A dual RNA-seq analyses revealed dynamic arms race during the invasion of walnut by Colletotrichum gloeosporioides.

BACKGROUND: Walnut anthracnose caused by Colletotrichum gloeosporioides seriously endangers the yield and quality of walnut, and has now become a catastrophic disease in the walnut industry. Therefore, understanding both pathogen invasion mechanisms and host response processes is crucial to defense against C. gloeosporioides infection. RESULTS: Here, we investigated the mechanisms of interaction between walnut fruits (anthracnose-resistant F26 fruit bracts and anthracnose-susceptible F423 fruit bracts) and C. gloeosporioides at three infection time points (24hpi, 48hpi, and 72hpi) using a high-resolution time series dual transcriptomic analysis, characterizing the arms race between walnut and C. gloeosporioides. A total of 20,780 and 6670 differentially expressed genes (DEGs) were identified in walnut and C. gloeosporioides against 24hpi, respectively. Generous DEGs in walnut exhibited opposite expression patterns between F26 and F423, which indicated that different resistant materials exhibited different transcriptional responses to C. gloeosporioides during the infection process. KEGG functional enrichment analysis indicated that F26 displayed a broader response to C. gloeosporioides than F423. Meanwhile, the functional analysis of the C. gloeosporioides transcriptome was conducted and found that PHI, SignalP, CAZy, TCDB genes, the Fungal Zn (2)-Cys (6) binuclear cluster domain (PF00172.19) and the Cytochrome P450 (PF00067.23) were largely prominent in F26 fruit. These results suggested that C. gloeosporioides secreted some type of effector proteins in walnut fruit and appeared a different behavior based on the developmental stage of the walnut. CONCLUSIONS: Our present results shed light on the arms race process by which C. gloeosporioides attacked host and walnut against pathogen infection, laying the foundation for the green prevention of walnut anthracnose.

A Prognostic Model for Survival in Patients with Gastric Signet Ring Cell Carcinoma.

INTRODUCTION: The objective of our study was to develop a nomogram to predict overall survival (OS) and cancer-specific survival (CSS) in patients with gastric signet ring cell carcinoma (GSRCC). METHODS: A total of 3,408 GSRCC patients between 1975 and 2017 were screened from the Surveillance, Epidemiology, and End Results (SEER) database and randomly divided into training and validation cohorts. Univariate and multivariate Cox analyses were conducted to identify independent prognostic factors for the construction of a nomogram. The performance of the model was then assessed by the concordance index (C-index), calibration plot, and area under the receiver operating characteristic curve (AUC). Then, the novel nomogram was further assessed by 64 GSRCC patients from our hospital as the external cohort. RESULTS: We identified age, tumor lymph node metastasis (TNM) staging system, surgery, and chemotherapy as significant independent elements of prognosis. On this basis, a nomogram was constructed, with a C-index of OS in the training and validation cohorts of 0.763 (95% CI: 0.751-0.774) and 0.766 (95% CI: 0.748-0.784) and a C-index of CSS of 0.765 (95% CI: 0.753-0.777) and 0.773 (95% CI: 0.755-0.791), respectively. The AUCs of the nomogram for predicting 2- and 5-year OS were 0.848 and 0.885, respectively, and those for predicting CSS were 0.854 and 0.899, respectively, demonstrating the excellent predictive value of the constructed nomogram compared to the traditional AJCC staging system. Similar results were also observed in both the internal and external validation sets. CONCLUSION: The nomogram provided an accurate tool to predict OS and CSS in patients with GSRCC, which can assist clinicians in making predictions about individual patient survival.

Theoretical investigation of the reaction mechanism of THP oxidative rearrangement catalysed by BBOX.

γ-Butyrobetaine hydroxylase (BBOX) is a non-heme FeII/2OG dependent enzyme that is able to perform two different kinds of catalytic reactions on 3-(2,2,2-trimethylhydrazinium) propionate (THP) to produce distinct catalytic products. Although the structure of BBOX complexed with THP has been resolved, the details of its catalytic mechanism are still elusive. In this study, by employing molecular dynamics (MD) simulations and density functional theory (DFT) calculations, the mechanism of the THP oxidative rearrangement reactions catalysed by BBOX was investigated. Our calculations revealed how the enzyme undergoes a conformational conversion to initiate the catalytic reactions. In the first catalytic step, BBOX performs hydrogen abstraction from the substrate THP as a common non-heme iron enzyme. Due to the structure of the substrate stabilizing the radical species and polarizing the adjacent N-N bond, in the next step, THP takes the pathway for N-N bond homolysis but not regular hydroxyl rebounding. The cleaved ammonium radical could either react with the hydroxyl group on the iron centre of the enzyme or recombine with the other cleaved fragment of the substrate to generate the rearranged product. This study revealed the catalytic mechanism of BBOX, detailing how the enzyme and the substrate regulated the hydroxyl rebound process to generate various products.

Rice yield benefits from historical climate warming to be negated by extreme heat in Northeast China.

Rice is currently benefiting from climate warming in Northeast China, but whether such positive effect will continue in the future remains unknown. Here, we evaluate the impacts of individual and combined climate variables on rice yields in Northeast China during 1980-2015. Results show that there is 10% yield increase induced by climate change in Northeast China since 1980. At present, the reduced chilling results in 5.4% yield increase (approximately 28,000 tons) and the higher growing degree-day contributes to 4.6% yield increase (approximately 24,000 tons), while the high-temperature extreme reduced yield by 0.054% (approximately 280 tons). However, with continuous warming, the harmful impact of such high-temperature extreme will outweigh other positive climate effects when the temperature increases by 3.36 °C. Therefore, high-temperature extremes cannot be ignored despite their influence on rice yield being quite limited at present in Northeast China. Climate change mitigation and heat tolerance breeding are thus necessary for rice production in Northeast China.

Theoretical investigation on the reaction mechanism of UTP cyclohydrolase.

Nucleoside triphosphate cyclohydrolase (UrcA) is a critical enzyme of the uracil catabolism pathway that catalyses the two-step hydrolysis of uridine triphosphate (UTP). Although the recently resolved X-ray structure of UrcA in complex with substrate analogue dUTP provided insights into the structural characteristics of the enzyme, the detailed catalytic mechanism, including how the reaction intermediate accomplishes conformational conversion in the active centre, remains unclear. In this study, extensive DFT calculations and MD simulations were performed to investigate the catalytic reaction process of UrcA. This study shows that the first hydrolytic reactions in UrcA follow a three-step mechanism, while the second hydrolytic reaction follows a two-step mechanism. Glu392 plays a critical role in deprotonating the lytic water in both hydrolytic reactions. The rate-limiting step of the first hydrolytic reaction lies in the cleavage of the uracil ring, in which an extraneous water molecule bridges the proton transfer from C6-OH to N1 to enable the reaction to go through a six-membered transition state with relatively low steric tension. In the second hydrolytic reaction, Glu392 abstracts protons from the lytic water and directly transfers them to the nitrogen atom of the cleaved C4-N3 bond so that the hydrolytic reaction is no longer rate-limited by the C-N bond cleavage step. MD simulations show that the reaction intermediate experiences spontaneous conformation overturn in the active site of UrcA under the assistance of the hydrogen bond interaction from Tyr307 to place its C4-N3 bond alongside the Zn2+ centre of the enzyme to trigger the second hydrolytic reaction.

Reorienting Mechanism of Harderoheme in Coproheme Decarboxylase-A Computational Study.

Coproheme decarboxylase (ChdC) is an important enzyme in the coproporphyrin-dependent pathway (CPD) of Gram-positive bacteria that decarboxylates coproheme on two propionates at position 2 and position 4 sequentially to generate heme b by using H2O2 as an oxidant. This work focused on the ChdC from Geobacillus stearothermophilus (GsChdC) to elucidate the mechanism of its sequential two-step decarboxylation of coproheme. The models of GsChdC in a complex with substrate and reaction intermediate were built to investigate the reorienting mechanism of harderoheme. Targeted molecular dynamics simulations on these models validated that harderoheme is able to rotate in the active site of GsChdC with a 19.06-kcal·mol-1 energy barrier after the first step of decarboxylation to bring the propionate at position 4 in proximity of Tyr145 to continue the second decarboxylation step. The harderoheme rotation mechanism is confirmed to be much easier than the release-rebinding mechanism. In the active site of GsChdC, Trp157 and Trp198 comprise a "gate" construction to regulate the clockwise rotation of the harderoheme. Lys149 plays a critical role in the rotation mechanism, which not only keeps the Trp157-Trp198 "gate" from being closed but also guides the propionate at position 4 through the gap between Trp157 and Trp198 through a salt bridge interaction.

Enhanced antitumor activity of inulin-capped Se nanoparticles synthesized using Jerusalem artichoke tubers.

An inulin polysaccharide with a molecular weight of ~ 2600 Da was derived from Jerusalem artichoke tubers and referred to as "JAP". Previous studies have shown that inulin can improve glucose tolerance and the liver lipid profile; however, its antitumor activity remains to be examined in detail. Therefore, to investigate the possible improvement of the antitumor activity of JAP, a novel nanostructured biomaterial was constructed by capping Se nanoparticles with JAP using sodium selenite, via a redox reaction with ascorbic acid, and referred to as "JAP-SeNPs". Transmission electron microscopy revealed that the average diameter of JAP-SeNPs is ~ 50 nm, and the C:Se mass ratio in JAP-SeNPs was found to be 15.4:1 by energy-dispersive X-ray spectroscopy. The well-dispersed JAP-SeNPs exhibited a significant in vitro antiproliferative effect on mouse forestomach carcinoma cells at a concentration of 400 µg/mL when incubated for 48 h, with an inhibition rate of 41.5%. Moreover, 38.9% of later apoptotic cells were observed. These results reveal that a combination of Se and JAP can effectively enhance the antitumor activity of polysaccharides obtained from Jerusalem artichoke tubers.

Interactive Regulation between Aliphatic Hydroxylation and Aromatic Hydroxylation of Thaxtomin D in TxtC: A Theoretical Investigation.

TxtC is an unusual bifunctional cytochrome P450 that is able to perform sequential aliphatic and aromatic hydroxylation of the diketopiperazine substrate thaxtomin D in two distinct sites to produce thaxtomin A. Though the X-ray structure of TxtC complexed with thaxtomin D revealed a binding mode for its aromatic hydroxylation, the preferential hydroxylation site is aliphatic C14. It is thus intriguing to unravel how TxtC accomplishes such two-step catalytic hydroxylation on distinct aliphatic and aromatic carbons and why the aliphatic site is preferred in the hydroxylation step. In this work, by employing molecular docking and molecular dynamics (MD) simulation, we revealed that thaxtomin D could adopt two different conformations in the TxtC active site, which were equal in energy with either the aromatic C20-H or aliphatic C14-H pointing toward the active Cpd I oxyferryl moiety. Further ONIOM calculations indicated that the energy barrier for the rate-limiting hydroxylation step on the aliphatic C14 site was 9.6 kcal/mol more favorable than that on the aromatic C20 site. The hydroxyl group on the monohydroxylated intermediate thaxtomin B C14 site formed hydrogen bonds with Ser280 and Thr385, which induced the l-Phe moiety to rotate around the Cß-Cγ bond of the 4-nitrotryptophan moiety. Thus, it adopted an energetically favorable conformation with aromatic C20 adjacent to the oxyferryl moiety. In addition, the hydroxyl group induced solvent water molecules to enter the active site, which propelled thaxtomin B toward the heme plane and resulted in heme distortion. Based on this geometrical layout, the rate-limiting aromatic hydroxylation energy barrier decreased to 15.4 kcal/mol, which was comparable to that of the thaxtomin D aliphatic hydroxylation process. Our calculations indicated that heme distortion lowered the energy level of the lowest Cpd I α-vacant orbital, which promoted electron transfer in the rate-limiting thaxtomin B aromatic hydroxylation step in TxtC.

N-Nitrosation Mechanism Catalyzed by Non-heme Iron-Containing Enzyme SznF Involving Intramolecular Oxidative Rearrangement.

The non-heme iron-dependent enzyme SznF catalyzes a critical N-nitrosation step during the N-nitrosourea pharmacophore biosynthesis in streptozotocin. The intramolecular oxidative rearrangement process is known to proceed at the FeII-containing active site in the cupin domain of SznF, but its mechanism has not been elucidated to date. In this study, based on the density functional theory calculations, a unique mechanism was proposed for the N-nitrosation reaction catalyzed by SznF in which a four-electron oxidation process is accomplished through a series of complicated electron transferring between the iron center and substrate to bypass the high-valent FeIV═O species. In the catalytic reaction pathway, the O2 binds to the iron center and attacks on the substrate to form the peroxo bridge intermediate by obtaining two electrons from the substrate exclusively. Then, instead of cleaving the peroxo bridge, the Cε-Nω bond of the substrate is homolytically cleaved first to form a carbocation intermediate, which polarizes the peroxo bridge and promotes its heterolysis. After O-O bond cleavage, the following reaction steps proceed effortlessly so that the N-nitrosation is accomplished without NO exchange among reaction species.

Microscale optoelectronic infrared-to-visible upconversion devices and their use as injectable light sources.

Optical upconversion that converts infrared light into visible light is of significant interest for broad applications in biomedicine, imaging, and displays. Conventional upconversion materials rely on nonlinear light-matter interactions, exhibit incidence-dependent efficiencies, and require high-power excitation. We report an infrared-to-visible upconversion strategy based on fully integrated microscale optoelectronic devices. These thin-film, ultraminiaturized devices realize near-infrared (∼810 nm) to visible [630 nm (red) or 590 nm (yellow)] upconversion that is linearly dependent on incoherent, low-power excitation, with a quantum yield of ∼1.5%. Additional features of this upconversion design include broadband absorption, wide-emission spectral tunability, and fast dynamics. Encapsulated, freestanding devices are transferred onto heterogeneous substrates and show desirable biocompatibilities within biological fluids and tissues. These microscale devices are implanted in behaving animals, with in vitro and in vivo experiments demonstrating their utility for optogenetic neuromodulation. This approach provides a versatile route to achieve upconversion throughout the entire visible spectral range at lower power and higher efficiency than has previously been possible.

Theoretical Studies on the Binding Mode and Reaction Mechanism of TLP Hydrolase kpHIUH.

In this work, we have investigated the binding conformations of the substrate in the active site of 5-HIU hydrolase kpHIUH and its catalytic hydrolysis mechanism. Docking calculations revealed that the substrate adopts a conformation in the active site with its molecular plane laying parallel to the binding interface of the protein dimer of kpHIUH, in which His7 and His92 are located adjacent to the hydrolysis site C6 and have hydrogen bond interactions with the lytic water. Based on this binding conformation, density functional theory calculations indicated that the optimal catalytic mechanism consists of two stages: (1) the lytic water molecule is deprotonated by His92 and carries out nucleophilic attack on C6=O of 5-HIU, resulting in an oxyanion intermediate; (2) by accepting a proton transferred from His92, C6-N5 bond is cleaved to completes the catalytic cycle. The roles of His7, His92, Ser108 and Arg49 in the catalytic reaction were revealed and discussed in detail.

ONIOM investigations of the heme degradation mechanism by MhuD: the critical function of heme ruffling.

The oxygen-dependent heme utilization degrading enzyme in Mycobacterium tuberculosis (MhuD) uniquely integrates monooxygenase and dioxygenase functions in a single active site. It cannot convert heme to biliverdin as canonical heme oxygenases but generates mycobilin without releasing carbon monoxide. Herein, by employing ONIOM calculations, we investigated the heme degradation mechanism of MhuD. Our calculations revealed that MhuD firstly follows a canonical monooxygenation mechanism to hydroxylate heme on the δ-meso carbon guided by the asparagine residue Asn7, which experiences a 21.2 kcal mol-1 energy barrier in the O-O cleavage rate-limiting step during the conversion process from ferric heme-hydroperoxy species to mycobilin. In the second degradation step, the ruffled conformation of oxoheme (oxoheme is the ferrous π radical complex formed by hydroxyheme experiencing deprotonation in the hydroxyl group and intramolecular electron transfer) imposed by the hydrophobic environment of the enzyme not only inhibits the continuing conversion of oxoheme to biliverdin but also endows the meso-carbons with radical characteristics, which turns the second degradation step to a dioxygenation reaction with 20.4 kcal mol-1 energy barrier. We further analysed the electronic structure change along the reaction process. Our calculation discovered that the ruffled structure of oxoheme is critical to the regiospecificity and even atom location selectivity, as well as the reaction mechanism of the degradation process.

Ruffling drives coproheme decarboxylation by facilitating PCET: a theoretical investigation of ChdC.

Coproheme decarboxylase (ChdC) is an essential enzyme in the coproporphyrin-dependent heme synthesis pathway, which catalyzes oxidative decarboxylation of coproheme at the positions p2 and p4 to generate heme b under the action of hydrogen peroxide. A mysterious characteristic of catalytic mechanism of ChdC is that both of the two decarboxylation sites are located remotely from the iron center of coproheme, which binds with hydrogen peroxide. By using density functional theory calculations, we have studied the coproheme decarboxylation mechanism of ChdC in detail. The calculation results show that in the first step of the catalytic reaction, H2O2 homolysis takes place synergistically with the proton coupled electron transfer process of a tyrosine (Tyr145) residing near p2 propionate. The produced reactive Tyr radical then abstracts a hydrogen atom from the ß carbon of the p2 propionate side chain, which is the rate-limiting step of the whole reaction with a 19.16 kcal mol-1 energy barrier. Finally, through intramolecular electron and proton rearrangement of coproporphyrin, decarboxylation of p2 propionate is accomplished. Our study revealed that the ruffled conformation of coproheme in ChdC is an important structural factor, which facilitates the decarboxylation reaction. We also found that the hydrogen bond chain located below the coproheme ring plays a role to regulate the PCET process of Tyr145. In addition, molecular dynamics simulations discovered that Lys149 is responsible for stabilizing the harderoheme III and positioning the second decarboxylation site p4 to the catalytic Tyr145 site in the decarboxylation reaction of the p4 site.

Theoretical study on the catalytic mechanism of human deoxyhypusine hydroxylase.

Deoxyhypusine hydroxylase is a critical enzyme for hypusination of eukaryotic translation initiation factor 5A (eIF5A). Human deoxyhypusine hydroxylase (hDOHH) has a nonheme diiron active site that resembles both in structure and function of those found in methane and toluene monooxygenases, bacterial and mammalian ribonucleotide reductases, and stearoyl acyl carrier protein Δ9-desaturase from plants. However, the detailed catalytic mechanism of hDOHH is still unclear. In this work, extensive DFT calculations reveal that the catalytic mechanism of hDOHH consists of four consecutive steps: (1) peroxo isomerization triggered by substrate binding; (2) rate-determining O-O bond cleavage and formation of the [FeIV2(µ-O)2]4+ compound; (3) H atom abstraction from the substrate; and (4) OH rebound to the substrate. This work not only rationalizes the exceptional stability of the diiron(iii)-peroxo complex in hDOHH, but also confirms that hDOHH uses a diamond shape [FeIV2(µ-O)2]4+ core to complete crucial H atom abstraction from the substrate. Our DFT calculations exclude the reaction pathway of hDOHH to use diiron(iii)-peroxo species to directly react with the substrate.

A Theoretical Study of the Recently Suggested MnVII Mechanism for O-O Bond Formation in Photosystem II.

The mechanism for water oxidation in photosystem II has been a major topic for several decades. The active catalyst has four manganese atoms connected by bridging oxo bonds, in a complex termed the oxygen-evolving complex (OEC), which also includes a calcium atom. The O-O bond of oxygen is formed after absorption of four photons in a state of the OEC termed S4. There has been essential consensus that in the S4 state, all manganese atoms are in the Mn(IV) oxidation state. However, recently there has been a suggestion that one of the atoms is in the Mn(VII) state. In the present computational study, the feasibility of that proposal has been investigated. It is here shown that the mechanism involving Mn(VII) has a much higher barrier for forming O2 than the previous proposal with four Mn(IV) atoms.

Impacts of the north and tropical Atlantic Ocean on the Antarctic Peninsula and sea ice.

In recent decades, Antarctica has experienced pronounced climate changes. The Antarctic Peninsula exhibited the strongest warming of any region on the planet, causing rapid changes in land ice. Additionally, in contrast to the sea-ice decline over the Arctic, Antarctic sea ice has not declined, but has instead undergone a perplexing redistribution. Antarctic climate is influenced by, among other factors, changes in radiative forcing and remote Pacific climate variability, but none explains the observed Antarctic Peninsula warming or the sea-ice redistribution in austral winter. However, in the north and tropical Atlantic Ocean, the Atlantic Multidecadal Oscillation (a leading mode of sea surface temperature variability) has been overlooked in this context. Here we show that sea surface warming related to the Atlantic Multidecadal Oscillation reduces the surface pressure in the Amundsen Sea and contributes to the observed dipole-like sea-ice redistribution between the Ross and Amundsen-Bellingshausen-Weddell seas and to the Antarctic Peninsula warming. Support for these findings comes from analysis of observational and reanalysis data, and independently from both comprehensive and idealized atmospheric model simulations. We suggest that the north and tropical Atlantic is important for projections of future climate change in Antarctica, and has the potential to affect the global thermohaline circulation and sea-level change.

Theoretical study of the mechanism of the manganese catalase KatB.

The mechanism of the H2O2 disproportionation catalyzed by the manganese catalase (MnCat) KatB was studied using the hybrid density functional theory B3LYP and the quantum chemical cluster approach. Compared to the previous mechanistic study at the molecular level for the Thermus thermophilus MnCat (TTC), more modern methodology was used and larger models of increasing sizes were employed with the help of the high-resolution X-ray structure. In the reaction pathway suggested for KatB using the Large chemical model, the O-O homolysis of the first substrate H2O2 occurs through a µ-η1:η1 coordination mode and requires a barrier of 10.9 kcal/mol. In the intermediate state of the bond cleavage, two hydroxides form as terminal ligands of the dimanganese cluster at the Mn2(III,III) oxidation state. One of the two Mn(III)-OH- moieties and a second-sphere tyrosine stabilize the second substrate H2O2 in the second-sphere of the active site via hydrogen bonding interactions. The H2O2, unbound to the metals, is first oxidized into HO2· through a proton-coupled electron transfer (PCET) step with a barrier of 9.5 kcal/mol. After the system switches to the triplet surface, the uncoordinated HO2· replaces the product water terminally bound to the Mn(II) and is then oxidized into O2 spontaneously. Transition states with structural similarities to those obtained for TTC, where µ-η2-OH-/O2- groups play important roles, were found to be higher in energy.

Carbon Nanoparticles Inhibit Α-Glucosidase Activity and Induce a Hypoglycemic Effect in Diabetic Mice.

New, improved therapies to reduce blood glucose are required for treating diabetes mellitus (DM). Here, we investigated the use of a new nanomaterial candidate for DM treatment, carbon nanoparticles (CNPs). CNPs were prepared by carbonization using a polysaccharide from Arctium lappa L. root as the carbon source. The chemical structure and morphology of the CNPs were characterized using Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, elemental analysis, and transmission electron microscopy. CNPs were spherical, 10-20 nm in size, consisting of C, H, O, and N, and featuring various functional groups, including C=O, C=C, C-O, and C-N. In vitro, the as-prepared CNPs could inhibit α-glucosidase with an IC50 value of 0.5677 mg/mL, which is close to that of the reference drug acarbose. Moreover, in vivo hypoglycemic assays revealed that the CNPs significantly reduced fasting blood-glucose levels in mice with diabetes induced by high-fat diet and streptozocin, lowering blood glucose after intragastric administration for 42 days. To the best of our knowledge, this is the first report of CNPs exhibiting α-glucosidase inhibition and a hypoglycemic effect in diabetic mice. These findings suggest the therapeutic potential of CNPs for diabetes.

Simulation of the isotropic EXAFS spectra for the S2 and S3 structures of the oxygen evolving complex in photosystem II.

Most of the main features of water oxidation in photosystem II are now well understood, including the mechanism for O-O bond formation. For the intermediate S2 and S3 structures there is also nearly complete agreement between quantum chemical modeling and experiments. Given the present high degree of consensus for these structures, it is of high interest to go back to previous suggestions concerning what happens in the S2-S3 transition. Analyses of extended X-ray adsorption fine structure (EXAFS) experiments have indicated relatively large structural changes in this transition, with changes of distances sometimes larger than 0.3 Å and a change of topology. In contrast, our previous density functional theory (DFT)(B3LYP) calculations on a cluster model showed very small changes, less than 0.1 Å. It is here found that the DFT structures are also consistent with the EXAFS spectra for the S2 and S3 states within normal errors of DFT. The analysis suggests that there are severe problems in interpreting EXAFS spectra for these complicated systems.

Cluster size convergence for the energetics of the oxygen evolving complex in PSII.

Density functional theory calculations have been made to investigate the stability of the energetics for the oxygen evolving complex of photosystem II. Results published elsewhere have given excellent agreement with experiments for both energetics and structures, where many of the experimental results were obtained several years after the calculations were done. The computational results were obtained after a careful extension from small models to a size of about 200 atoms, where stability of the results was demonstrated. However, recently results were published by Isobe et al., suggesting that very different results could be obtained if the model was extended from 200 to 340 atoms. The present study aims at understanding where this difference comes from. © 2017 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.