Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains. Thermobifida fusca NRPS condensation domain bound to the substrate-bearing peptidyl carrier protein (PCP) domain provide insight into the mechanisms of substrate selectivity and engagement within the catalytic pocket.Non-ribosomal peptide synthetases (NRPSs) are multi-modular enzymes assembling complex natural products. Here, the structures of a  The diversity of non-ribosomal peptides is due to the combination of an ability to incorporate an expanded range of monomers compared to ribosomal peptide biosynthesis together with extensive modifications of the peptide both during and after chain assembly2. This is enabled by the modular architecture of NRPSs, which use repeating groups of catalytic domains to install one monomer into the growing peptide  domains, which typically accept two PCP-bound substrates and catalyze peptide bond formation through the attack of the downstream acceptor substrate upon the thioester of the upstream donor substrate  enzyme fold, with key catalytic residues forming a conserved HHxxxDG motif located at the interface between the two subdomains. In addition, it was shown that C domains harbor two catalytic tunnels that lead from the donor-PCP and acceptor-PCP domain binding sites to the active site and represent the access route for the donor and the acceptor substrates, respectively. This architecture has since been confirmed by other structures16. While the conserved central histidine (HHxxxDG) is generally thought to act as the primary catalytic residue that promotes deprotonation of the α-amino group in the acceptor aminoacyl-PCP as it attacks the thioester, this remains a matter of debate3. Perhaps more importantly, the role C domains play in determining NRPS specificity is unclear, in part due to the lack of structural characterization of relevant PCP-bound C domain complexes.Non-ribosomal peptide synthetases (NRPSs) are important biosynthetic enzymes for the production of highly diverse and extensively modified peptideside Fig. . Within ate Fig. 3. The fi19, such efforts have not always been successful. This can be attributed to the complexity of the machinery combined with the necessity for non-native substrates to pass through multiple catalytic domains, each of which imparts a degree of specificity. A pertinent example of this is the recent recognition of the diverse functions of C domains in peptide biosynthesis, extending their well-established role in controlling peptide stereochemistry (working in concert with epimerization (E) domains) to gating in trans modifications, recruiting trans-acting enzymes and performing additional chemical transformations of their substrates during peptide bond formation  are less dynamic and can be more readily studied by methods such as X-ray crystallography28.The structural analysis of key domains, complexes and complete modules has made major contributions to our understanding of how selectivity is achieved by NRPS assembly lines29. However, C domains and C domain-containing complexes have proved more challenging to structurally characterize, with fewer examples reported to date  of the fuscachelin NRPS from the thermophilic organism Thermobifida fusca  using xenon-derivatized crystals (see Methods section). The crystals belonged to the P212121 space group, with the unit cell comprising two highly similar copies of the PCP2-C3 construct  0.74 Å).To elucidate the structure of a C domain with a PCP domain bound in the acceptor site, we screened several systems including a thermophilic example of a PCP2-C3 didomain structure we obtained from these experiments was solved at a resolution of 2.2 Å ; the serine residue that is the site of 4′-phosphopantetheine (PPant) attachment is located at the start of helix 2  1.3 Å, 32% sequence similarity, see Supplementary Table 3 domain of the didomain resembles other members of its class 12 highlights this, with a pronounced difference in displacements observed when comparing the fuscachelin C3 domain and Srf-A domain  on the C3 domain from the second chain in the asymmetric unit. The interface between the PCP2 domain and C3 domain is mostly hydrophobic in nature (537/510 Å2 buried surface area (chain A/B) excluding PPant), with the side chains of V2534, L2515, L2518, F2508, and F2538 of the PCP domain playing a major role in the interaction along with residues A2907, V2908, V2584, L2580, and W2579 of the C domain 12, AB3403 (PDB ID 4ZXH)10; see also Izoré et al.26). These interfaces center around a hydrophobic residue (L2515) immediately following the serine to which the PPant is attached (S2514) and at least one hydrophobic residue ~20 amino-acids after the serine residue. R2906 also plays an important role in positioning the PCP domain via interactions with the phosphate moiety of the PPant arm. In the PCP2-C3 structure, these residues are V2534 and the aliphatic moiety of R2535 that interacts with V2908 of the C domain. The overall orientation of the PCP domain relative to the C domain is similar to what has been observed in the structures of SrfA-C12 and ObiF1 (PDB ID 6N8E)8 7; Supplementary Fig. In the PCP2 domain precedes the C3 domain in the fusachelin NRPS, we had expected that the PCP2 domain would be positioned at the donor-PCP binding site of the C3 domain. We were surprised, therefore, to find that this construct crystallized with the PCP2 domain positioned at the acceptor-PCP binding site of the C3 domain of the second chain in the asymmetric unit  to be highly similar to the PCP2 domain  2 Å; Fig. 3 domain onto the acceptor-PCP binding site of the C3 domain showed that it binds in an almost identical orientation to the PCP2 domain in the structure of the PCP2-C3 didomain  revealed extra density extending from the conserved Ser (S2514) at the beginning of helix 2 of the PCP domain. This serine residue is the target of phosphopantetheinyl transferases, a class of enzymes that attach the essential PPant moiety to PCP domains. Mass spectrometric analysis of the PCP2-C3 didomain construct revealed a 340 Dalton mass increase, consistent with attachment of PPant to S2514, likely installed by the phosphopantetheinyl transferase EntD that phosphopantetheinylates some PCP domains when they are expressed in E. coli. Indeed, expression of the PCP2-C3 didomain construct in an entD mutant31 showed no increase in mass, supporting this hypothesis. Having confirmed the presence of a PPant arm, we modeled this into the electron density observed in our structure. Interestingly, we found that this did not extend into the active site of the C domain, but instead curled back towards the outer surface of the C domain  highlight the intrinsically dynamic nature of the acceptor substrate channel and the important role that R2577 has in modulating its shape and size , resulting in channels that do not block PPant access. Next, we identified all available C-domains from the MiBiG database and computed multiple sequence alignments  in order to discern the typical amino acid found at this position. Interestingly, this Arg residue appears largely conserved in LCL domains (73% harbor an Arg at this position), but is not seen in DCL domains (Gly (80%) or Ala (4%) are found instead  to ensure homogeneous PPant loading. Similar to the wild-type construct, the protein expressed well and crystallized in the same conditions. Crystals diffracted to 2 Å and the structure was phased using molecular replacement with the previous model  1.2 Å compared to wild type). The first noticeable difference is a small rotation of the PCP domain in relation to the C domain and slight alterations in the PCP interacting regions of the C domain, likely attributable to the R2577G mutation allowing the first helix of the C domain to sit deeper in the acceptor channel , due to interactions that interfere with crystallization when the substrate is not bound in the acceptor channel of the C domain. To further explore this mechanism, we next turned to the characterization of the PCP2-C3 construct with an aminoacyl group appended to the PPant thiol group.To verify the role of the R2577 in controlling access to the catalytic channel, we generated the Arg to Gly mutant (R2577G) of the Cite Fig.  in a sim2 domain, we attempted to load the apo-PCP2C3 didomain using Sfp and the CoA thioester of glycine. Crystals in the same space group were readily obtained using the same method as for the two previously described structures. Somewhat surprisingly, in this structure it was clear that the electron density corresponding to the PPant did not sit in the acceptor channel but rather followed the same path as the substrate-free PPant, appearing to be repelled by R2577. However, upon refinement it became clear that the glycyl thioester had been hydrolyzed during crystallization. This forced us to explore alternatives to thioester-tethered amino acids, and we chose to use an analog of the aminoacyl-CoA with a thioether, hence removing the reactive carbonyl that makes the thioester susceptible to nucleophilic attack. This results in a non-hydrolyzable substrate analog that is still tethered to the PPant via a C–S bond and has a very similar structure to the real substrate  bound, we again used Sfp to attach PPant-Glystab to the PCP domain. This construct was then crystallized as previously, resulting in diffraction to a resolution of 1.9 Å  excluding PPant). In the Glystab structure, however, the density for the PPant extends through the acceptor channel of the C domain into the active site, as observed in the structure of the R2577G mutant  to this amino group, where it likely forms a hydrogen bond. In order to determine whether the intrinsic mechanistic preference of the amide bond-forming reaction is stepwise or concerted, we calculated the reaction of a model donor, acceptor, and imidazole base in solution with density functional theory  with the nitrogen atom of Glystab (2.6 Å). It is important to note that Glystab sits in a different position to the aminoacyl mimic in a previous model of a C domain bound to the acceptor substrate – in these structures the aminoacyl mimic does not enter into the active site as far as observed in our GlyStab-PCP2-C3 complex -d-Arg-Gly donor substrate) and PCP3-Gly constructs. This experiment revealed no elongation when these constructs were incubated together. Thus, we turned to the use of a fused PCP2-C3-PCP3 construct, albeit one in which the PCP-constructs could be separately loaded with substrates prior to generation of the fused complex  has been separated from the rest of the domain . Isopeptide bond formation involving residues on both SpyCatcher/SpyTag components then results in the covalent bonding of the two fragments35. This system allows for the separate loading of the substrates on the PCP domain of each construct using Sfp and synthetic CoA substrates whilst also allowing the reconstitution of the NRPS assembly line.To test the activity and selectivity of the C domain, as well as the effect of mutating key residues, we first needed to generate an activity assay for the C domain using the PCPlex Fig. . To accod-Arg-Gly tripeptide into the Gly-extended tetrapeptide, as determined by high-resolution LC-MS/MS experiments  and hence we retained this simplified substrate for all subsequent assays. With a functional condensation assay in hand, we first could verify that the stabilized Glystab acceptor substrate was a functional mimic of Gly in this C domain , although this was reduced compared to the wild-type C domain (32%), possibly due to the loss of stabilizing interactions with the PPant arm  levels of conversion. In order to rationalize these differences, we analyzed the structures and performed molecular docking of these alternate substrates  into the structure of the C3-domain. First, assuming that the position of Glystab in our GlyStab-PCP2-C3 complex represents that catalytically competent conformation, and that alternate amino acid acceptor substrates must bind in a way that positions the terminal amine group in a similar position, we identified several residues in the central cavity that would likely interact with the side chain of an alternate acceptor substrate. In particular, the side chains of M2917, S2919, Q2921, P2941, and E2950 could contribute a putative side chain binding pocket for this C3-domain, in a manner reminiscent of A-domains  to position the acceptor substrate, the role of this glutamate (E2702) could be to stabilize and orient the acceptor substrate amine group to ensure an efficient nucleophilic attack of the donor substrate thioester. To observe how this motif is connected to acceptor substrate size, we extracted all C-domain sequences with known downstream A-domain specificity from the MiBiG database. Indeed, an analysis of these C domains demonstrated that there is a higher proportion of modified motifs where the acceptor substrate is small as opposed to traditional HHxxxDG containing C domains  of l-Phe, this appears likely to be due to the significant difference between the small, flexible Gly substrate and l-Phe, with its large, rigid side chain. The influence of the atypical HHxxxDE motif of this C-domain is also seen on lower levels of acceptance of larger amino acids (such as Leu), which can be released upon conversion of the motif into the typical HHxxxDG sequence. This demonstrates the versatile nature of C domains for tolerating active site modifications, some of which can play important additional roles in supporting catalysis24.The structural characterization and bioinformatics analysis we have performed of PCP-bound acceptor complexes in this work shows no general correlation between the size or chemical nature of the acceptor amino acid side chain and potential side chain binding residues in the C domain. Whilst C domain selectivity has recently been characterized in glycopeptide antibiotic biosynthesis26. Access of the PPant arm to the C domain active site appears to be gated by R2577, which repels the unmodified PPant arm  in favor of the aminoacyl-PPant. Whilst this residue is largely conserved in LCL domains, it is typically Gly or other small residues in DCL domains, which we have confirmed allows the unmodified PPant into the C-domain active site. One hypothesis for the role of this residue would be to prevent the unwanted “pass-through” of donor substrates without elongation (e.g. from PCP2 to PCP3). Examples of NRPS-dependent pathways in which CP-bound substrate transfer could occur reveals that the C domains implicated bear the Arg to  amino acid mutation (e.g. burkholdac biosynthesis)38, which provides some support for this hypothesis. For DCL domains, mutation of this Arg residue could be a requirement due to the need for E domain-catalyzed inversion of stereochemistry prior to chain elongation, as we note that the Arg to  mutation generally appears to be somewhat deleterious to peptide conversion levels, possibly due to a lack of interactions between the Arg and PPant arm in these C-domains. We anticipate that the structural snapshots presented here will pave the way for studies to probe the roles of this Arg residue as well as other active site residues in C domain catalysis, which is important due to the ever-increasing roles of C-type domains in non-ribosomal peptide biosynthesis.Within the active site, the amino group of the aminoacyl acceptor lies close to the central histidine residue, with calculations suggesting that this residue could indeed act as a base to deprotonate the zwitterionic intermediate. Further characterization of the PCP-C complex shows that the PCP binding site of the C domain is, as anticipated, dominated by hydrophobic interactions and is one that is relatively flexible with regards to the PCP domainThermobifida fusca (ATCC 27730) genomic DNA using primers #1 and #2 for PCP2-C3 and #3 and #4 for PCP3 (Supplementary Table 2-C3 cloned into pOPINS-S and PCP3 cloned into pET28a). In-Fusion® cloning reactions were incubated for 15 min at 50 °C, then placed on ice and 2.5 μL of the reaction mixture was used to transform E. coli StellarTM cells (Takara Bio). After overnight growth on LB-agar plate supplemented with kanamycin, colonies were screened by sequencing. The PCP2-C3 R2577G mutant was generated via standard Quick-Change site-directed mutagenesis procedures using primers #7 and #8 (Supplementary Table Gene fragments encoding the desired regions of FscG (UniProt ID Q47NR9) were amplified by PCR from 2-C3 SpyCatcher construct, we used an InFusion cloning reaction. The PCP2-C3 pOPINS plasmid was linearized using primers #21 and #22 (Supplementary Table E. coli StellarTM cells (TakaraBio). After overnight growth on LB-agar plate supplemented with kanamycin, colonies were screened by sequencing.To generate the PCP2-C3 SpyCatcher mutants were generated using standard Quick-Change site-directed mutagenesis procedures using primers listed in Supplementary Table PCP3 SpyTag construct, the PCP3 fragment was first cloned into the pHIS17 vector using an InFusion reaction. This step was necessary to introduce a His-tag at the C-terminus of the protein, thus allowing the subsequent addition of the SpyTag to the N-terminus. The pHIS17 vector was then linearized using primers #17 and #18 and the PCP3 region of FscG amplified using primers #5 and #6. After the InFusion reaction was completed, 2.5 μL of the reaction mixture was used to transform E. coli StellarTM cells (Takara Bio). After overnight growth on LB-agar plate supplemented with ampicillin, colonies were screened by sequencing. A positive clone was then linearized with primers #25 and #26, while the SpyTag insert was amplified from the Addgene plasmid #35050 “pET28a-SpyTagMBP” using primers #23 and #24. After following the same InFusion and transformation procedure described above, colonies were sent for sequencing.To generate the PCP2-C3 wild-type proteins, PCP2-C3 mutant proteins (cloned in pOPIN-S vector) and PCP3 proteins (cloned in pHIS17 vector) was performed as follows. A plasmid encoding the protein of interest (pOPIN-S or pHIS17) and pRARE plasmid were co-transformed into chemically competent E.coli BL21(DE3) (entdD-) cell and colonies were allowed to develop overnight at 37 °C on agar plate supplemented with the relevant antibiotics . Expression of all proteins was performed in 20 L TB media supplemented with the relevant antibiotic. Cells were incubated at 37 °C with shaking at 180 rpm until the OD600 nm reached 0.4–0.6. Protein expression was induced by the addition of IPTG (0.1 mM); cultures were subsequently grown overnight at 18 °C before being harvested by centrifugation.Production of PCPg for 20 min at 4 °C. Next, the cell pellet was resuspended in Ni-NTA buffer A  supplemented with protease inhibitor cocktail tablets  and benzonase (Sigma-Aldrich). The cells were lysed by a cell disruptor  operating at 14,000–19,000 psi, and the lysate was clarified by centrifugation at 22,680 g for 45 min at 4 °C. The supernatant was incubated at 4 °C for 1 h with 2 mL of equilibrated (Ni-NTA buffer A) Ni-NTA beads (Macherey-Nagel) with gentle stirring. After incubation, the beads were washed with 20 bed volumes of Ni-NTA buffer A. Subsequently, bound protein was eluted with 5 bed volumes of Ni-NTA buffer B .All proteins in this study were purified according to the following protocol. Cells were harvested by centrifugation at 3064 × For pOPIN-S derived proteins, the SUMO tag was cleaved with sentrin-specific protease (SENP) overnight while being dialyzed in a buffer composed of 50 mM Tris–HCl, pH 8.0; 300 mM NaCl, 1 mM DTT at 4 °C. The protein was subsequently incubated with 2 mL of equilibrated (Ni-NTA buffer A) Ni-NTA beads with gentle stirring for 10 min. The unbound, cleaved protein was washed with two bed volumes of Ni-NTA buffer A and used for further purification (uncut protein and the cleaved tag remain associated to the Ni-NTA beads). The protein was then incubated with 2 mL of GST agarose beads that had been previously equilibrated in PBS buffer with gentle stirring for 10 min to remove excess SENP. The unbound protein was washed with two bed volumes of PBS buffer that was then further purified.2-C3 constructs and 3 kDa MWCO for PCP3 constructs, Merck Millipore)). Protein concentration was determined by measuring protein absorbance at 280 nm using a NanoDrop One microvolume UV-vis spectrophotometer (Thermo Scientific). Protein was concentrated to 30 mg/mL for all PCP2-C3 and 8 mg/ml for PCP3 constructs, aliquoted (50 μL) into chilled 0.2 mL PCR tubes, flash frozen in liquid nitrogen, and stored at −80 °C.In the case of proteins expressed with a hexa-histidine tag (pHIS17 and pET28a constructs), the tag was not cleaved. In all cases, the protein of interest was further purified after Ni-NTA purification by gel-filtration chromatography using a SRT 10 SEC 300 (105 mL) column (Sepax Technologies) connected to an ÄKTA PURE system . The column was first equilibrated with 1.2 column volumes of gel-filtration buffer . Subsequently, the protein was concentrated and injected onto the column, and the eluate fractionated into 1.5 mL fractions. Elution fractions containing monomeric protein were analyzed by SDS-PAGE, and appropriate fractions were combined and concentrated using centrifugal filter units , N,N‐dimethylformamide (DMF), methanol, acetonitrile (MeCN), diethyl ether, and water were purchased from Fisher Scientific.1H NMR spectra were recorded in D2O and/or d4‐MeCN on the following Bruker Avance instruments: BACS‐400 400 MHz or BACS600 600 MHz. NMR spectra are shown in Supplementary Figs. 2O, 95:2.5:2.5 v/v′/v″, 5 mL) with shaking at room temperature for 1.5 h. The resin was removed by filtration and washed twice with TFA. The filtrate was then concentrated under a stream of N2 to ∼1 mL, the peptide precipitated with ice-cold diethyl ether (∼9 mL) and collected by centrifugation in a flame-resistant centrifuge. The crude peptide was purified using preparative RP-HPLC (using a gradient of 0−40% MeCN over 30 min). Purified hydrazide peptides were then dissolved in buffer 1  to a final concentration of 5 mM. The solution was cooled to −15 °C using a salt/ice bath, 0.5 M NaNO2 (0.95 eq.) was added and the mixture was stirred for 10 min. CoA  was then added to the reaction. The pH was slowly adjusted to 6.5 using KH2PO4/K2HPO4 buffer . The reaction mixture was stirred at −15 °C for additional 2 h, before the final peptidyl-CoA product was purified using preparative RP-HPLC (gradient 0−40% MeCN over 30 min)40. For characterization see Supplementary Figs. Peptidyl-CoAs were synthesized manually on solid phase at 0.05 mmol scale with subsequent hydrazide activation and displacement to generate the desired CoA thioesters. In all, 2-chlorotrityl chloride resin (200 mg) was swelled in DCM , washed three times with DMF, and incubated with a 5% hydrazine solution in DMF . The resin was washed three times with DMF, and a solution of DMF/triethylamine (TEA)/methanol  was added to cap unreacted 2-chlorotrityl groups. The first Fmoc-protected amino acid (0.05 mmol) was coupled to the resin overnight using O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate  and diisopropylethylamine . After that, unreacted hydrazine groups were capped with Boc−glycine (0.15 mmol) that had been activated prior to addition using HCTU (0.15 mmol) and DIPEA (0.15 mmol) for 1 h. Subsequent Fmoc removal was performed using a 20% piperidine solution in DMF  followed by coupling of the desired Fmoc- or Boc-protected amino acid (0.15 mmol) after pre-activation with HCTU (0.15 mmol) and DIPEA (0.15 mmol) for 1 h. Cleavage of the hydrazide peptide from resin and removal of side chain protecting groups was accomplished using trifluoroacetic acid/triisopropylsilane/water  was dissolved in 10 mL of buffer 2 . Tris (2-carboxyethyl)phosphine  was added and the mixture stirred for 30 min. Alkyl bromide (3 eq.) was dissolved in MeCN (2 mL) and added to the CoA solution, which was then stirred at room temperature overnight. The desired compound was concentrated and purified by preparative RP-HPLC purification (MeCN gradient 0–40% over 30 min)2O and the pellet collected using centrifugation in a flame-resistant centrifuge. The addition of Et2O and subsequent centrifugation was repeated three times to wash the sample. The crude product was purified by preparative RP-HPLC (MeCN gradient 0-40% over 30 min). Cleavage of the Boc group was performed using a mixture of TFA/ TIS/ H2O  for 1 h and the solution was concentrated under a stream of N2 before precipitation of the peptide was performed by addition of ice-cold Et2O, followed by subsequent washing (3x)33. For characterization see Supplementary Figs. Boc-amino acid (2 eq.), TEA (2 eq.) and (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate  were dissolved in DMF and stirred in an ice bath for 30 min before the dropwise addition of a solution of DMF containing CoA (1 eq.). The mixture was then stirred overnight at room temperature. Crude Boc-aminoacyl-CoA was precipitated by the addition of ice-cold EtCompound purification was performed using a Shimadzu High Performance Liquid Chromatograph equipped with a SPD-M20A Prominence Photo Diode Array Detector and two LC-20AP pumps. Purification used a Waters XBridge BEH300 Prep C18 column  at a flow rate of 10 mL/min. The solvents used were water + 0.1% TFA (solvent A) and ACN + 0.1% TFA (solvent B).32. The loading reaction utilized a 1:2:0.1 molar ratio of the PCP domain, peptidyl-/aminoacyl-CoA and Sfp (R4-4 mutant), respectively. Peptidyl-CoA (200 µM) was loaded onto the PCP-containing construct (100 µM) for 1 h at 30 °C using the Sfp (10 µM) in PCP-loading buffer . After the loading reaction, the remaining peptidyl-CoA was removed by three concentration/ dilution steps using centrifugal concentrators  or 3 kDa MWCO for PCP3 constructs, Merck Millipore) in gel-filtration buffer . Holo-PCP constructs were then immediately used for in vitro reconstitution assays or crystallization experiments.All proteins containing PCP-domains were expressed and purified in their apo form, which were converted into their holo form using the phosphopantetheinyl transferase Sfp (R4-4 mutant) and desired CoAs2-C3 Spy-Catcher construct was incubated with unloaded PCP3 Spy-tag construct (both 100 µM) for 10 min at 30 °C, which was followed by loading of the desired aminoacyl-CoA on the PCP3 as described above. The reaction was then incubated for an additional 1 h at 30 °C to allow for the condensation reaction to occur. For thioether tethered amino acid loaded PCP3 substrates, reaction mixtures were directly analyzed using nano LC ESI MS (see below Ppant ejection section)42. For thioester-tethered amino acid loaded PCP3 substrates, chemical cleavage by an addition of 15 μL of methylamine liberated the methylamide peptides; reaction mixtures were incubated for 15 min at room temperature. The peptide products were then purified from the reaction mixture using solid phase extraction . Before loading the sample, cartridges were activated with 0.1% formic acid (FA) in methanol (1 mL) and subsequently equilibrated with 0.1% FA in water. Samples were loaded onto equilibrated cartridges and the solution passed through the column bed under gravity. Once the samples were loaded, the cartridge was washed with 0.1% FA in water (1 mL) before the peptides were eluted with 0.1% FA in MeCN/water . The samples were then dried by freeze dryer at −50 °C and analyzed by HRMS.The peptide loaded PCP−1. Full scan MS was performed in the Orbitrap at 60,000 nominal resolution, with targeted MS2 scans of peptides of interest acquired at 15,000 nominal resolution in the Orbitrap using HCD with stepped collision energy (24 ± 5% NCE). QualBrowser  was used to view spectra and generate extracted ion chromatograms for the singly charged species at 20 ppm. The level of peptide extension in the assays shown in Fig. 2 fragments were generated with MS-Product  and manually assigned to spectra43. See Supplementary Figs. High-resolution mass spectrometry measurements were performed on an Orbitrap Fusion mass spectrometer (Thermo Scientific) coupled online to a nano-LC  via a nanospray source. Peptides were separated on a 50-cm reverse-phase column  after binding to a trap column . Elution was performed on-line with a gradient from 6% MeCN to 30% MeCN in 0.1% formic acid over 30 min at 250 nL minMass spectrometry measurements were performed on a MicroTOFq mass spectrometer  coupled online to a 1200 series capillary/nano-LC (Agilent Technologies) via a Bruker nano ESI sprayer. Proteins were separated on a 150-mm reverse-phase column  after binding to a trap column . Elution was performed on-line with a gradient from 4% MeCN to 60% MeCN in 0.1% FA over 30 min at 300 nL/min. Proteins >20 kDa were separated on a MabPac SEC-1 5 µm 300 Å 50 × 4 mm (Thermo Scientific) column with an isocratic gradient of 50% MeCN, 0.05% TFA and 0.05% FA at a flow rate of 50 µL/min. The protein was eluted over a 20-min run-time monitored by UV detection at 254 nm. After 20 min the flow path was switched to infuse Low concentration Tune mix  to calibrate the spectrum post acquisition. The eluent was nebulized and ionized using the Bruker electrospray source with a capillary voltage of 4500 V dry gas at 180 °C, flow rate of 4 L/min and nebulizer gas pressure at 0.6 bar. MSMS spectra were acquired by manual selection of isolation mass and isolation width with a collision energy of 32. The spectra were extracted and deconvoluted using Data explorer software version 3.4 build 192 . For analysis see Supplementary Fig. 44 partner repository with the dataset identifier PXD024004.The HRMS and PPant ejection data have been deposited to the ProteomeXchange Consortium via the PRIDE2-C3 affording the holo forms of PCP2-C3 and concentrated to a final concentration of 30 mg/mL in gel-filtration buffer. Initial screening was performed at the Monash Molecular Crystallisation Facility (MMCF) with subsequent optimization performed in 48-well sitting-drop plates. Crystallization trials of PCP2-C3 at a concentration of 30 mg/mL in a 1:1 ratio (v/v) with the crystallization solution (2 µL drops) led to a condition composed of 18–22% v/v PEG 3350 and 0.17–0.3 M magnesium); crystals formed overnight at room temperature. Crystals were cryoprotected by transferring in a drop made of the reservoir solution supplemented with glycerol . Crystals were collected in cryoloops and flash frozen in liquid nitrogen.Aminoacyl-CoAs were loaded onto PCP3SO4. Sitting drops were made of 1 μL of PCP3 at a concentration of 11 mg/mL and 1 µL of the crystallization solution. Crystals formed overnight at room temperature. Crystals were cryoprotected by transferring in a drop comprising reservoir solution supplemented with glycerol  and flash frozen in liquid nitrogen.Initial screening was performed at the Monash Molecular Crystallisation Facility (MMCF) with subsequent optimization performed in 48-well sitting-drop plates (MRC Maxi plates (molecular dimensions)). After optimization, the best crystallization condition was composed of 500 µM Bis-Tris, pH 5.5, 1.8 M NH45  and MX2  equipped with an Eiger detector (Dectris) at 100 K46. Data processing was performed using XDS47 and AIMLESS as implemented in CCP448. Phases for the PCP2-C3 constructs were obtained from a single wavelength anomalous diffraction experiment (SAD) using xenon-derivatized crystals. In brief, crystals were mounted into a cryo-loop and briefly exposed to xenon gas using the Hampton Research Xenon Chamber available at the Australian Synchrotron and flash frozen in liquid nitrogen. The SAD dataset was then reduced with XDS47 and the phases obtained using HKL2MAP49. The initial model generated by HKL2MAP was subsequently used in molecular replacement experiments to obtain phases for the other datasets using PHENIX in-built Phaser module50. The crystals belonged to the P212121 space group, with the unit cell comprising 2 highly similar copies of the PCP2-C3 construct  on beamlines MX152 for model building and PHENIX-refine for refinement50. Ramachandran statistics : WT PCP2-C3 PPant , R2577G PCP2-C3 PPant , WT PCP2-C3 Glystab , PCP3 . The model quality of each structure assessed by Molprobity (score/percentile): WT PCP2-C3 PPant , R2577G PCP2-C3 PPant , and WT PCP2-C3 Glystab  PCP3 . Similar structures were identified by DALI53, and the PCP/C-domain interface analyzed using PISA54. All graphics were generated with Pymol (Schrödinger LLC) or UCSD Chimera55.Structural models were built and refined using COOT56, accessed on 03.03.2020. Domain sequences, specificities and other information were extracted from the MiBiG entries’ gbk and json files by parsing for keywords. We identified 2049 C-domains with known selectivity (1456 LCL and 593 DCL), of which downstream A domain specificity was known in 488 sequences. The C-A linkers, which include the possible “pocket” residues were defined as the sequences that start at the end of a C domain and end at the beginning of the downstream A domain within the same NRPS gene. 401 such regions with known A domain specificity were isolated and used in the corresponding analysis. The conserved HHxxxDG motifs were analyzed from 481 C domain sequences with known downstream A domain specificity, this time including starter C domains and ones with dual selectivity.All sequences used for statistics and correlation analyses were isolated from the MiBiG database57 tool (version 3.8.31) with default settings. Sequence logos  of the SciPy59 python library (version 1.4.1), by using the sum of molecular weights of the “pocket” residues and the mass of the acceptor substrate. Principal Component Analysis (PCA) was conducted with the PCA function (sklearn.decomposition module) of the scikit-learn60 python library (version 0.22.2.post1) while taking into account each residue’s molecular mass. The PCA graph  and from the NumPy62 python library (version 1.18.1). All scripts were implemented with Python version 3.7.6.All Multiple Sequence Alignments (MSAs) used for statistics and correlation analyses were produced with the MUSCLE63 using a probe radius of 0.7 Å, shell radius of 4 Å, and shell depth of 4 Å. The clustering threshold was set at 3.5. The starting point was defined as the point between His2697, Glu2702, and Pro2841.Protein tunnels were identified and assessed using Caver 3.065 at pH 7.0, and the restrained minimization was performed using the OPLS3e force field66 (converging heavy atoms to RMSD of 0.30 Å).Computational protein–protein docking, substrate docking, and molecular dynamics (MD) simulations were performed using Schrödinger Release 2019-1. In all cases, protein structures were prepared using the Protein Preparation Wizard in Maestro. Following pre-processing of the pdb files (including addition of hydrogens), all water molecules and small molecules were removed, and alternate confirmations were restricted to the most probable rotamer. Hydrogen bonds were optimized using ProPKA33 and the C3 domain from the unloaded PCP2-C3 didomain structure  were performed using the protein–protein docking wizard in Maestro, which uses the PIPER docking algorithm67. In order to constrain the docking of PCP3 to the acceptor PCP binding site, a distance restraint was set between Ser3558 of the PCP3 and Tyr2585 of the C3 domain . During rigid-body protein-protein docking, 70000 ligand rotations were probed, and the top 30 poses were refined prior to analysis.Protein–protein docking between the PCP3 domain  from unloaded PCP2-C3 didomain and Glystab-PCP2-C3 didomain structures. Protein structures were prepared using the Protein Preparation Wizard as described above, then placed in an orthorhombic box with a buffer of 10 Å around the protein molecule and periodic boundary conditions (PBCs) were applied. This provided sufficient distance between neighboring protein molecules once PBCs were applied (~20 Å); this distance was significantly larger than the 9 Å electrostatic cut-off used during simulations. Each system was solvated with SPC water molecules, and the system was neutralized through the addition of Na+ ions. Following the default Desmond relaxation protocol, 100 ns production runs were performed in triplicate using the default Desmond settings. Snapshots were recorded every 0.5 ns and were analyzed using CAVER 3.0, as described above. Dihedral angles of Arg2577 were measured using the simulation event analysis wizard in Schrödinger. The OPLS3e force field66 was used at all stages of the simulation. The OPLS3e force field is the default force field in Desmond and performs well against other force fields for the simulation of protein molecules66.Molecular dynamics (MD) simulations were performed in Desmond (Schrödinger Release 2019-1). Simulations were initiated from the structures of the C68. The C3 domain from the Glystab-bound PCP2-C3 didomain structure  was used as the receptor for docking studies and prepared as described above. The PPant from the Glystab-bound PCP2-C3 didomain structure was used as a template from which alternate substrates were modeled. Alternate ligands were constructed using the 3D builder tools in Maestro. The LigPrep wizard was used to prepare these ligands using the OPLS3e force field and possible ionization states (pH 7.0 ± 2) were generated using Epik. Ligands were computationally docked using the “standard protocol” option in the ligand docking tool. The central phosphorous of the phosphate moiety was restrained to the position of the phosphorus in the structure of the PCP2-C3 didomain in complex with Glystab (sphere of radius 3 Å around this position).Docking of PPant-linked substrates was performed in Schrödinger using the ligand docking wizard and Glide algorithms3 domain from the Glystab-bound PCP2-C3 didomain structure  as a template, the mutation was introduced using the mutation tool in Maestro. A basic local minimization step was performed, followed by Protein Preparation Wizard’s restrained minimization, as described above.The E2702G mutant was modeled in silico; using the C69. The B3LYP-D3 functional74 and 6-31 G(d) basis set were used, in conjunction with the SMD model75 of implicit diethyl ether . Transition states were characterized by the presence of a single imaginary vibrational frequency corresponding to the reaction coordinate. Intrinsic reaction coordinate77 calculations were also performed to identify the local minima situated on either side (reactant and product) of transition states. The DFT computations were carried out to determine whether the attack of the amine on the thioester has an intrinsic preference for a stepwise or concerted mechanism. They did not attempt to model the exact binding orientation of the substrates within the enzyme active site.Density functional theory (DFT) computations were performed in Gaussian 16Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


These small molecules trigger mobilization of intracellular Ca2+ and promote transcription factor transport into the nucleus launching the adaptive immune response. The TEC family kinases are responsible for phosphorylating and activating PLCγ, and our group aims to understand mechanisms that regulate immune cell signal transduction by focusing on this kinase/phospholipase axis in T-cells and B-cells. Here, we review the current molecular level understanding of how the TEC kinases (ITK and BTK) and PLCγ1/2 are autoinhibited prior to activation of cell surface receptors, how TEC kinases are activated to specifically recognize the PLCγ substrate, and how conformational changes induced by phosphorylation trigger PLCγ activation.Cell surface receptors such as the T-cell receptor (TCR) and B-cell receptor (BCR) engage with external stimuli to transmit information into the cell and initiate a cascade of signaling events that lead to gene expression that drives the immune response. At the heart of controlling T- and B-cell cell signaling, phospholipase Cγ hydrolyzes membrane associated PIP In the absence of activating stimuli, TEC kinases adopt an autoinhibited conformation that limits unwarranted catalytic activity. TEC family kinases have been notoriously resistant to crystallizing in their full-length form leaving open many questions regarding the molecular details of regulation.Tyrosine kinases are first responders that propagate cellular signals immediately following membrane receptor engagement. In a subset of immune cells including T- and B-cells, the TEC family kinases are the third in a series of proximal non-receptor tyrosine kinases that are activated by phosphorylation. Activated TEC family kinases ITK and BTK are assembled within the membrane associated scaffolding protein complex and phosphorylate their substrate phospholipase Cγ (PLCγ) a. In the2) to generate the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG) (b) triggering further signaling events that ultimately result in changes in gene transcription, that for T- and B-cells, result in activation of the adaptive immune response.PLC’s are a central node in controlling many aspects of cell signaling and gene expression. The PLCγ subtype consists of PLCγ1 and PLCγ2 isoforms that are expressed in T-cells and B-cells respectively among other cell types. Activated PLCγ1/2 hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIPol (DAG) b triggerThis graphical review directs its attention to the conformational changes and molecular binding events that control the transition between autoinhibited and activated TEC kinases and phospholipase Cγ. Throughout, we highlight available data to build a structural understanding of the molecular events that regulate the TEC kinases and the phospholipase C gamma family; sequence and structural homology between BTK and ITK, as well as PLCγ1 and PLCγ2, suggest that structural features determined for one family member are likely shared across the protein family.Non-receptor tyrosine kinases (NRTKs) are cytosolic enzymes that are activated following receptor engagement. The well-studied SRC family kinases are the prototypical example of NRTKs and make up the largest family of NRTKs with 9 distinct kinases. The SRC kinase sequences all include a conserved C-terminal regulatory tail that contains an inhibitory tyrosine phosphorylation site (Y527 in SRC). The precise regulatory role of SRC Y527 became clear when the first SRC kinase was crystalized in 1997 . Phosphoa,b). The SRC-module (SH3-SH2-kinase fragment) crystallized as a domain swapped dimer with domain swapping occurring in the SH2 domain. While the functional significance of the domain swapped dimer is unknown, each half of the domain swapped dimer adopts an autoinhibitory conformation quite similar to that observed for the SRC family. The SH3 and SH2 domains assemble on the ‘back’ of the kinase domain with the SH2-linker region contacting both the SH3 and kinase domain N-lobe . This domain arrangement is similar to SRC kinases despite the absence of the SRC regulatory tail in the TEC family. Solution studies of BTK . In addition, interactions between the SH2-kinase linker and the N-lobe of the kinase assemble a hydrophobic stack  which stabilizes the autoinhibited structure and also allosterically communicates with the active site; formation of the inhibitory hydrophobic stack leads to selective binding of ADP over ATP in the kinase domain, an observation first established with ITK and SRC , TEC & BMX) are expressed primarily in cells of hematopoietic lineage and none of the TEC kinases contain the C-terminal regulatory phosphorylation site that is so critical in controlling the function of the SRC family kinases. To date, a crystal structure of any full-length autoinhibited TEC kinase is lacking but in 2015, a fragment containing just three of the domains in the TEC family kinase BTK was crystallized  represenFig. 2s of BTK , reveale and SRC . This wo and SRC .Fig. 2Do, center). Accurately defining the autoinhibitory contacts for the PHTH domain and the role of the PRR has been challenging (c) and reveals a contact between the PHTH α2 helix and N-lobe of the kinase domain (c) partially overlaps with the SH3 domain in the structure of the autoinhibited SRC module of BTK (a) and so these structures are mutually exclusive. It is possible that this binding mode represents an intermediate on the activation pathway instead of a stable autoinhibited structure. An alternative autoinhibitory position for the PHTH domain (d) has been characterized based on nuclear magnetic resonance (NMR) data and hydrogen–deuterium exchange applied to the full-length BTK molecule  it is likely that the PHTH domain is dynamic and possibly sampling a range of conformational states with respect to the autoinhibited SRC module (e).A distinguishing feature of the TEC family is the Pleckstrin-Homology Tec-Homology (PHTH) domain followed by a long linker region that contains a proline-rich region (PRR) that matches the canonical SH3 ligand sequence , center.llenging . A crystllenging c and reve domain . The pose domain c partiale of BTK a and so H domain d has beemolecule . These smolecule c,d it isC module e., center) might serve to ‘prime’ the full-length kinase for activation (Current models of autoinhibited full-length BTK do not resolve the proline-rich region (PRR). Available data suggest that this long linear motif situated between PHTH and SH3 domains , center tivation . The cantivation . It is aStudies have shown that mutants of both PLCγ1 and PLCγ2 have been implicated in driving human diseases such as leukemias and lymphomas, as well as being associated with dysregulated immune responses and resistance to inhibitors. Therefore, numerous experimental approaches have been carried out to structurally characterize the PLCγ isozyme .a). The cSH2 binding pocket also showed electron density for the structure of unphosphorylated PLCγ1 nSH2-cSH2-linker, which the authors suggest that the Y783 containing linker binds to the cSH2 domain even in the absence of phosphorylation. Using a combination of NMR and small angle X-ray scattering (SAXS), the same authors produced a structural model of the entire γSA. In this model, the sPH-SH3 segment contacts the cSH2 domain (via an sPH/cSH2 interface) involving the βD-βE strands of the cSH2 domain . Split PH domains are characterized by insertions of one or more autonomously folded protein modules in the middle of pH domain sequences, which is an uncommon feature for PH domains making split PH domains quite unique . These inhibitory interfaces latch the γSA to the top of the catalytic core and block access to the PIP2 substrate. The cSH2-linker region of PLCγ1 is not evident in the full-length crystal structure and was in fact shortened considerably in order to obtain crystals of the full-length PLCγ1. The cSH2/core interface is coincident with the binding site of the phosphorylated Y783 linker suggesting that release of the cSH2 domain from the core is triggered upon phosphorylation. In addition, the association of the non-phosphorylated Y783 containing linker with cSH2  show two distinct arrangements of the sPH/SH3 cassette with respect to the nSH2-cSH2 segment. It is suggested that a large conformational change occurs in the γSA region upon PLCγ1 activation (d) and the putative active state . In essence, the models suggest that activation via phosphorylation of Y783 competes with the cSH2/core interaction allowing the sPH/SH3 domains to swing down and interact with a different surface on cSH2. The conformational adjustment initiated by pY783 binding to the cSH2 domain would result in release of γSA from the PLCγ core followed by steric occlusion of the autoinhibitory surface of γSA. This activation mechanism may, in part, serve to prevent re-association of cSH2 with the PLCγ core to maintain the active state of PLCγ as it encounters its substrate at the membrane. Additional experiments are needed to test this idea and gain a deeper appreciation for the dynamics that control activation/autoinhibition of the PLCγ family.Comparison of the two available structural models for the PLCγ1 γSA region f show twtivation  and so ttivation d and theAdaptor proteins/ membrane association/substrate priming and docking , Fig. 5Fa). As depicted in 3, the headgroup of PIP3, IP4 or the soluble phosphoinositol IP6 (b). BTK dimerization and the presence of multiple ligand binding sites may confer a switch-like activation of BTK .Interactions of the regulatory domains of TEC kinases with their ligands leads to activation and recruitment to the receptor signaling complex a. As depitol IP6 , show thitol IP6 b. BTK din of BTK  by requiiposomes . The actiposomes  revealediposomes a, 5.a,c). Receptor activation also leads to phosphorylation of adaptor proteins such as SLP-76 (pY145) which recruits ITK into the scaffolded complex via a canonical ITK SH2/pY145 interaction that disrupts the autoinhibitory interaction between SH2 and kinase domain .SH3 ligands (PXXP motifs) within the adaptor proteins can displace the SH2-kinase linker bound to the SH3 domain in the autoinhibited conformation stabilizing the active kinase within the signaling complex a,c. Recee domain a,d.Interestingly, BTK is the target of Ibrutinib and other second-generation inhibitors used in the treatment of B-cell chronic lymphocytic leukemia (CLL). BTK is prone to mutations that elicit drug resistance and in at least one case the mutation shifts the conformational equilibrium toward the active state of the kinase thereby increasing cellular BTK activity promoting escape from drug induced inhibition . As well2 (2 derived second messengers IP3 and DAG on calcium channels and the PKC/Ras pathway drive changes in cell morphology and gene transcription ultimately generating the adaptive immune response.Once activated, BTK/ITK is assembled within the membrane associated adaptor protein complex. Into this environment enters PLCγ1/2, which exits its autoinhibited conformation to associate with the kinase domain and specific regions of adaptor/scaffold proteins . Here, T2 . The nexThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Here, we show that sequence-specific DNA-binding proteins with this bipartite domain structure have resulted from dozens of independent hAT domestications in different eukaryotic lineages. CGGBPs display a wide range of sequence specificity, usually including preferences for CGG or CGC trinucleotides, whereas some bind AT-rich motifs. The CGGBPs are almost entirely nonsyntenic, and their protein sequences, DNA-binding motifs, and patterns of presence or absence in genomes are uncharacteristic of ancestry via speciation. At least eight CGGBPs in the coelacanth Latimeria chalumnae bind distinct motifs, and the expression of the corresponding genes varies considerably across tissues, suggesting tissue-restricted function.The human transcription factor (TF) CGGBP1 (CGG-binding protein) is conserved only in amniotes and is believed to derive from the zf-BED and Hermes transposase DNA-binding domains (DBDs) of a  The hAT repeats . The ZBEptations . Human Cxpansion , 1997 anon , suggesting that the CGGBP1 domain model corresponds only to specific hAT subtypes. There is considerable overlap between species containing hAT-19 RepeatMasker hits and those with host genes encoding CGGBPs  designs (hATs (E-scores” (Determining what sequences DBPs bind is often a starting point in their characterization . We quer designs . The two designs  each con designs  online. ns (hATs  online. -scores”  as well -scores” .hAT proteins (including variants of hAT-19_Crp) displayed clear sequence preferences, that is, a set of high-scoring 8-mers with related sequences, that were shared between the two array designs and yielded a clear motif. These 22 proteins all had relatively long N-terminal extensions (and thus a longer linker between the GST tag and the CGGBP domain), and most contained both Cys and His residues that complex zinc in the zf-BED, a leucine two positions N-terminal to the zf-BED, and two prolines that are widely conserved in the Hermes domain, suggesting that these residues are required for DNA sequence specificity. Eighteen of the CGGBPs and four hAT-encoded CGGBPs have high-scoring matches within subterminal regions of the corresponding Repbase model  and Locusta migratoria (migratory locust), and the one CGGBP-containing protein in the fungus Rhizophagus irregularis , we examined the homology of nearby genes at their chromosomal locations, because common descent is often reflected in shared ordering of genes (synteny). Gene order is typically preserved between human and bony fish, for example . In conthAT integration events. Several other observations are consistent with this notion. One is that the intron/exon structures of the CGGBPs differ from each other  inhabit very different environments. Second, the narrow coverage of the CGGBP domain model among hATs (29/824) raises the possibility that domain models corresponding to the same regions of other hAT subclasses may uncover additional classes of host DBPs.Finally, the fact that the CGGBP domain model represents only a small subset of A. limnaeus, or the NCBI-annotated identity of inferred kanglang fish genes.We designed and built expression constructs for the 114 CGGBP proteins to include the entire domain plus 50 flanking AAs on both sides (unless the end of the protein was reached), fused to an N-terminal GST tag and driven by a T7 promoter. Three proteins with unknown AA residues (X) were run as all possible variants consistent with corresponding codon sequences. PBM data were generated as described , with moMolecular Biology and Evolution online.msab007_Supplementary_DataClick here for additional data file.


We demonstrated numerically the skyrmion formation in ultrathin nanodisks using a magnetic force microscopy tip. We found that the local magnetic field generated by the magnetic tip significantly affects the magnetization state of the nanodisks and leads to the formation of skyrmions. Experimentally, we confirmed the influence of the local field on the magnetization states of the disks. Micromagnetic simulations explain the evolution of the magnetic state during magnetic force microscopy scanning and confirm the possibility of skyrmion formation. The formation of the horseshoe magnetic domain is a key transition from random labyrinth domain states into the skyrmion state. We showed that the formation of skyrmions by the magnetic probe is a reliable and repetitive procedure. Our findings provide a simple solution for skyrmion formation in nanodisks. Magnetic skyrmions are circular domains surrounded by a single chirality domain wall ,2,3. TheThe challenge of the experimental study of skyrmions is to find an efficient technique for forming stable skyrmions. Recently, individual skyrmion manipulation in a Pt/CoFeB/MgO multilayer using a magnetic force microscopy (MFM) tip was reported . This meIn this study, we demonstrated experimentally the switching of domain states to a single-domain state by a local field induced by an MFM tip. The numerical simulations showed a possible evolution of the domain state to a single-domain state. Further, induction of the skyrmions from the domain state was demonstrated by numerical simulations when the pinning centers were introduced at the boundary of the dot.−5 Pa; working gas was Ar at 0.175 Pa). The thicknesses of the deposited layers were defined by the rotation speed of the substrate over the magnetrons, and they were as follows: Pt = 1.5 nm, Co = 1.2 nm, Au = 1.5 nm. Then electron-beam lithography with a positive tone resist was used to define multiple arrays of dots with variable diameters varied from 150 to 525 nm. After the development, a 15 nm thick titanium layer was evaporated and lifted off to reveal Ti circle-shaped mask patterns. Finally, ion-beam etching was used to transfer the Ti-mask pattern into the Pt/Co/Au multilayer resulting in formation of stacked nanodots. The total height of the nanodots was 45, 52, 58 and 64 nm for samples with 4, 5, 6 and 7 repetitions of the Pt/Co/Au trilayer  with an in-plane magnetic field up to 2T. The obtained value of the magnetization saturation was about Ms = 1.1 × 10SWf between the counter-propagating spin waves at the specific value of the spin-wave wavenumber SWk. The value of ΔSWf corresponds to the shift between the Stokes and anti-Stokes frequencies, and then the value of DMI constant D can be directly estimated as:γ = 176 GHz/T is the gyromagnetic ratio. The measured values of frequency shift and DMI constant for the spin waves with the wavevector SWk = 0.011 nm−1 were calculated using Equation (1) and are summarized in The DMI strength was evaluated by the Brillouin light scattering (BLS) measurements in the Damon–Eshbach configuration ,22,23. Bx-axis—from left to the right side of a dot array—then was returned back to the beginning of the same line. Within the second pass, the tip was lifted up (in z-direction) at a distance of 15 nm above the sample to lower Van der Waals forces and to image only magnetic forces. The tip’s motion was repeated according to sample topography. Next, the tip position was shifted along the y-axis by 15 nm to continue scanning on the next line. Note that in the two-pass MFM method, tip scans over each line four times.The magnetic states of multilayered nanodots were investigated and induced by MFM with low-magnetic-moment tip (LM) and high-magnetic-moment tip (HM), respectively. The measurements were performed at room temperature with tip scanning speed of 5 μm/s. In both LM and HM scans, a standard two-pass MFM method was used. At first, sample topography was recorded in semi-contact mode (1st pass). During this scan, MFM tip was moving along the TM, Neuchatel, Switzerland), with tip radius below 15 nm optimized for high-resolution MFM imaging. The spatial resolution of the LM probe was evaluated from fast Fourier transform analysis according to [4 A/m, which helps to minimize the invasive interaction between the tip and sample. The scans taken with LM probe revealed initial magnetic states in as-fabricated dots without affecting them by measurements. Next, HM probe with approximately four times higher magnetic moment  was magnetized in an external magnetic field in order to set it into a single-domain state. Subsequently, the same dot array was scanned by the HM probe whose stray field affected magnetization in dots while scanning. Finally, new magnetic states were measured by the LM probe again. Four identical dot arrays on each sample were scanned to check the reproducibility of the process.All samples were firstly scanned by LM super-sharp probes ; one of them is presented in −11 to 1.55 × 10−11 J/m with step 0.05 × 10−11 J/m. Similarly, FFT was used to estimate the domains’ width. The value of A = 1.0 × 10−11 J/m is the value that leads to the best correspondence between experiment and simulations. The simulated domain corresponding to this value is presented in The micromagnetic simulations of magnetic states in nanodots were performed using Boris spintronics solver . This soWe compared the results of LM MFM measurements of magnetic states before and after HM tip scan . In the next part of our manuscript, the magnetic field induced by the tip is called the tip field. 5 J/m3 with a standard deviation of 35% [Due to the fact that a local MFM tip was able to remagnetize magnetic samples to a single-domain state in large dots (up to 475 nm), we assume that the local pinning of the sample plays an important role in the process of inducing a single-domain state. To mimic the polycrystalline nature of the sample, a Voronoi tessellation was added in the micromagnetic simulations where, to each grain of 6 nm in size, a slightly different perpendicular magnetic anisotropy was assigned. The anisotropy value was chosen randomly from a normal distribution centered around a mean value of Ku = 1.26 × 10n of 35% . In the simulations, we reproduced the MFM scan path by scanning each horizontal line twice—left to right and right to left—and then moving to the next line, bottom to top. The horizontal and vertical step was set to 15 nm. To reproduce this procedure in simulations, we relaxed magnetization for each position of the tip field. A characteristic time scale over which a perturbed magnetic domain will relax is in the range of a few nanoseconds, much shorter than the scanning time of a single point (approx. 3 ms). We performed simulations for various tip field amplitudes, from 100 to 1500 mT, to find the minimal strength and size of the magnetic field induced by the MFM tip and verify the necessity of local pinning sites in the sample. We found that when the magnetic field is weak (below 200 mT), the influence of the tip field is not sufficient to influence the domain structure, neither by remagnetization nor moving of domains. The tip field with a strength of 200 mT is strong enough to move and annihilate domains at the boundaries. However, it is not sufficient to re-magnetize the large-domain or single-domain state with opposite polarity. A field of 1150 mT is needed to re-magnetize a domain of any size . The evolution of the exemplary random magnetic domains under the influence of the tip field with a strength value of 350 mT is represented in Taking into account the non-uniformities of the sample (g)–(l) allows the creation of strong pinning centers for the domain, and it prevents shrinking of the red domain when the tip is outside of the disk. Therefore the domains are successively moved by the tip field during the scan and are annihilated at the upper boundary. Domain behavior at these selected fields observed in the simulations may differ in relation to experimental measurements, taking into account values of magnetic material parameters or heterogeneity of magnetic parameters ,29,30. TNext, we performed numerical simulations to demonstrate the potential of MFM-tip-induced formation of the isolated skyrmion in the dots with uniform properties inside the area of the dot (without the pinning centers) and non-uniformities at the dot boundaries. The origin of the boundary pinning might be due to non-uniformities at the boundaries ,32, thicIn the case when the value of the tip field was sufficiently strong to move the domains (over 200 mT), the MFM scan nucleated the skyrmion. We observed that the process of domain shifting and domain formation depends on the initial state of magnetization in the nanodot. Exemplary evolution of the magnetization in 400 nm nanodots during the HM scan is shown in Depending on the initial magnetization state, the first MFM scan does not always lead to skyrmion formation. We performed simulations for 20 random initial states see k or a skIn summary, we demonstrated that the local magnetic field induced by the MFM tip can influence the magnetic state of a nanodisk. The experimentally measured disks were remagnetized from the domain state to the single-domain state. The numerical simulations indicated the important role of pinning centers in that process. Further, with numerical simulations, we showed that control of pinning centers within the area of the dot and at the boundary of the dot can lead to a reproducible method of skyrmion nucleation in the nanodots. We demonstrated the evolution of remagnetization from the domain state to skyrmion and identified the important role of the intermediate horseshoe state in that process.


The FUSION-X-US-II prototype was developed to combine 3D automated breast ultrasound (ABUS) and digital breast tomosynthesis in a single device. We evaluated the performance of ABUS and tomosynthesis in a single examination in a clinical setting.In this prospective feasibility study, digital breast tomosynthesis and ABUS were performed using the FUSION-X-US-II prototype without any change of the breast position in patients referred for clarification of breast lesions with an indication for tomosynthesis. The tomosynthesis and ABUS images of the prototype were interpreted independently from the clinical standard by a breast diagnostics specialist. Any detected lesion was classified using BI-RADS® scores, and results of the standard clinical routine workup (gold standard) were compared to the result of the separate evaluation of the prototype images. Image quality was rated subjectively and coverage of the breast was measured.One hundred one patients received both ABUS and tomosynthesis using the prototype. The duration of the additional ABUS acquisition was 40 to 60 s. Breast coverage by ABUS was approximately 80.0%. ABUS image quality was rated as diagnostically useful in 86 of 101 cases (85.1%). Thirty-three of 34 malignant breast lesions (97.1%) were identified using the prototype.The FUSION-X-US-II prototype allows a fast ABUS scan in combination with digital breast tomosynthesis in a single device integrated in the clinical workflow. Malignant breast lesions can be localized accurately with direct correlation of ABUS and tomosynthesis images. The FUSION system shows the potential to improve breast cancer screening in the future after further technical improvements.• The FUSION-X-US-II prototype allows the combination of automated breast ultrasound and digital breast tomosynthesis in a single device without decompression of the breast.• Image quality and coverage of ABUS are sufficient to accurately detect malignant breast lesions.• If tomosynthesis and ABUS should become part of breast cancer screening, the combination of both techniques in one device could offer practical and logistic advantages. To evaluate a potential benefit of a combination of ABUS and tomosynthesis in screening-like settings, further studies are needed. Mammography is the basis of breast cancer screening worldwide. Early detection and treatment of breast cancer can result in reduced mortality –3. StillTomosynthesis, a 3D mammogram created from low-dose digital X-ray projections, has been shown to increase sensitivity and specificity of breast cancer detection compared to 2D-mammography –17. SeveThis was a prospective single-center study at our breast unit performed in 2019. Patients were eligible for the study if they presented for clarification of breast lesions with an indication for tomosynthesis. Exclusion criteria were previous breast surgeries in the examined breast, pregnancy, age of less than 18 years, and inability to give informed consent. Patients were recruited non-selectively if the prototype and the instructed personnel were available. The standard diagnostic workup included clinical examination, 2D-digital mammography , HHUS using an ACUSON S2000 or S3000 ultrasound unit with an 18-MHz transducer , and tomosynthesis using the FUSION-X-US-II prototype . The study-specific additional ABUS was obtained in the same setting directly after tomosynthesis by a radiologic technologist. No sonographer or physician is needed to perform the examination . An ultrThe study protocol was accepted by the appropriate ethics committee  in concordance with the Health Information Portability and Accountability Act of 1996 (HIPPA). Informed consent was obtained from every patient enrolled in the study.The FUSION-X-US-II prototype is based on the ACUSON S2000 Automated Breast Volume Scanner  and the MAMMOMAT Inspiration , which are both FDA approved and CE certified. The research device combining both is not commercially available. The prototype was developed from the FUSION-X-US-I prototype with several technical improvements. In comparison to the previous FUSION-X-US-I prototype, the compression paddle was adapted in terms of weight and size to provide better positioning. The breast is compressed using a specially woven gauze, which provides sufficient ultrasound coupling through more constant tension and equal pressure distribution for ABUS and X-ray permeability for tomosynthesis. An improved tightening mechanism of the gauze enables a more conform compression. A new feature to improve contact between the breast surface and the ultrasound probe has been implemented: A special air cushion attached to the housing of the X-ray detector can be inflated to push the peripheral parts of the breast homogenously towards the gauze. The inflation is controlled manually by the radiologic technologist.Apart from the inflation of the air cushion, the performance of tomosynthesis including the positioning of the patient and compression of the breast does not differ from routine examinations. After tomosynthesis, the breast remains compressed in the same position until the ABUS scan is completed. ABUS and tomosynthesis images are transferred to a digital workstation where the corresponding coordinate systems are aligned so that both modalities are linked and can be analyzed side by side.2 with a maximum penetration depth of 10 cm, resulting in 585 slices for one volume. In comparison, standard ABUS covers an area of 16.8 × 15.4 cm2 with a penetration depth of 6 cm and 318 slices.ABUS acquisition by the prototype differs from a regular ABUS by covering an increased area of maximum 30 × 15.4 cmIn the study setting, a physician with more than 10 years of experience with ABUS systems evaluated the tomosynthesis and ABUS scans using the syngo.breast ultrasound software  together with an additional software tool for side-by-side matching of ABUS and tomosynthesis slides . He was blinded to the results of the standard diagnostic workup. Image quality of ABUS was rated subjectively by the physician on a scale ranging from 1 to 5. Categories 1 and 2 represent a quality below diagnostic applicability without (category 1) or with (category 2) identifiable breast structures. Categories 3 to 5 depict a sufficient quality for diagnostic applicability with a quality that is lower than that in HHUS (category 3), close to/comparable to HHUS (category 4), and equal or higher compared to HHUS (category 5). All detected lesions were measured and classified using BI-RADS® scores (18). To quantify the breast coverage in ABUS and tomosynthesis, the depicted breast area at the level of the nipple region was measured using the software Fiji  in both techniques. The breast coverage of tomosynthesis was used as a reference standard for the coverage of ABUS.The primary endpoint was the breast cancer detection rate in the study setting. Secondary endpoints were the detection and classification of benign findings, image quality, breast coverage, and the time of performance and interpretation of ABUS and tomosynthesis.p values are not corrected for multiplicity and must be interpreted descriptively. RStudio was used for statistical analysis . Mean with standard deviation or median with quartiles are given for values with normal or non-normal data distribution. To assess the primary endpoint, we correlated the malignant breast lesions described in the standard diagnostic workup with the findings from the study setting and compared localization, size, and BI-RADS® classifications. The difference in quality over the projections was tested for significance with the two-sided Fisher’s exact test, and the difference in the breast coverage of tomosynthesis and ABUS was tested for significance with the Mann-Whitney U test. Significance was set as α = 0.05.This explorative study is based on descriptive statistical methods. The One hundred fifty-two included patients received tomosynthesis using the prototype , 42 in mediolateral (ML), and one in craniocaudal (CC) projection, and were included in the analysis. The applied compression force was on average 106.30 ± 22.47 N and resulted in an averaged compression thickness of 51.83 ± 12.71 mm. The scanning time of ABUS ranged between 40 and 60 s, depending on the breast volume. The total time of performing ABUS and tomosynthesis ranged from 90 to 130 s. The average time for the interpretation of both ABUS and tomosynthesis images was 277 ± 113 s.2  and 123.37 cm2  in ABUS with a median coverage in ABUS of 80.0%  of the tomosynthesis coverage. The area covered in tomosynthesis was significantly larger than that in ABUS with a median difference of 31.57 cm2  .The median breast area measured in tomosynthesis was 162.54 cmp = 0.29, see Table All cases were rated as at least category 2, so tissue structures were identifiable in all cases with a median quality of 3 . In 86 of 101 cases (85.1%), ABUS image quality was rated as diagnostically useful. In 18 of 101 scans (17.8%), image quality was described as being close to HHUS (category 4). None of the scans was rated as 5 . There was no significant difference in the rating of image quality over the three projections  were classified as likely benign (BI-RADS® 1–3). Forty-eight of 101 cases (47.5%) were classified as unclear or suspicious (BI-RADS® 4–5) with the recommendation for histological confirmation by core-cut biopsy, which was performed in 47 of 101 patients (46.5%). Thirty-four carcinomas  were diagnosed in the study population  were identified and classified as suspicious (BI-RADS® 4 or 5) or unclear with the need for further diagnostic workup (BI-RADS® 0). Twenty-six of these 33 carcinomas (78.8%) were described in tomosynthesis and ABUS  detected in ABUS were completely visualized, two of 27 carcinomas (7.4%) were partly visualized due to proximity to the thoracic wall. Three of 27 carcinomas (11.1%) were classified as BI-RADS® 0 due to low image quality (category 2). One lesion was described in ABUS as suspicious (BI-RADS® 4) and was not seen in tomosynthesis. A core-cut biopsy confirmed the diagnosis of an invasive lobular carcinoma in the lesion not seen in tomosynthesis. In three cases, which were histologically confirmed as non-special type carcinoma (NST), ABUS led to a correct upgrading of the BI-RADS® classification of tomosynthesis: twice from BI-RADS® 4b to BI-RADS® 5 and once from BI-RADS® 4a to BI-RADS® 4b.Retrospective evaluation (by the unblinded physician in knowledge of the standard diagnostic workup) of the seven carcinomas not detected in ABUS revealed that five carcinomas (71.4%) were located outside of the area covered by the ABUS images. One of these carcinomas was not detected, by neither ABUS nor tomosynthesis. Two of seven carcinomas (28.6%) were not described in the interpretation of ABUS images in the study setting but could be localized in a retrospective second-look evaluation as discreet architectural distortions.n = 41), morphologically likely benign lesions with recommendation for follow-up , or were histologically benign after core-cut biopsy had been recommended based on imaging . One patient declined the biopsy. Ten of the twelve patients with recommendation for follow-up showed no evidence for malignant disease in the 12 months follow-up at our breast unit. Two patients failed to follow-up.Sixty-seven of 101 cases (66.3%) were classified as unsuspicious in the standard diagnostic workup, containing no or surely benign lesions  were rated concordantly in the standard diagnostic workup and the study setting. In six cases, lesions were classified as unclear in tomosynthesis (BI-RADS® 0) in the study setting, but could be correctly downgraded through ABUS (BI-RADS® 2). Further, 19 lesions were rated as BI-RADS® 3 or 4 in both the standard diagnostic workup and the study setting. In this group, four cases were classified as BI-RADS® 4 in tomosynthesis and as BI-RADS® 3 in ABUS in the study setting. Formally, ABUS led to a correct downgrading of these cases in the study setting.Twelve cases were described as unclear or suspicious (BI-RADS® 0 or 4) in the study setting, but were unsuspicious (BI-RADS® 1–3) in the standard diagnostic workup.Overall, the combined performance of tomosynthesis and ABUS led to a sensitivity of 97.1%  and a specificity of 59.7%  in the study setting (see Table n = 101 vs. n = 30) than the FUSION-X-US-I study [This is the first larger prospective cohort study on the use of a prototype combining ABUS and tomosynthesis in a clinical setting. The prototype worked technically reliable in most cases and the completed scans showed a high detection rate of carcinomas . The one carcinoma missed by the FUSION-X-US-II prototype was localized close to the thoracic wall in HHUS in the standard diagnostic workup. ABUS coverage of the breast in the FUSION-X-US-II prototype was improved by 21% compared to the previous prototype. Besides this technical aspect, the present study was performed in a much larger cohort , one should keep in mind that the cohort had a high prevalence of breast lesions to be described and classified. In a screening-like situation with a lower prevalence of breast lesions, the average time for the interpretation will be substantially shorter.Overall, in 85.1% of the cases, ABUS quality was rated category 3 or higher, which means that tissue structures were clearly visible and ABUS images were of diagnostic use. Image quality differed largely depending on the individual breast shape. In general, small breasts were harder to position under the compression paddle and therefore reaching adequate contact with the ultrasound transducer was difficult in some cases. On the hardware level, image resolution of ABUS is limited by the technical specifications of the ultrasound transducer. So far, no specific ultrasound transducer has been designed for the prototype. Adapting current high-end transducers to fit the requirements of the special prototype setup, e.g., regarding penetration depth and resolution, is necessary to reach higher image quality. Importantly, the prototype is not intended to replace high-resolution HHUS as a tool for clarification of unclear or suspicious lesions. It is rather meant to provide additional ABUS in the same orientation as tomosynthesis in a large number of patients who would otherwise not undergo any sonographic examination in a screening setting.Tomosynthesis and ABUS coverage were sufficient to cover the whole breast of all patients except for one patient with macromastia. In this case, we obtained two scans of both tomosynthesis and ABUS to cover the whole breast. This was no study-specific problem, so we did not exclude the scans in the analysis.ABUS coverage, which was 66.0% in the FUSION-X-US-I prototype, has been improved and now reaches a median coverage of 80.0%  of the volume covered in tomosynthesis . The incThe prototype showed a high detection rate of carcinomas . One of the carcinomas was only seen in ABUS and not in tomosynthesis. In this case, the combined analysis of ABUS and tomosynthesis showed a clear advantage over tomosynthesis alone see Fig. . In the Benign lesions were not assessed systematically in the study, because unsuspicious masses  are not routinely described in detail in the reports of the standard diagnostic workup. Regarding the BI-RADS® classifications on the case level, ABUS in the prototype setting led to a correct downgrading of ten cases, which were falsely described as suspicious or as unclear in tomosynthesis. On the other hand, the prototype decreased the diagnostic precision or formally led to a false upgrading of twelve cases compared to the standard diagnostic workup.This is the first larger prospective cohort study on the use of a prototype combining ABUS and tomosynthesis in a clinical setting. The prevalence of malignant lesions in the study cohort  was high enough to evaluate the detection of malignant lesions using the prototype.The results of this study show that a combined performance of ABUS and tomosynthesis in a clinical setting using the FUSION-X-US-II prototype is feasible and time efficient. The examination was fully integrated into the clinical workflow, so the clinical applicability of the results is given. Since the examination is performed by a radiologic technologist and the physician can afterwards interpret the images separately, there is a potential cost-saving factor. In the future, automatization and standardization to acquire clinical image data might gain further importance when image recognition algorithms are used to support clinical decision-making. Our analysis on automized and standardized ABUS combined with tomosynthesis is an important step to enable development of such algorithms in the near future.In most current screening programs, HHUS is only performed in patients with unclear mammographic findings. Hybrid devices like this prototype could potentially allow a higher number of patients to receive supplemental ABUS to mammography/tomosynthesis. Previous studies suggest that a widespread use of ABUS can lead to the detection of additional carcinomas , but furThe inclusion criteria led to a high prevalence of breast lesions (selection bias), which could be expected by the examiner (observer bias). To limit the observer bias, the examiner was blinded to the results of the standard diagnostic workup. In a screening population with much lower prevalence of breast lesions, the detection rate using the prototype will likely be lower than that in the study setting, which has to be considered regarding the potential applicability of the prototype for screening-like situations.We did not separate the readings of tomosynthesis and ABUS, because we tried to simulate a clinical workflow as realistically as possible where one would always aim to have as much comprehensive information available at the same time to give a diagnosis based on all diagnostic procedures. So, only the reading of tomosynthesis alone can clearly be separated from ABUS, while the reading of ABUS might have had a recall bias.As the scans were evaluated by only one observer, we were not able to calculate interobserver agreement, which is an important question regarding the applicability in screening-like situations and therefore should be assessed in the following studies. The image quality was also only assessed subjectively by one examiner, whose impression of image quality is influenced by several factors, e.g., image resolution, presence of artefacts, coverage, and personal experience leading.Similarly, the assessment of breast coverage is difficult, because there was no tool to measure the three-dimensional extension of each breast. Therefore, the measurements of tomosynthesis were used as a reference standard for the coverage of ABUS.Overall, this study has shown that a combined performance of tomosynthesis and ABUS with the FUSION-X-US-II prototype can be successfully implemented in a clinical workflow. Malignant lesions were accurately detected. For a clinical application, image quality and coverage of the prototype need to be improved. Further studies are needed to evaluate the potential benefit in a screening collective.


Dacryodes edulis (L.) powder as a substitute for margarine in the production of functional rice biscuits. The effect of substituting margarine and refined wheat flour with D. edulis powder locally called safou and rice flour, respectively, at different proportions was assessed for the sensory and physicochemical properties of the formulated biscuits. For this, statistical models were developed, validated, and optimized using the response surface methodology with the Doehlert design as a tool. The results showed that an increase in the substitution rate of margarine with of D. edulis powder enhanced the aroma while the substitution of refined wheat flour with rice flour led to an improvement of the overall quality of the biscuits. The optimal composition of dough for the production of biscuits with satisfying sensory properties was 20.24% of wheat flour, 24.51% of rice flour, 19.09% of margarine, and 2.47% of D. edulis powder. The optimized biscuit which scored the highest overall acceptability contained proteins (10.33 g/100 g DM), fat (27.66 g/100 g DM), crude fibers (2.5 g/100 g DM), ash (3.55 g/100 g DM), and carbohydrates (54.01 g/100 g DM). It has an energy density of 506.3 ± 0.1 kcal/g and could therefore be suitable for the management of malnutrition. Mineral analysis revealed that the biscuit contained sodium (0.200 mg/100 g), potassium (0.192 mg/100 g), phosphorus (0.123 mg/100 g), iron (33.60 ppm), and zinc (26.81 ppm) at levels satisfying the recommended daily intakes. The results of this study demonstrated the suitability of safou as substitute of margarine in the rice biscuit formulation and suggests the potential of the formulated biscuits in the management of malnutrition and noncommunicable diseases such as hypertension, obesity, and cardiovascular diseases.Trends in the food industry are nowadays directed towards the reduction of the level of trans fatty acids in food intended for human consumption. The present study was designed and aimed at valorizing  Bakery products with a good nutritional profile, sensory characteristics, and texture are nowadays most popular among all age groups and appreciated by consumers . Due to Wheat flour is a basic ingredient in the preparation of biscuits . HoweverD. edulis (L.)) a highly perishable local food [Besides rice, other local ingredients for which a growing attention is paid nowadays in biscuit formulation are those rich in health-promoting compounds. Pasqualone et al.  demonstrcal food  stands acal food , and sevcal food , 14. Safcal food . Hence, cal food , 17. In cal food . It systcal food . The objD. edulis (4 months) were harvested at the experimental farm of the Institute of Agricultural Research for Development (IRAD), located at Nkolbisson in the city of Yaoundé, Centre Region of Cameroon. The safou fruits were blue in colour, with a length and midpoint diameter of 10.0 cm and 5.4 cm, respectively. Mature sour fruits of Fine broken samples of rice, of TOX-3, and 145-TOC-38-2-3 varieties were collected from millers. These two varieties were chosen because they are mainly cultivated in the Ndop Rice Development Hub (rice hub), North West and West Regions of Cameroon .2 and dried in a Heraeus® D-6450 Hanau drying oven for 24 h at 55–60°C. The chips were removed, cooled at room temperature, crushed using a grinder, and sieved (1 mm mesh sizes). The powder obtained was packaged into plastic bags and stored at 4°C until use. The chemical composition of safou powder is presented in The method described by Eyenga et al.  was usedμm, and its chemical composition previously determined [Rice flour was prepared following the method of Eyenga et al. . Samplestermined  is consiThe method described by Eyenga et al.  was usedThe response surface methodology through a Doehlert design was used to perform the experiments. The independent variables and their ranges of variation were wheat flour 120–1200 g, rice flour 120–1200 g, margarine 60–600 g, and safou powder 60–600 g. The responses measured were hardness, aroma, crispness, granular, and overall acceptability of the biscuits.The independent variables were related to the assessed responses through a second-order polynomial model using the following equation. R2), the adjusted R2, the absolute analysis of average deviation (AAD), the bias factor (Bf), the accuracy factor (Af), and the lack of fit. Adequacy was considered when R2 ≥ 80%, adj − R2 ≥ 80%, AAD⟶0, 0.75 < Af < 1.25, and 0.75 < Bf < 1.25 [The indicators used for model validation were coefficient of determination  trained panelists were recruited from the staff of the Institute of Agricultural Research for Development (IRAD), Yaoundé, Cameroon. All panelists were volunteers and signed an informed consent sheet. A six-point scale was used to evaluate the biscuits from “0 = very low” to “5 = very high”. Panelists were recruited based on their regular consumption frequency of biscuits and their nonallergy to any foods. The panelists were asked to describe the intensity of the following descriptors: the flavor, hardness, granular, crispness, and overall quality of the biscuits. Samples were identified with three-digit code numbers and presented in a random sequence to the panelists. Water was provided to rinse the mouth between evaluations.The nutritional composition of the optimized biscuit was assessed. The water content of the biscuit was determined by the gravimetric method . The lipt-test was used to compare values and the statistical significance was set at p < 0.05.All experiments were performed in triplicates and values were expressed as means ± standard deviation. The data obtained was subjected to analysis of variance and regression analysis using Minitab software version 18.1 . Graphs were plotted using SigmaPlot 12.5 version 12.5.0.38 . The Student Y1 = crispness, Y2 = hardness, Y3 = aroma, Y4 = granulometry, Y5 = overall quality, X1 = wheat flour, X2 = rice flour, X3 = margarine, and X4 = safou powder.The results presented in R2), the adjusted R2, the absolute analysis of average deviation (AAD), the bias factor (Bf), the accuracy factor (Af), and the lack of fit.The adequacy of these models to explain the variability in the different responses was evaluated by the coefficient of determination  between the predicted and the experimental values as observed in The adequacy of the model equations for predicting the response values was tested by comparing the experimental and predicted values at optimum conditions. The results showed that there is no significant difference . The presence of crude fibers in the biscuit suggests their therapeutic potential as fibers are useful in the prevention of heart diseases, colon cancer, digestive disorders, and diabetes [The proximal composition of optimized biscuit illustrated in diabetes . The ashdiabetes . The enhdiabetes . The autGlobally, the nutritional value of the optimized biscuit was improved. The improvement observed could be attributed to safou powder used as substitute of margarine. In studies conducted by Ene-Obong et al.  and EyenThe mineral profile of the optimized biscuit was determined using atomic absorption spectrophotometry . Ca, Mg,The required daily intake (RDI) values for minerals in foods intended to human consumption were established by the United State Department of Agriculture . For K, In this study, a new product made with wheat and rice flours, margarine, and safou powder was developed using response surface methodology. The optimum composition of dough leading to a biscuit with the most appreciated granulometry, crispness, hardness, aroma, and overall quality was 20.24% wheat flour, 24.51% rice, 19.09% margarine, and 2.47% safou powder. The physicochemical analyses of the optimized biscuit revealed its richness in nutrients and its high energy density. The results of this study demonstrated the suitability of safou as substitute of margarine in the production of rice and suggests the potential of the formulated biscuits in the management of malnutrition and noncommunicable diseases such as hypertension, obesity, and cardiovascular diseases.