Coevolutionary Information Captures Catalytic Functions and Reveals Divergent Roles of Terpene Synthase Interdomain Connections.

Inferring the historical and biophysical causes of diversity within protein families is a complex puzzle. A key to unraveling this problem is characterizing the rugged topography of sequence-function adaptive landscapes. Using biochemical data from a 29 = 512 combinatorial library of tobacco 5-epi-aristolochene synthase (TEAS) mutants engineered to make the native major product of Egyptian henbane premnaspirodiene synthase (HPS) and a complementary 512 mutant HPS library, we address the question of how product specificity is controlled. These data sets reveal that HPS is far more robust and resistant to mutations than TEAS, where most mutants are promiscuous. We also combine experimental data with a sequence Potts Hamiltonian model and direct coupling analysis to quantify mutant fitness. Our results demonstrate that the Hamiltonian captures variation in product outputs across both libraries, clusters native family members based on their substrate specificities, and exposes the divergent catalytic roles of couplings between the catalytic and noncatalytic domains of TEAS versus HPS. Specifically, we found that the role of the interdomain connectivities in specifying product output is more important in TEAS than connectivities within the catalytic domain. Despite being 75% identical, this property is not shared by HPS, where connectivities within the catalytic domain are more important for specificity. By solving the X-ray crystal structure of HPS, we assessed structural bases for their interdomain network differences. Last, we calculate the product profile Shannon entropies of the two libraries, which showcases that site-site connectivities also play divergent roles in catalytic accuracy.

Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes.

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.

Overcoming resolution attenuation during tilted cryo-EM data collection.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

Prevalence, correlates and explanatory models of cognitive deficits in patients with schizophrenia-A cross sectional study.

Background: Schizophrenia may cause significant impairment in social and economic aspects of a patient's life. Current evidence suggests that cognitive deficits may affect the functioning of a person with schizophrenia more than positive or negative symptoms. There is a lack of literature on explanatory models of cognitive deficits in schizophrenia that can influence help-seeking behavior. Objectives: This study aimed to estimate the prevalence of cognitive deficits and assess their relationship with socio-demographic and clinical characteristics among patients with schizophrenia. We also planned to explore the explanatory models of cognitive deficits in these patients. Methods: Consecutive outpatients with schizophrenia who met eligibility criteria were recruited after obtaining informed consent. The Addenbrooke's Cognitive Examination Tamil version (ACE III) and Observable Social Cognition - A Rating Scale (OSCARS) and Positive and Negative Symptom Scale (PANSS) were used to assess cognitive functioning and symptom profile, respectively. Beliefs about illness were recorded using the modified Short Explanatory Model Interview (SEMI). Socio-demographic and treatment-related details were collected with a structured proforma. Statistical analysis was done using SPSS for Windows (version 16.0.1). Results: One hundred and forty patients participated in the study. The prevalence of cognitive deficits was 75.7% using ACE-III scores, 19.3% on OSCARS, and 40% based on subjective reports. Though the majority (81.4%) of patients reported a medical explanatory model for cognitive impairment, a significant number of them (70.7%) also held non-medical models simultaneously. Conclusion: Cognitive deficits are prevalent in the majority of patients with schizophrenia. Poor test performance on cognitive testing was observed in those with a significant family history. Multiple contradictory explanatory models for the causation of cognitive deficits were reported.

Cardiac Autonomic Activity, Personality Traits, and Academic Performance in First-Year Medical Students: A Gender-Specific Relation.

Background It is not always the sincere or hardworking or intelligent student that gets the highest grades. Exploring unknown dimensions that may distinguish academic performance in adolescents/youth migrating from a high school study environment to that of a professional school and in a learning environment without parental supervision for the first time remains important. We hypothesized that cardiac autonomic activity influenced by cognitive domain factors and emotions would predict academic success in them. Further, we investigated which of their personality traits related to academic performance. Exploratory gender-based analysis was included. Methods A prospective cohort study measured first-year medical students' resting heart rate, heart rate variability (HRV), and personality traits (from the self-reported NEO Five-Factor Inventory-3). Spearman's correlation coefficient tested the correlation between the year-end final aggregate marks and assessed parameters, including subgroup analysis based on gender. Regression analyses of variables with academic marks were performed in the entire cohort. Results The aggregate marks of 81 volunteering students (Mage = 18.7, SD = 0.8 years; 42 females, 39 males) as a cohort did not correlate with their resting heart rate or HRV indices. Subgroup analysis revealed a positive correlation between marks and high-frequency power (r = 0.33, p= 0.03) and total power (r = 0.37, p= 0.02) of HRV in females. The marks positively correlated with the personality conscientiousness score (r = 0.32, p= 0.04) and extraversion score (r = 0.34, p= 0.03) in females. Multivariable regression analysis in the entire cohort revealed no significant interactions. Conclusion Academic performance was significantly related to cardiac autonomic modulation and personality traits of conscientiousness and extraversion in female but not male first-year medical students. These results indicate a gender-specific difference in the relation between scholastic performance and HRV in adolescents/youth transiting from high school to professional schools and entering a study environment without parental supervision for the first time. Further our data expands the knowledge base of educational psychology among them.

Masked cerulenin enables a dual-site selective protein crosslink.

Protein-reactive natural products such as the fungal metabolite cerulenin are recognized for their value as therapeutic candidates, due to their ability to selectively react with catalytic residues within a protein active site or a complex of protein domains. Here, we explore the development of fatty-acid and polyketide-synthase probes by synthetically modulating cerulenin's functional moieties. Using a mechanism-based approach, we reveal unique reactivity within cerulenin and adapt it for fluorescent labeling and crosslinking of fatty-acid and iterative type-I polyketide synthases. We also describe two new classes of silylcyanohydrin and silylhemiaminal masked crosslinking probes that serve as new tools for activity and structure studies of these biosynthetic pathways.

Biochemistry and Protein Interactions of the CYREN Microprotein.

Over the past decade, advances in genomics have identified thousands of additional protein-coding small open reading frames (smORFs) missed by traditional gene finding approaches. These smORFs encode peptides and small proteins, commonly termed micropeptides or microproteins. Several of these newly discovered microproteins have biological functions and operate through interactions with proteins and protein complexes within the cell. CYREN1 is a characterized microprotein that regulates double-strand break repair in mammalian cells through interaction with Ku70/80 heterodimer. Ku70/80 binds to and stabilizes double-strand breaks and recruits the machinery needed for nonhomologous end join repair. In this study, we examined the biochemical properties of CYREN1 to better understand and explain its cellular protein interactions. Our findings support that CYREN1 is an intrinsically disordered microprotein and this disordered structure allows it to enriches several proteins, including a newly discovered interaction with SF3B1 via a distinct short linear motif (SLiMs) on CYREN1. Since many microproteins are predicted to be disordered, CYREN1 is an exemplar of how microproteins interact with other proteins and reveals an unknown scaffolding function of this microprotein that may link NHEJ and splicing.

Overcoming Resolution Attenuation During Tilted Cryo-EM Data Collection.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle for dataset acquisition. These data reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

Role of anchoring groups on the light harvesting and optoelectronic properties of triphenylamine derivatives: insights from theory.

BACKGROUND: In the present work, DFT and time-dependent DFT calculations were performed to investigate the role of anchoring groups on the photophysical properties and reveal structure-property correlations of triphenylamine (TPA) derivatives. The selected anchoring groups are tetrazole, acrylamide, hydantoin, and rhodanine. RESULTS: Our results show that the different anchoring groups employed alter the planarity, intramolecular charge transfer properties, and HOMO-LUMO gap and hence influence the optoelectronic properties of the dyes. Although all molecules fulfill the basic requirements with suitable energy levels, band gap, absorption, and charge transfer properties, the dye with rhodanine acceptor (TPA4) was the most promising candidate due to its lowest HOMO-LUMO gap, red-shifted highest λmax absorption value, better ICT pattern, low total reorganization energy, and good electron injection properties. Overall, it is anticipated that the results of this investigation will point to new avenues for the experimental fabrication of remarkably effective metal-free organic dyes for solar cell applications.

Mechanism-based cross-linking probes capture the Escherichia coli ketosynthase FabB in conformationally distinct catalytic states.

Ketosynthases (KSs) catalyse essential carbon-carbon bond-forming reactions in fatty-acid biosynthesis using a two-step, ping-pong reaction mechanism. In Escherichia coli, there are two homodimeric elongating KSs, FabB and FabF, which possess overlapping substrate selectivity. However, FabB is essential for the biosynthesis of the unsaturated fatty acids (UFAs) required for cell survival in the absence of exogenous UFAs. Additionally, FabB has reduced activity towards substrates longer than 12 C atoms, whereas FabF efficiently catalyses the elongation of saturated C14 and unsaturated C16:1 acyl-acyl carrier protein (ACP) complexes. In this study, two cross-linked crystal structures of FabB in complex with ACPs functionalized with long-chain fatty-acid cross-linking probes that approximate catalytic steps were solved. Both homodimeric structures possess asymmetric substrate-binding pockets suggestive of cooperative relationships between the two FabB monomers when engaged with C14 and C16 acyl chains. In addition, these structures capture an unusual rotamer of the active-site gating residue, Phe392, which is potentially representative of the catalytic state prior to substrate release. These structures demonstrate the utility of mechanism-based cross-linking methods to capture and elucidate conformational transitions accompanying KS-mediated catalysis at near-atomic resolution.

Structural Basis of Stereospecific Vanadium-Dependent Haloperoxidase Family Enzymes in Napyradiomycin Biosynthesis.

Vanadium-dependent haloperoxidases (VHPOs) from Streptomyces bacteria differ from their counterparts in fungi, macroalgae, and other bacteria by catalyzing organohalogenating reactions with strict regiochemical and stereochemical control. While this group of enzymes collectively uses hydrogen peroxide to oxidize halides for incorporation into electron-rich organic molecules, the mechanism for the controlled transfer of highly reactive chloronium ions in the biosynthesis of napyradiomycin and merochlorin antibiotics sets the Streptomyces vanadium-dependent chloroperoxidases apart. Here we report high-resolution crystal structures of two homologous VHPO family members associated with napyradiomycin biosynthesis, NapH1 and NapH3, that catalyze distinctive chemical reactions in the construction of meroterpenoid natural products. The structures, combined with site-directed mutagenesis and intact protein mass spectrometry studies, afforded a mechanistic model for the asymmetric alkene and arene chlorination reactions catalyzed by NapH1 and the isomerase activity catalyzed by NapH3. A key lysine residue in NapH1 situated between the coordinated vanadate and the putative substrate binding pocket was shown to be essential for catalysis. This observation suggested the involvement of the ε-NH2, possibly through formation of a transient chloramine, as the chlorinating species much as proposed in structurally distinct flavin-dependent halogenases. Unexpectedly, NapH3 is modified post-translationally by phosphorylation of an active site His (τ-pHis) consistent with its repurposed halogenation-independent, α-hydroxyketone isomerase activity. These structural studies deepen our understanding of the mechanistic underpinnings of VHPO enzymes and their evolution as enantioselective biocatalysts.

Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon.

Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure determination by SPA relies upon obtaining multiple distinct views of a macromolecular object vitrified within a thin layer of ice. Ideally, a collection of uniformly distributed random projection orientations would amount to all possible views of the object, giving rise to reconstructions characterized by isotropic directional resolution. However, in reality, many samples suffer from preferentially oriented particles adhering to the air-water interface. This leads to non-uniform angular orientation distributions in the dataset and inhomogeneous Fourier-space sampling in the reconstruction, translating into maps characterized by anisotropic resolution. Tilting the specimen stage provides a generalizable solution to overcoming resolution anisotropy by virtue of improving the uniformity of orientation distributions, and thus the isotropy of Fourier space sampling. The present protocol describes a tilted-stage automated data collection strategy using Leginon, a software for automated image acquisition. The procedure is simple to implement, does not require any additional equipment or software, and is compatible with most standard transmission electron microscopes (TEMs) used for imaging biological macromolecules.

A Simplified Measurement Configuration for Evaluation of Relative Permittivity Using a Microstrip Ring Resonator with a Variational Method-Based Algorithm.

In this paper, we present a simple yet efficient method for determination of the relative permittivity of thin dielectric materials. An analysis that led to definition of the proper size and placement of a sample under test (SUT) on the surface of a microstrip ring resonator (MRR) was presented based on the full-wave simulations and measurements on benchmark materials. For completeness, the paper includes short descriptions of the design of an MRR and the variational method-based algorithm that processes the measured values. The efficiency of the proposed method is demonstrated on 12 SUT materials of different thicknesses and permittivity values, and the accuracy between 0% and 10% of the relative error was achieved for all SUTs thinner than 2 mm.

Nature and Correlates of Executive Dysfunction in Schizophrenia: An Exploratory Study.

BACKGROUND: Executive function (EF) impairment has been demonstrated in patients with schizophrenia. This study attempted to examine the clinical and demographic correlates associated with the different components of EF in these patients using a comprehensive battery of neuropsychological tests. MATERIALS AND METHODS: Consecutive inpatients with schizophrenia in remission were recruited. The following instruments were administered: (a) Positive and Negative Syndrome Scale (PANSS), (b)World Health Organization Disability Assessment Schedule 2.0, (c) Tower of London, (d) Stroop Test, (e) Controlled Oral Word Association Test, (f) Animal Names Test, and (g) Verbal N-Back Test. Sociodemographic and clinical details were also recorded. Data was analyzed using standard bivariate and multivariate statistics. RESULTS: A total of 50 patients were recruited. The mean age of the population was 30 years (standard deviation [SD]: 7.74). The majority were male, literate, single, from a rural background, from a middle socioeconomic background, and unemployed. The mean dose of antipsychotic medication was 618.57 mg (SD: 282.08) of chlorpromazine equivalents per day. Impairment was found in the different sub-components of EF. On multivariate analysis, factors significantly associated with executive dysfunction were lower education, unemployment, lower income, positive PANSS score, higher antipsychotic dose, and history of treatment with electroconvulsive therapy. CONCLUSION: EFs encompass a wide range of cognitive processes that influence an individual's ability to adapt and function in the society. These are often impaired in patients with schizophrenia. Clinicians need to be aware of these deficits and factors associated with them, to plan appropriate and effective remedial measures.

Breast ultrasound tumor image classification using image decomposition and fusion based on adaptive multi-model spatial feature fusion.

BACKGROUND AND OBJECTIVE: Breast cancer is a fatal threat to the health of women. Ultrasonography is a common method for the detection of breast cancer. Computer-aided diagnosis of breast ultrasound images can help doctors in diagnosing benign and malignant lesions. In this paper, by combining image decomposition and fusion techniques with adaptive spatial feature fusion technology, a reliable classification method for breast ultrasound images of tumors is proposed. METHODS: First, fuzzy enhancement and bilateral filtering algorithms are used to process the original breast ultrasound image. Then, various decomposition images representing the clinical characteristics of breast tumors are obtained using the original and mask images. By considering the diversity of the benign and malignant characteristic information represented by each decomposition image, the decomposition images are fused through the RGB channel, and three types of fusion images are generated. Then, from a series of candidate deep learning models, transfer learning is used to select the best model as the base model to extract deep learning features. Finally, while training the classification network, adaptive spatial feature fusion technology is used to train the weight network to complete deep learning feature fusion and classification. RESULTS: In this study, 1328 breast ultrasound images were collected for training and testing. The experimental results show that the values of accuracy, precision, specificity, sensitivity/recall, F1 score, and area under the curve of the proposed method were 0.9548, 0.9811, 0.9833, 0.9392, 0.9571, and 0.9883, respectively. CONCLUSION: Our research can automate breast cancer detection and has strong clinical utility. When compared to previous methods, our proposed method is expected to be more effective while assisting doctors in diagnosing breast ultrasound images.

Automatic segmentation for ultrasound image of carotid intimal-media based on improved superpixel generation algorithm and fractal theory.

OBJECTIVE: Carotid atherosclerosis (CAS) is the main reason leading to cardiovascular conditions such as coronary heart disease and cerebrovascular diseases. In the carotid ultrasound images, the carotid intima-media structure can be observed in an annular narrow strip, which its inner contour corresponds to the carotid intima, and the outer contour corresponds to the carotid extima. With the development of carotid atherosclerosis, the carotid intima-media will gradually thicken. Therefore, doctors can observe the carotid intima-media so as to obtain the pathological changes of the internal structure of the patient's carotid arteries. However, due to the presence of artifacts and noises the quality of the ultrasound images are degraded, making it difficult to obtain accurate carotid intima-media structures. This article presents a novel self-adaptive method to enable obtaining the carotid intima-media through carotid intima/extima segmentation. METHOD: After preprocessing the ultrasound images by homomorphic filtering and median filtering, we propose an improved superpixel generation algorithm that employs the fusion of gray-level and luminosity-based information to decompose the image into numerous superpixels and later presents the carotid intima. Meanwhile, based on the features of the carotid artery, the initial position of the carotid extima is located by the normalized cut algorithm and later the fractal theory is employed to segment the carotid extima. RESULTS: The proposed method for segmenting carotid intima obtained mean values of the DICE true positive ratio (TPR), false positive ratio (FPR), precision scores of 97.797%, 99.126%, 0.540%, 97.202%, respectively. Further from the segmentation method of the carotid extima the performance measures such as mean DICE, TPR, accuracy, F-score obtained are 95.00%, 92.265%, 97.689%, 94.997%, respectively. CONCLUSION: Comparing with traditional methods, the proposed method performed better. The experimental results indicated that the proposed method obtained the carotid intima-media both automatically and accurately thus effectively assist doctors in the diagnosis of CAS.

Structure and mechanistic analyses of the gating mechanism of elongating ketosynthases.

Ketosynthases (KSs) catalyze carbon-carbon bond forming reactions in fatty acid synthases (FASs) and polyketide synthases (PKSs). KSs utilize a two-step ping pong kinetic mechanism to carry out an overall decarboxylative thio-Claisen condensation that can be separated into the transacylation and condensation reactions. In both steps, an acyl carrier protein (ACP) delivers thioester tethered substrates to the active sites of KSs. Therefore, protein-protein interactions (PPIs) and KS-mediated substrate recognition events are required for catalysis. Recently, crystal structures of Escherichia coli elongating type II FAS KSs, FabF and FabB, in complex with E. coli ACP, AcpP, revealed distinct conformational states of two active site KS loops. These loops were proposed to operate via a gating mechanism to coordinate substrate recognition and delivery followed by catalysis. Here we interrogate this proposed gating mechanism by solving two additional high-resolution structures of substrate engaged AcpP-FabF complexes, one of which provides the missing AcpP-FabF gate-closed conformation. Clearly defined interactions of one of these active site loops with AcpP are present in both the open and closed conformations, suggesting AcpP binding triggers or stabilizes gating transitions, further implicating PPIs in carrier protein-dependent catalysis. We functionally demonstrate the importance of gating in the overall KS condensation reaction and provide experimental evidence for its role in the transacylation reaction. Furthermore, we evaluate the catalytic importance of these loops using alanine scanning mutagenesis and also investigate chimeric FabF constructs carrying elements found in type I PKS KS domains. These findings broaden our understanding of the KS mechanism which advances future engineering efforts in both FASs and evolutionarily related PKSs.

Nipple Localization in Automated Whole Breast Ultrasound Coronal Scans Using Ensemble Learning.

Nipple is a vital landmark in the breast lesion diagnosis. Although there are advanced computer-aided detection (CADe) systems for nipple detection in breast mediolateral oblique (MLO) views of mammogram images, few academic works address the coronal views of breast ultrasound (BUS) images. This paper addresses a novel CADe system to locate the Nipple Shadow Area (NSA) in ultrasound images. Here the Hu Moments and Gray-level Co-occurrence Matrix (GLCM) were calculated through an iterative sliding window for the extraction of shape and texture features. These features are then concatenated and fed into an Artificial Neural Network (ANN) to obtain probable NSA's. Later, contour features, such as shape complexity through fractal dimension, edge distance from the periphery and contour area, were computed and passed into a Support Vector Machine (SVM) to identify the accurate NSA in each case. The coronal plane BUS dataset is built upon our own, which consists of 64 images from 13 patients. The test results show that the proposed CADe system achieves 91.99% accuracy, 97.55% specificity, 82.46% sensitivity and 88% F-score on our dataset.

Plant-based CO2 drawdown and storage as SiC.

Since the 1950's the Earth's natural carbon cycle has not sufficiently sequestrated excess atmospheric CO2 contributed by human activities. CO2 levels rose above 400 ppm in 2013 and are forecasted to exceed 500 ppm by 2070, a level last experienced during the Paleogene period 25-65 MYA. While humanity benefits from the extraction and combustion of carbon from Earth's crust, we have overlooked the impact on global climate change. Here, we present a strategy to mine atmospheric carbon to mitigate CO2 emissions and create economically lucrative green products. We employ an artificial carbon cycle where agricultural plants capture CO2 and the carbon is transformed into silicon carbide (SiC), a valuable commercial material. By carefully quantifying the process we show that 14% of plant-sequestered carbon is stored as SiC and estimate the scale needed for this process to have a global impact.

Interfacial plasticity facilitates high reaction rate of E. coli FAS malonyl-CoA:ACP transacylase, FabD.

Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP-AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the X-ray crystal structure of a type II ACP-AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long-timescale molecular dynamics (MD) simulations. Together, these complementary approaches reveal key catalytic features of FAS ACP-AT interactions. These mechanistic inferences suggest that AcpP adopts multiple, productive conformations at the AT binding interface, allowing the complex to sustain high transacylation rates. Furthermore, MD simulations support rigid body subdomain motions within the FabD structure that may play a key role in AT activity and substrate selectivity.