dataesr/scientific-paragraphs-categorization huggingface dataset:
The cDNAs of PSD-95, SAP97, SAP102 and PSD-93 (gift from Nathalie Sans, University of Bordeaux) were all from the rat species. Boundaries used for the first two PDZ domains (tandem) are the following residues 61-249 for PSD-95 (PSD-95-12), 145-333 for SAP102 (SAP102-12), 220-408 for SAP97 (SAP97-12) and 93-282 for PSD-93 (PSD-93-12). Boundaries for the isolated PDZ domains 1 and 2 of PSD-95 were, respectively, residues 61-152 and 155-249. Xph0 was synthesized as described above (Genscript) and Xph clones were directly PCR-amplified from the isolated colonies obtained at the end of the selection after sequencing. The S63K mutation in Xph clones (the numbering corresponds to the one used for the wild-type 10 FN3, PDB ID 1FNA), F119R in PSD-95 and R278F in SAP97 were inserted by site-directed mutagenesis.
After a last centrifugation of 30 s, the eluant containing the purified dU-ssDNA was saved. The ssDNA concentration was determined with a Nanodrop spectrophotometer (Thermo Scientific) and the quality was evaluated by electrophoresing 3 μL on a 0. 8% TAE/agarose gel at 100 V for 1 h. Combinatorial libraries in which the BC and FG loops of 10 FN3 were diversified were constructed by using pFunkel mutagenesis  33  , inspired by Kunkel mutagenesis. Five phosphorylated oligonucleotides were designed to introduce from 5 to 9 NNK random codons at position 25 in the 10 FN3 gene and seven phosphorylated oligonucleotides were designed to introduce 7 to 13 NNK random codons at position 75 corresponding to the BC and the FG 10 FN3 loops, respectively (see Supplementary Note 2 for the oligonucleotides sequence; the numbering corresponds to the one used for the wild-type 10 FN3, PDB ID 1FNA).
The entire remaining volume was then plated on dishes (Greiner bio-one, 145 mm × 145 mm × 20 mm) containing 2x YT agar-plate with 50 μg mL -1 carbenicillin, 10 mM glucose and incubated overnight at 37 °C. Each plate was recovered with 4 mL of 2x YT medium and scraped to recover librarycontaining TG1 cells. Two more washes with 2 mL of 2x YT medium were made to ensure maximal library recovery. Bacteria were centrifuged at 5000 × g for 10 min at 4 °C and resuspended in fresh 2x YT medium for two times. Bacteria were then flash-frozen with liquid nitrogen after the addition of 20% sterile glycerol and were kept at -80 °C for later use. Sequencing analysis software. SynDivA is a tool implemented in Python for analyzing multiple sequencing results in the context of directed evolution projects using diversified gene libraries.
SynDivA works as follows. First, SynDivA preprocesses the sequencing input data using BioPython  68. It determines the orientation of the sequences and their reading frame, translates the nucleotide sequences into protein sequences and then filters the sequences by removing the ones that do not have pre-defined restrictions sites or contain stop codons in variable regions. Pairwise alignment is performed on the valid sequences using NW-align (http:// zhanglab. ccmb. med. umich. edu/NW-align). Sequences are clustered using MCL algorithm  69  and multiple sequence alignment is done using Clustal Omega  70. SynDivA outputs all the results in an HTML report. SynDivA is available for use on the instance of Galaxy hosted on CBiB's server via https://services. cbib. u-bordeaux. fr/galaxy/?tool_id=fibronectin&version=1.
Next, 100 μL of 1-Step Ultra (3,3′,5,5′-tetramethylbenzidine) TMB-ELISA was added and the reaction was stopped with 100 μL of 1 M H 3 PO 4. Absorbance was read at 450 nm on a POLARstar Omega plate reader. The sequence of each clone was determined by performing Sanger sequencing of the 96 selected colonies. Analysis of the sequences was performed using SynDivA software. GST pull-down assay. Plasmids containing Xph clones in a pET-IG vector were transformed into BL21-CodonPlus (DE3)-RIPL competent cells and overnight precultures started from a single colony were used to inoculate 60 mL of LB. The cells were grown at 37 °C until an OD 600 of 0. 6 was reached. Induction was performed by adding 1 mM of IPTG and the cells were grown for an additional 16 h at 16 °C. The cells were then harvested by centrifuging 50 mL of the cultures at 3000 × g for 30 min at 4 °C.
Both the LED and the intensifier were modulated at frequency up to 100 MHz. A series of 12 images was recorded for each sample. By varying the phase shifts (12 times) between the illuminator and the intensifier modulation, we could calculate the phase and modulation for each pixel of the image. Consequently, we could determine the sample fluorescence lifetime image using the manufacturer's software LI-FLIM software. Lifetimes were referenced to a 1-μM solution of fluorescein in Tris-HCl (pH 10) or a solution of erythrosin B (1 mg mL -1 ) that was set at 4. 00 ns lifetime (for fluorescein) or that was set at 0. 086 ns (for erythrosin B)  39. Experiments other than Fig. 2f  were obtained using this method. For competition experiments, only cells presenting a high level of expression of the competitor or control as measured by mIRFP670 fluorescence were taken into consideration.
Datasets were analysed by an ANOVA test followed by a Tukey's Multiple Comparison Test 2 by 2. NMR spectroscopy. NMR spectra were recorded at 298 K using Bruker Avance III 700 MHz or 800 MHz spectrometers equipped with a triple resonance gradient standard probe or cryoprobe, respectively. Topspin versions 2. 1, 3. 2 and 3. 5 (Bruker BioSpin) were used for data collection. Spectra processing used NMRPipe  71  followed by analysis with Sparky 3 (T. D. Goddard and D. G. Kneller, University of California) or CARA (RLJ. Keller, ETH Zurich). Backbone chemical shift assignment of PSD-95. Spectra for the assignment of backbone 1 H N ,  13  C',  13  C α ,  13  C β and 15 N H nuclei of the first PDZ domain of rat PSD-95 (residues 61-152 with an N-terminal Ser-Gly-Ser-remaining after cleavage by TEV protease) were collected on a 345 μM 13 C,  15  N-labelled sample in PBS with 10% D 2 O added for the lock.
NMR assignment used 2D 15 N-HSQC, 3D HNCO, 3D HNCACO, 3D HNCA, 3D CBCACONH and 3D HNCACB spectra. The backbone assignment is nearly complete, except for missing assignments for the initial Ser-Gly-remaining from the His-tag, the backbone carbonyl for Asp91, and the amide 1 H N and 15 N H chemical shifts for Asn72, Ser73, Leu75 and Glu122. Also assigned are sidechain 15 N δ2 , 1 H δ2 and 13 C γ nuclei from Asn72, Asn85, Asn114 and Asn121, as well as sidechain  15  N ε2 , 1 H ε2 and 13 C δ nuclei from Gln107. Chemical shift assignments for PSD-95 PDZ1 were deposited in the Biological Magnetic Resonance Data Bank (BMRB) as entry 27309. An annotated  15  N-HSQC is included in Supplementary Fig. 13a. Spectra for the assignment of backbone 1 H N ,  13  C',  13  C α , 13 C β and 15 N H nuclei of the second PDZ domain of rat PSD-95 (residues 155-249 with an N-terminal Ser-Gly-Ser-remaining after cleavage by TEV protease) were collected on a 196-μM 13 C,  15  N-labelled sample in PBS with 10% D 2 O added for the lock.
NMR assignment used 2D  15  N-HSQC, 3D HNCO, 3D HNCACO, 3D HNCA, 3D CBCACONH and 3D HNCACB spectra. The backbone assignment is nearly complete, except for missing assignments for the initial Ser-Gly-Ser-remaining from the His-tag, all nuclei for Pro16, and the amide 1 H N and 15 N H chemical shifts for Lys168, Leu170 and Ser217. Also assigned are sidechain 15 N δ2 , 1 H δ2 and 13 C γ nuclei from Asn180, Asn187, Asn216, Asn234 and Asn248, as well as sidechain  15  N ε2 , 1 H ε2 and 13 C δ nuclei from Gln181 and Gln207. Chemical shift assignments for PSD-95 PDZ2 were deposited in the Biological Magnetic Resonance Data Bank (BMRB) as entry 27310. An annotated  15  N-HSQC is included in Supplementary Fig. 13b. Spectra for the assignment of backbone 1 H N ,  13  C',  13  C α , 13 C β and 15 N H nuclei of the tandem PDZ1-PDZ2 domains of rat PSD-95 (residues 61-249 with an Nterminal Ser-Gly-Ser-remaining after cleavage by TEV protease) were collected on an 80 μM 2 H,  13  C,  15  N-labelled sample in PBS with 10 % D 2 O added for the lock.
NMR assignment used 2D 15N-TROSY, 3D TROSY-HNCO, 3D TROSY-HNCACO, 3D TROSY-HNCA, 3D TROSY-CBCACONH, and 3D TROSY-HNCACB spectra. Assignments are missing for all nuclei in Pro153, Pro154, Lys157, Pro167 and the initial Ser-Gly-remaining from the His-tag. Amide 1 H N and 15 N H chemical shifts are missing for Asn72, Ser73, Leu75, Ser217, Asn121, Glu122, Ala155, Lys168 and Leu170, and the backbone carbonyl is missing for Asn72, Asp91, Asn121, Ile183, His225 and Glu226. Residues His225, Glu226, Asp227, Leu232 and Tyr236 lack an assigned  13  C α , and residues His225, Glu226, Asp227, Ala228 and Lys233 lack an assigned  13  C β. Chemical shift assignments for PSD-95 PDZ1-PDZ2 were deposited in the Biological Magnetic Resonance Data Bank (BMRB) as entry 27308. An annotated  15  N-HSQC is included in Supplementary Fig.
The top and bottom plugs were inserted and the 1 J H-N + 1 D H-N values were measured by again using interleaved spin state-selective TROSY experiments, averaged from two separate measurements. Subtraction of average 1 J H-N from average 1 J H-N + 1 D H-N yielded 1 D H-N values for 60 residues (24 from PDZ1, 36 from PDZ2). Docking-based models. Models were generated by using HADDOCK  73  as implemented in the HADDOCK2. 2 webserver 74 made available by WeNMR  75. For Xph15, an homology model was first generated by using SWISS-MODEL with the template PDB entry 3RZW, and the PDZ1 domain of PSD-95 was taken from the PDB entry 3GSL. The HADDOCK protocol used the WeNMR GRID-enabled docking server with the Easy interface and 1000 calculated structures. Active residues were identified by chemical shift perturbation analysis (Supplementary Figs.
14  and 17 ) and predicted solvent accessibility, with passive residues automatically generated. Active residues included residues 28-31, 33, 55, 56, 58 and 79-82 from Xph15, and residues 62-65, 67, 118, 119, 121, 124, 145, 147 and 149 from PSD-95-1. From the 1000 calculated structures, 200 structures were further refined in explicit water, and 186 of these final structures (93. 0%) were clustered into six clusters, from which the top cluster based on Z-score (-1. 8) was taken as the predicted ensemble. For Xph20, the protocol was similar and used an homology model for Xph20 based on PDB entry 3K2M. Active residues were again selected based on chemical shift perturbation (Supplementary Figs. 15  and 18 ) coupled with solvent accessibility. Active residues on Xph20 included residues 29-31, 33, 57, 58,  60, 81 and 83-88, and active residues from PSD-95-1 included 62-65, 67, 118, 119,  121, 124, 126, 143, 145, 147, 149 and 150.
From the 200 water-refined structures, 174 (87. 0%) were clustered into 11 clusters, with the top cluster selected as the predicted ensemble (Z-score of -1. 7). For Xph18, the homology model was based on PDB entry 3RZW but the model calculation strategy was carried out in two steps. Unlike Xph15 and Xph20, initial chemical shift perturbation studies using Xph18 with isolated PSD-95-1 and PSD-95-2 did not reveal binding to either domain. Upon repeating the study with doubled protein concentrations, numerous 1 H,  15  N crosspeaks for PSD-95-1 displayed perturbation or peak broadening, with still no observed binding to PDZ2 (see Supplementary Fig. 16 ). Therefore, an initial HADDOCK docking used Xph18 and PDZ1, with active residues for PSD-95-1 including residues 61, 63, 65, 67, 90, 91, 119, 124, 127, 145, 147, 149 and 151.
Active residues for Xph18 comprised solvent accessible residues from the BC, DE and FG loops (residues 28, 29, 30, 31, 32, 33, 34, 35, 57, 58, 59, 80, 81, 82, 83, 48, 85,  86, 87, 88 and 89). The main cluster represented 123 of the 200 calculated models, with a HADDOCK score of -115. 9 and a Z-score of -2. 5. The lowest energy model from this cluster was then used to guide structure calculation within ARIA2. 3/CNS1. 2 for the complete PSD-95-12:Xph18 complex, by maintaining the structure of Xph18:PDZ1 complex with synthetic NOE distances derived from the HADDOCK model. Additional NOE distances and dihedrals were obtained for all non-hydrogen atoms in PDB entry 3GSL, while leaving linker residues 151 to 155 free of any restraints. HADDOCK-type restraints were included for contacts between PDZ2 and Xph18.
For X100 experiments, settings only differed for the dissociation phase (170 s). Regeneration of the functionalized surface was achieved with the standard regeneration solution supplied with the Biotin CAPture kit. All sensorgams were double referenced prior to analysis by first subtracting data from a reference flow cell functionalized with the capture reagent but on which no ligand was attached and then subtracting a blank cycle where buffer was injected instead of the protein sample. The kinetic data were analysed using a 1:1 Langmuir binding model of the BIAevaluation software with the bulk refraction index (RI) kept constant and equal to 0 and the mass transfer constant (t c ) kept constant and equal to 10 8. Affinity from steadystate (equilibrium) was obtained by fitting a plot of the response at equilibrium against the concentration with the one site binding (hyperbola) equation from GraphPad Prism 7.
04. Isothermal titration calorimetry. ITC experiments were performed in PBS 1 × buffer pH 7. 4 for Xph15 and Xph20 and in PBS 1 × pH 7. 4 with 0. 01% Tween 20 using a MicroCal iTC200 (GE Healthcare). The titration sequence included a single 0. 5-μL injection followed by 29 injections, 1 μL each, with a spacing of 180 s between the injections. Concentrations of the protein used for the titrations (Xph) were in the range of 18-40 μM, while the ligand in the syringe (PSD-95-12) was in the range of 0. 25-0. 44 mM. All experiments were performed at 25 °C. As a blank, an independent experiment with only buffer in the calorimeter's cell was performed with the same ligand solution of PSD-95-12 in the syringe to determine the corresponding heats of dilution. The experimental thermograms were baseline corrected, blank subtracted and the peaks were integrated to determine the binding heats produced by each ligand injection with the OriginLab Software (OriginLab, Northampton, MA).
Uncropped gels are provided in the Supplementary Information. The gels were then analysed with the ImageLab software (lanes and bands detections) and the intensity of the band corresponding to the tandem PDZ domains without competitor was taken as the 100% intensity. The relative intensities were reported in GraphPad Prism 7. 02 software in function of the logarithm of the competitor concentration and the data were fitted to the competition binding equation "One site -Fit Ki" with constrained values for the concentration of labelled ligand (tandem PDZ domains, in nanomolar) and for the equilibrium dissociation constant of the labelled ligand (K I for [Stg 15 ] 2 determined by fluorescence polarization in nanomolar). Pull-down on brain lysates. Adult rat brain lysates were produced as previously described  25.
For the anti-PSD-95 coIP, 10 μg of the starting material and all Co-IP elution were loaded on SDS-PAGE, transferred to nitrocellulose membrane. Dried membrane was blocked with Odyssey blocking buffer and incubated overnight with a mouse anti-PSD-95 (Millipore-Merck, MAB1596) 1/1000 and with a mouse anti-Histidine tag (Sigma-Aldrich, H1029) 1/1000. Following incubation with secondary antibody anti-mouse light chain IRDye-800 (Jackson ImmunoResearch, 115-655-174) (1/15,000), the blots were washed, dried and imaged using an Odyssey Imaging System. Analysis was done using the Odyssey Image Studio Lite Ver5. 2 software and quantification of the co-immunoprecipitated Xph20-ETWV fraction normalized to the IP fraction of PSD-95 on the same lane was performed using the average intensity of each single band on linear range values.
Peptide mixture was analysed on an Ultimate 3000 nanoLC system (Dionex, Amsterdam, The Netherlands) coupled to an Electrospray mass spectrometer. For the analysis of pull-down material, 10 μL of peptide digests were loaded onto a 300-μm-inner diameter × 5-mm C 18 PepMapTM trap column (LC Packings) at a flow rate of 10 μL min -1. The peptides were eluted from the trap column onto an analytical 75-mm-inner diameter × 50-cm C 18 Pep-Map column (LC Packings) with a 4-40% linear gradient of solvent B in 105 min (solvent A was 0. 1% formic acid and solvent B was 0. 1% formic acid in 80% ACN). The separation flow rate was set at 300 nL min -1. The mass spectrometer operated in positive ion mode at a 1. 8-kV needle voltage. Data were acquired on an Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer (Thermo Fisher Scientific, San Jose, CA) using Xcalibur 4.
The peptides were eluted from the trap column onto an analytical 75-mm-inner diameter × 25-cm C 18 Pep-Map column (LC Packings) with a 4-40% linear gradient of solvent B in 108 min (solvent A was 0. 1% formic acid in 5% ACN, and solvent B was 0. 1% formic acid in 80% ACN). The separation flow rate was set at 300 nL min -1. The mass spectrometer operated in positive ion mode at a 1. 8-kV needle voltage. Data were acquired on an Electrospray Q-Exactive quadrupole Orbitrap benchtop mass spectrometer (Thermo Fisher Scientific, San Jose, CA) using Xcalibur 2. 2 software in a data-dependent mode. MS scans (m/z 350-1600) were recorded at a resolution of R = 70,000 (at m/z 200) and an AGC target of 3 × 10 6 ions collected within 100 ms. Dynamic exclusion was set to 30 s and top 12 ions were selected from fragmentation in HCD mode.
MS/MS scans with a target value of 1 × 10 5 ions were collected with a maximum fill time of 100 ms and a resolution of R = 17,500. Additionally, ions with 2-7 charges were selected for fragmentation. Others settings were as follows: no sheath nor auxiliary gas flow, heated capillary temperature, 250 °C; normalized HCD collision energy of 27% and an isolation width of 2 m/z. For the pull-down material analysis, data were searched by SEQUEST through Proteome Discoverer 2. 3 (Thermo Fisher Scientific Inc. ) against three bait protein sequences imbedded in the Rattus norvegicus Reference Proteome Set (from Uniprot 2019-05; 29938 entries). Spectra from peptides higher than 5000 Da or lower than 350 Da were rejected. The search parameters were as follows: mass accuracy of the monoisotopic peptide precursor and peptide fragments was set to 10 ppm and 0.
Quantitative data were considered for proteins quantified by a minimum of two peptides, fold changes above 2 and a statistical p-value < 0. 05. For the photocrosslinking experiments, data were exported as mgf files, and StavroX 3. 6 (Michael Götze, University of Halle-Wittenberg, Department of Biochemistry & Biotechnology, General Biochemistry)  79  was used for the detection of crosslinks between ligands incorporating pAzF and every possible amino acids in a database consisting of PDZ domains of PSD-95 embedded in ten yeast protein sequences. Stavrox analysis was performed with the following settings: proteolytic cleavage: C-terminal at Lys and Arg with 3 and 2 missed cleavages, respectively, minimum peptide length: 5, static modification: alkylation of Cys by IAA, variable modification: oxidation of M, cross-linker: AzidoPhe (-N 2 ), precursor and fragment mass accuracy: 10 ppm, signal-to-noise ratio: 2, precursor mass correction activated, prescore cut-off at 10% intensity, FDR cut-off: 5%.
Images of LRRTM2 and PSD-95 were acquired using the MetaMorph software (Molecular Devices) under the same acquisition parameters. Analysis was performed using a custom-made macro on MetaMorph. Briefly, images of AP-LRRTM2 and PSD-95-eGFP were segmented using a wavelet-based segmentation method to identify clusters. Segmented images from the AP-LRRTM2 signal were used to draw regions of LRRTM2 clusters. These regions were transferred onto the segmented images of PSD-95 to determine the percentage of LRRTM2 clusters containing PSD-95 (apposed and colocalized clusters). Datasets were analysed by a non-parametric ANOVA test followed by a Dunn's Multiple Comparison Test 2 by 2. Molecular graphics. All figures depicting the protein structures were generated with PyMOL (Version 2. 0. 7 Schrödinger, LLC).
, 13  C and 15  N chemical shift assignments for PSD-95-12, PSD-95-1 and PSD-95-2 were deposited in the Biological Magnetic Resonance Data Bank (BMRB) as entries 27308, 27309 and 27310, respectively. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD015313 (cellular target identification) and PXD015366 (photocrosslinking). Key plasmids constructed in this study are available directly from Addgene at https://www. addgene. org/Matthieu_Sainlos/. The authors declare that the data supporting the findings of this study are available within the paper, its Supplementary Information file and Source Data file. Additional raw data and other materials are available from the corresponding authors upon reasonable request.
Acknowledgements This research was financially supported by grants from the  Centre National de la Recherche Scientifique , the  Conseil Régional de la Nouvelle Aquitaine , the  National Infrastructure France BioImaging  (grant  ANR-10INBS-04-01 ), the  Agence Nationale de la Recherche  ( CheMoPPI ,  ANR-13-BS07-0019-01 ) to C. M. , C. P. and M. S. , the  European Research Council  advanced grants nano-dyn-syn (232942) and ADOS ( 339541 ) to D. C. , the  Labex BRAIN  ( ANR-10-LABX-43 ) to C. R. and a  Marie-Curie postdoctoral training grant  to  D. G. B. (neuroCHEMbiotools,  FP7-PEOPLE-2010-IEF  project # 273817 ). We also thank the  Biochemistry and Biophysics Core Facility of the Bordeaux Neurocampus  funded by the  Labex BRAIN  ( ANR-10-LABX-43 ) and  J. M. Blanc  and  Y. Ruffin  for technical assistance as well as the  Structural Biophysico-Chemistry plateform  ( UMS3033/US001 ) of the  Institut Européen de Chimie et Biologie (Pessac, France ) for access to the  T200 Biacore instrument  and  Laetitia Minder  for technical assistance and the IINS cell culture facility and  Emeline Verdier  for technical assistance.
Financial support from the  IR-RMN-THC Fr3050 CNRS  for conducting the research is gratefully acknowledged. The  FP7 WeNMR  (project#  261572 ),  H2020   West-Life  (project # 675858 ) and the  EOSC-hub  (project # 777536 ) European e-Infrastructure projects are acknowledged for the use of their web portals, which make use of the EGI infrastructure with the dedicated support of  CESNET-MetaCloud, INFN-PADOVA, NCG-INGRID-PT, TW-NCHC, SURFsara  and  NIKHEF , and the additional support of the national  GRID Initiatives of Belgium, France, Italy, Germany , the  Netherlands, Poland, Portugal, Spain, UK, Taiwan  and the  US Open Science Grid.
Backbone 1 H 1 , 13  C and 15  N chemical shift assignments for PSD-95-12, PSD-95-1 and PSD-95-2 were deposited in the Biological Magnetic Resonance Data Bank (BMRB) as entries 27308, 27309 and 27310, respectively. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD015313 (cellular target identification) and PXD015366 (photocrosslinking). Key plasmids constructed in this study are available directly from Addgene at https://www. addgene. org/Matthieu_Sainlos/. The authors declare that the data supporting the findings of this study are available within the paper, its Supplementary Information file and Source Data file. Additional raw data and other materials are available from the corresponding authors upon reasonable request.