protein can be irreversibly trapped into a misfolded species through the formation of its sole intramolecular SS-bond. The misfolded species had a much lower solubility than the native one at pHs higher than about 7, and circular dichroism measurements clearly indicated that its secondary structure content was different from the native species. Furthermore, the peaks in the Heteronuclear Single Quantum Correlation spectroscopy (HSQC) spectrum of DEN4 ED3 from the supernatant fraction were sharp and well dispersed, reflecting the beta-sheeted native structure, whereas the spectrum of the precipitated fraction showed broad signals clustered near its center suggesting no or little structure and a strong tendency to aggregate. The two species had distinct biophysical properties and could interconvert into each other only by cleaving and reforming the SS-bond, strongly suggesting that they are topologically different. This phenomenon can potentially happen with any single SS-bonded protein, and our observation emphasizes the need for assessing the conformation and biophysical properties of bacterially produced therapeutic proteins in addition to their chemical purities.

Correlation between Aortic Calcification Score and Biochemical Parameters in Hemodialysis Patients.

BACKGROUND: The purpose of this study was to determine the correlation between aortic calcification and demographic and biochemical parameters in hemodialysis patients. SUMMARY: Calcification scores of the aortic arch and abdominal aorta were determined from multi-slice computed tomography scans and evaluated according to the Agatston score. The associations between demographic and biochemical parameters and aortic calcification score were determined. In total, 190 patients were included in the study. There was a significant positive correlation between aortic calcification scores and age, duration of hemodialysis, cardiothoracic ratio, normalized protein catabolic rate, brachial-ankle pulse wave velocity (baPWV), serum markers of mineral metabolism, and inflammation. A significant negative correlation was found between aortic calcification scores and platelet count. Multivariate analysis showed that age, duration of hemodialysis, baPWV, phosphate, calcium and phosphate (Ca×P) product, parathyroid hormone, and C-reactive protein levels were independent risk factors for calcification of the aortic arch, while baPWV and Ca×P product were independent risk factors for calcification of the abdominal aorta. Key Messages: Aortic calcification scores correlate with age, duration of hemodialysis, and several biochemical parameters of inflammation and mineral metabolism.

Efficacy of L-carnitine supplementation for improving lean body mass and physical function in patients on hemodialysis: a randomized controlled trial.

BACKGROUND: Carnitine deficiency is common in patients on hemodialysis. However, the efficacy of L-carnitine supplementation for improving lean body mass (LBM) and physical function has not yet been evaluated. METHODS: In this multicenter, prospective, parallel, randomized, controlled trial, 91 patients on hemodialysis who developed carnitine deficiency were randomly assigned to receive injections of 1,000 mg L-carnitine 3 times per week after each hemodialysis session (L-carnitine group) or no injections (control group) with monitoring for 12 months. RESULTS: The data for 84 of the 91 patients were available for analysis (L-carnitine group, n = 42; control group, n = 42). Dry weight and body mass index did not significantly change in the L-carnitine group, but significantly decreased in the control group. Arm muscle area (AMA) did not change significantly in the L-carnitine group but decreased significantly in the control group; the difference in mean AMA between the groups was 6.22% (95% confidence interval [CI] 1.90-10.5; P = 0.037). Hand grip strength did not change significantly in the L-carnitine group, but decreased significantly in the control group. The difference in change in hand grip strength between the groups was 4.27% (95% CI 0.42-8.12; P = 0.030). Furthermore, LBM did not change significantly in the L-carnitine group but decreased significantly in the control group; the difference in mean LBM between the groups was 2.92 % (95% CI 1.28-4.61; P = 0.0007). CONCLUSIONS: L-carnitine supplementation is useful in patients who develop carnitine deficiency on hemodialysis because it maintains physical function and LBM.

Genome Sequences of 12 Mycobacteriophages Recovered from Archival Stocks in Japan.

Using Mycobacterium smegmatis mc2155, 12 siphoviruses were recovered from long-term archival stocks stored in Japan. Their genome sequences were 46.0 to 61.3 kbp with 63 to 68% G+C contents, which allowed them to be categorized within cluster W and subclusters A1, A2, B3, A7, I1, and K4.