dataesr/scientific-paragraphs-categorization huggingface dataset:
The third section involves a covalent linkage between the Ig-like domain and the hinge region. Two disulfide bonds (Cys124-Cys147 and Cys128-Cys153) connect a loop in the hinge region, which is stabilized by electrostatic interactions between Gln131 and main chain functional groups, to a linker that leads into the first strand of the Iglike domain. SPACA6 is structurally similar to IZUMO1. The SPACA6 ectodomain structure and the separate 4HB and Ig-like domain structures were used to search the Protein Data Bank for structurally similar entries  42. We identified matches with high Dali Zscores, small root mean square deviations, and large LALI scores (the latter indicates the number of structurally equivalent residues). While the top 10 hits from the full ectodomain search (Table  S1 ) have reasonable Z scores of >8  42  , searches of only the 4HB or Ig-like domains revealed that the majority of these hits align only to the β-sandwich, a ubiquitous fold in many proteins.
The secondary structure element of the β-sheet is shared between IZUMO1 and SPACA6, although the strands are much longer in IZUMO1, and the 3 10 helix (Helix 5) is unique to SPACA6. These discrepancies result in different domain orientations of the two otherwise similar proteins. SPACA6 and IZUMO1 are founding members of a conserved protein superfamily. Our Dali server search revealed that SPACA6 and IZUMO1 are the only two experimentally determined structures deposited in the Protein Data Bank that share this particular 4HB fold (Table  S1 ). Recently, DeepMind (Alphabet/Google) developed AlphaFold, a neural network-based system that accurately predicts protein 3D structure from a primary sequence  48. Shortly after we solved the SPACA6 structure, the AlphaFold Database was released, providing predicted structural models that cover 98.
Methods Cloning and expression of recombinant SPACA6 ectodomain. The DNA sequence corresponding to the predicted ectodomain of human SPACA6 (NCBI Accession Number NP_001303901. 1; residues 27-246) was codon optimized for expression in Drosophila melanogaster S2 cells and synthesized as a gene fragment (Eurofins Genomics) with an encoded Kozak sequence, BiP secretion signal, and appropriate 5′ and 3′ overhangs for ligation-independent cloning of the gene into the metallothionein promoter-based pMT expression vector modified for puromycin selection (pMT-puro). The pMT-puro vector encodes for a thrombin cleavage site followed by a C-terminal 10x-His tag (Fig. S2 ). Stable transfection of the SPACA6 pMT-puro vector into D. melanogaster S2 cells (Gibco) was performed similar to protocols used for IZUMO1 and JUNO  43.
Four scans were taken per temperature point, averaged, and baseline corrected. The resultant change in ellipticity was normalized between 0 (folded) and 1 (unfolded) and fit to a non-linear biphasic sigmoidal curve using the program GraphPad (version 8. 4. 3) to determine the apparent melting temperature (T m ). Secondary structural composition (helices, strands, turns and coils) was estimated using the CONTIN method and the Set3 library (185-240 nm) using the Dichroweb server  67. SEC-MALS analysis. The oligomeric state of tag-removed, fully glycosylated SPACA6 was assessed by SEC-MALS. 0. 14 mg Bovine Serum Albumin (BSA) and 0. 14 mg SPACA6 were prepared in 1X PBS at a concentration of 1. 2 mg mL -1. A Superdex 75 10/300 GL size-exclusion column (Cytiva/GE) was equilibrated overnight with 5 CV PBS.
Monomeric BSA (MW = 66,432 Da) was used as a reference calibration standard. Prior to SEC-MALS analysis, each sample was centrifuged at 15,000 × g for 15 min at 4 °C and then the supernatant was loaded onto the size-exclusion column on an AKTA Pure FPLC (Cytiva) at 0. 2 mL min -1. Triple detection was performed by measuring absorbance at 280 nm using the integrated UV monitor on the AKTA Pure, three-angle light scattering using the miniDAWN TREOS MALS detector (Wyatt) and refractive index (RI) using Optilab T-rEX RI detector (Wyatt). The data were processed, and weight-averaged molecular mass was calculated using the ASTRA software package (version 7. 0. 2. 11). Sparse matrix crystallization and data collection. Sitting-drop sparse matrix crystallization screening was performed using a Douglas Instrument Oryx8 liquid handling system.
The crystals used to solve the structure were obtained by the microseeding protocol using a precipitant solution composed of 200 mM ammonium chloride, 20% (w/v) PEG 3350, and 34 mM FOS-choline-8. Crystals were looped and coated with perfluoropolyether cryo oil (Hampton Research) before being flash cooled in liquid nitrogen (100 K). Multiple native datasets were collected at the AMX (17ID-1) beamline at the National Synchrotron Light Source-II (NSLS-II) at Brookhaven National Laboratory (Upton, NY). Data were indexed and integrated using DIALS  68  and averaged and scaled using Aimless in the CCP4 suite  69.
Structure determination. No usable molecular replacement model was available, be it solved structure of AlphaFold model. Therefore, attempts to solve the structure of SPACA6 via SAD phasing were made, with multiple datasets collected at the NE-CAT (24-ID-C) beamline at the Advanced Photon Source (Argonne National Laboratory, Lemont, IL); phasing attempts with tantalum clusters and/or intrinsic sulfurs were fruitless. Eventually, initial phases were determined using the human IZUMO1 model (PDB: 5JK9) as a starting template for ARCIMBOLDO_SHREDDER  70, 71. ARCIM-BOLDO_SHREDDER carried out expected log-likelihood gain-guided molecular replacement of template-generated fragments using the program Phaser  72. During molecular replacement, additional refinement, such as gyre refinement against the rotation function, and gimble refinement after translation, was employed to refine the internal geometry of the fragments, allowing additional degrees of freedom by subdivision in rigid groups  73.
Consistently placed and refined fragments were combined in reciprocal space with ALIXE  74. Best-scored phase sets were subject to density modification and autotracing in SHELXE  75. The solution from ARCIMBOLDO_SHRED-DER provided clear density for ~65% of the structure, successfully built with a CC of 41. 6%. X-ray data collection statistics are presented in Table  1. To complete the model, Br-SAD phasing was performed and combined with the phases from the ARCIMBOLDO_SHREDDER model. SPACA6 crystals were soaked in 200 mM ammonium chloride, 20% (w/v) PEG 3350, 34 mM FOScholine-8, 25% (v/v) glycerol, and 1 M sodium bromide for 10 s before flash cooling directly into liquid nitrogen. Data were collected on the NE-CAT (24-ID-C) beamline at the Advanced Photon Source (Argonne National Laboratory, Lemont, IL).
Data were indexed and integrated using DIALS  68  , followed by scaling and merging using Aimless from the CCP4 suite  69. X-ray data collection statistics are presented in Table  1. The Br-SAD dataset is not isomorphous to the native dataset, likely due to cryo solution-induced dehydration of the crystal. PHENIX_Autosol was used to carry out a combination of molecular replacement and SAD phasing  76, 77. The atomic positions of ten bromide ions were found using a hybrid substructure search in PHENIX_Autosol. Model phases calculated from a molecular replacement solution using the ARCIMBOLDO model was combined with the anomalous signal from the Br-SAD data. PHENIX_AutoBuild was used to build the majority (~80%) of the SPACA6 model. The final PHENIX_AutoBuild model was subjected to an initial round of simulated annealing torsion angle refinement starting at 5000 K using PHENIX_refine  78.
The complete SPACA6 model was built with iterative rounds of manual model building followed by coordinate refinement using Coot  79  and PHENIX_refine  78  , respectively. Riding hydrogens and Translation/Liberation/Screw groupings were included to improve the structure. The program PDB-REDO  80  was used to provide a final geometry clean up prior to model validation using Ramachandran, geometry, and rotamer analysis, difference map peak searches in Coot  79  , and MolProbity 81 via PHENIX. All structural images were generated using The PyMOL Molecular Graphics System (Version 2. 0 Schrödinger, LLC). Electrostatic calculations were done using the APBS web server  82. Biolayer interferometry. The binding affinities of SPACA6 to IZUMO1 and JUNO were measured by BLI using a single-channel BLItz instrument (FortéBio/Sartorius).
Subsequently, the streptavidin-coated biosensor was immersed into BLI kinetics buffer for 120 s to dissociate the analyte. BSA and BLI kinetics buffer only against streptavidincoated biosensors loaded with biotinylated bait were used to quantify non-specific binding. The data were analysed and sensorgrams were step corrected, reference corrected, and fit to a 1:1 binding model. Reported results are representative of independent duplicate trials. Pull down assay. Bait protein (IZUMO1 or JUNO) tagged with 10x-His tags were mixed with target protein (JUNO or SPACA6) in a 1:1 molar ratio in Buffer B at a total volume of 100 µL. The samples were incubated on a rotator for 1 h at room temperature and then incubated with 15 µL of a 25% slurry of nickel-charged MagBeads (Genscript) for an additional 1 h on the rotator at room temperature.
5 mL min -1 on a Shimadzu bio-inert HPLC system for in-line SAXS measurements. Flow from the column was split 2:1 using a passive splitter between the X-ray scattering measurements and the UV/Vis and refractive index detectors. Subsequent buffer subtraction, peak selection, and profile analysis was undertaken using Lixtools  83  and the ATSAS (3. 0. 3) software package  86. Ab initio reconstructions of SPACA6 were performed using DAMMIN  87, 88. Twenty independent DAMMIN models were superimposed and averaged using DAMAVER  89  to obtain a consensus molecular envelope. The final SPACA6 structure was superimposed onto the SAXS molecular envelope using SUPALM function as part of the SASpy PyMOL plugin  90. Hydrogen/deuterium-exchange mass spectrometry. Deuterium exchange was initiated by mixing 25 µL of untagged SPACA6 ectodomain (4 mg mL -1 ) with 75 µL D 2 O buffer (8.
3 × 50 mm, 130 A, 17 µm) using a linear gradient of acetonitrile from 5% to 45% over 30 min. MS analysis was performed using the OrbiTrap Elite Mass Spectrometer (Thermo Scientific) with a capillary temperature of 200 °C. Data were acquired in both data-dependent MS/MS mode and MS1 profile mode, and the data were analysed by Proteome Discoverer software and HDExaminer (Sierra Analytics Inc. ). Statistics and reproducibility. For CD thermal melts, the results are presented as mean±standard deviation (SD). Independent triplicates, n = 3, were performed for all CD spectroscopy studies. Independent duplicates, n = 2, were performed for BLI and pull downs studies.
Acknowledgements We thank  Dr. Vitor Hugo B. Serrão  and  Dr. Karen K. Siu  for their assistance with the biophysical studies and the crystallography, respectively. We thank the staff on  Beamline 16ID  (LiX) and 17ID-1 (AMX) at the  National Synchrotron Light Source II (NSLS-II ), and Beamline  24ID-C (NE-CAT ) at the  Advanced Photon Source (APS)  for synchrotron access and support. This work was supported by funding from the  Canadian Institutes of Health Research (CIHR ;  PJT-153281 ) and  Canada Research Chair  ( CRC-2017-00140 ) to J. E. L. and by a joint grant to J. E. L. and A. Z. from the  New Frontiers in Research Fund  ( NFRFE-2019-00230 ). Biophysics and structural biology infrastructure were supported by funding from the  Canada Foundation for Innovation-John R. Evans Leaders Fund.
T. D. R. V. was supported by a  CIHR   Postdoctoral Fellowship. The phasing of the structure is based upon research conducted at the  Northeastern Collaborative Access Team beamlines , which are funded by the  National Institute of General Medical Sciences  from the  National Institutes of Health  ( P30 GM124165 ). This research used resources of the  Advanced Photon Source , a  U. S. Department of Energy (DOE) Office of Science User Facility  operated for the  DOE Office of Science by Argonne National Laboratory  under Contract No. DE-AC02-06CH11357. Support for work performed at the  Center for Biomolecular Structure beamline LIX  ( 16ID) | AMX (17ID-1 ) | FMX ( 17ID-2 ) at NSLS-II is provided by  NIGMS-1P30GM133893  and  BER-BO 070. NSLS-II  is supported by  DOE ,  BES-FWP-PS001.
Data availability Further information and requests for resources and reagents are available from the corresponding author on reasonable requests. Atomic coordinates and structure factors for SPACA6 have been deposited in the Protein Data Bank (PDB) with the accession code: 7TA2. SAXS data have been deposited in the Small Angle Scattering Biological Data Bank (SASBDB) with the accession code: SASDNM3. Source data are provided with this paper, including unedited/uncropped gels in Fig. S15.
32 R merge (%) a,b 6. 6 (65. 4) 10. 8 (71. 6) R meas (%) a,c 8. 8 (91. 8) 11. 6 (81. 5) R pim (%) a,d 4. 6 (48. 5) 4. 1 (30. 3) CC1/2 a 0. 997 (0. 675) 0. 998 (0. 871) R work (%) a,e 27. 6 (29. 3) 20. 1 (22. 0) R free (%) a 29. 8 (30. 5) f 25. 5 (30. 1) g Number of non- 1592 1811 hydrogen atoms macromolecules 1592 1736 bromide N/A 10 solvent 0 65 RMSD (bonds; Å) 0. 010 0. 005 RMSD (angles; °) 1. 2 1. 0 Ramachandran 90. 3 95. 4 favored (%) Ramachandran 6. 6 4. 6 allowed (%) Ramachandran 3. 1 0. 0 outliers (%) Clashscore 9. 6 1. 8 Average B-factor (Å 2 ) 44. 5 44. 0 macromolecules 44. 5 44. 3 bromide N/A 50. 9 solvent N/A 34. 4 Number of TLS groups N/A 7 PDB accession number 7TA2 a Statistics for the highest-resolution shell are shown in parentheses. b R merge = Σ hkl Σ j |I j -<I>|/Σ hkl Σ j |<I>|, where I j and <I> represent the diffraction intensity values of the individual measurements and the corresponding mean values, respectively.
Acknowledgements This work is supported by a public grant overseen by the  French National Research Agency (ANR)  as part of the  "Investissements d'Avenir" program  (reference:  ANR-11-LABX-0039 ,  Labex CHARMMMAT). We thank  Prof. Joanne Xie  and  Jonathan Long  for fruitful discussions, and  Dr. Stéphane Maisonneuve  and  Jean-Pierre Baltaze  for their help with the NMR experiments. We also gratefully acknowledge one of the reviewers for his constructive comments and suggestions to improve this manuscript.
To reproduce the band shape the vertical transitions from each snapshot were convoluted using Gaussian functions of full-width at half length (FWHL) of 0. 15 eV. The nature of excited states was assessed using Natural Transition Orbitals (NTOs) and ΦS indices, defining the overlap between the attachment and the detachment densities that is, the electron density removed/rearranged during the excitation. NTOs and ΦS were obtained with the Nancy_EX code  53, 54  and visualized with the Avogadro 55 software package. Other parameters describing the degree of overlap between HOMO and LUMO orbitals, such as D-index, S-index and t-index were computed using the Multiwfn 56 software package. SOC elements between S1, and Tn states involved in RISC mechanism were calculated with Amsterdam Density Functional Code (ADF),  57  at the M06-2X/DZP  36, 39  level of theory.
Acknowledgements This work is partially supported by the LPCT resources,  TUBITAK ULAKBIM High Performance and Grid Computing Center  (TRUBA resources) and  National Center for High Performance Computing of Turkey (UHEM). SC and PU thank  TUBITAK  (Project Number:  118Z914 ) and  BAP-M (16863)  for financial support. A. M. also thanks  ANR  and  CGI (Commissariat à l'Investissement d'Avenir ) for their financial support of this work through  Labex SEAM (Science and Engineering for Advanced Materials and devices) ,  ANR 11 LABX 086  and  ANR 11 IDEX 05 02. The support of the IdEx " Université Paris 2019 "  ANR-18-IDEX-0001  and of the Platform  P3MB  is gratefully acknowledged.
Acknowledgements M. W. , X. -L. S. , and Z. -H. Z. contributed equally to this work. This work was supported by the  National Natural Science Foundation of China  (Grant No. 11604212 ), the  National Natural Science Foundation of Guangdong Province of China  ( 2022A1515010929 ),  Science and Technology plan project of Shenzhen  ( 20200811230408001 ). ZGC thanks the financial support from the  Australian Research Council ,  HBIS-UQ Innovation Centre for Sustainable Steel  project, and  QUT   Capacity Building Professor Program. The authors are thankful for the assistance on STEM-HAADF observation received from the  Electron Microscope Center of the Shenzhen University. Open access publishing facilitated by  Queensland University of Technology , as part of the  Wiley -Queensland University of Technology agreement via the Council of Australian University Librarians.
Funding: This research was supported by the  National Science Foundation  through  NSF-DMR 2003057 , and by the  Agence Nationale de la Recherche  ( HEROES ANR-17-CE09-0010-01 ) is acknowledged as well as the  CNRS, University of Bordeaux , and  Nouvelle Aquitaine Region. This work of the  Interdisciplinary Thematic Institute QMat , as part of the  ITI 2021 2028 program  of the  University of Strasbourg ,  CNRS  and  Inserm , was supported by  IdEx Unistra  ( ANR 10 IDEX 0002 ), and by  SFRI STRAT ' US  project ( ANR 20 SFRI 0012 ) and  EUR (QMat- ANR-18-EUR-0016 ) under the framework of the  French Investments for the Future Program. K. P. M. thanks the  Foundation for Frontier Research in Chemistry of Strasbourg  for support, B. D. thanks the  Institut Universitaire de France (IUF)  for financial support.
Small-Angle X-Ray Scattering SAXS data were recorded at the SWING beamline (SOLEIL, Saint-Aubin, France) using a wavelength of 1. 03 Å. Scattering data were collected from a 5 mg/ml sample of CDC48A (from the standard purification after freeze-thawing once) saturated with 2 mM of ATP (Sigma-Aldrich), with the sample passed through Bio SEC-3 HPLC (Agilent) SEC prior to analysis. The distance of the sample to the detector was 1. 8 m, resulting in the momentum transfer of a range of 0. 1 1/Å < q < 0. 5 1/Å. Buffer data were calculated from the buffer [20 mM BisTris (pH 6. 5), 150 mM NaCl, 1 mM TCEP] eluted before proteins, and subtracted from the protein data using SWING's on-site software. Data were analyzed using PRIMUS, as part of the ATSAS software package  (Manalastas-Cantos et al. , 2021) , and models were fit to the scattering curve using FOXS  (Schneidman-Duhovny et al.
, 2010). The monomeric CDC48A model was generated by AlphaFold  (Jumper et al. , 2021) , and the hexameric models were generated using SWISS-MODEL  (Waterhouse et al. , 2018)  with p97 as a template  (Banerjee et al. , 2016; PDB ID 5FTM, and 5FTN). Missing flexible N-terminal, and C-terminal residues were modeled using MultiProt (https://github. com/ strubelab/multiprot) in several configurations, and the best fitting models were overlaid on the SAXS profile.
Next, ~5 µl of 2% uranyl acetate was added to the grid, and immediately blotted. Then, ~5 µl of 2% uranyl acetate was added again, and left to stain for 1 min. Grids were then blotted, and airdried. For cryogenic grid preparation at KAUST, samples were prepared using the Vitrobot Mark IV (Thermo Fisher Scientific) at 22 °C with 100% humidity. CDC48A samples were prepared as previously described, applying 2 µl of sample, and blotting for 3 s. Samples from the initial CDC48A dataset, and optimized datasets 1 and 2 were prepared on glow discharged Cu grids (C-flat Cu 300 mesh R1. 2/1. 3, Protoships Inc. ), and samples from optimized datasets 3 and 4 were prepared on glow discharged Au grids (C-flat Au 300 mesh R1. 2/1. 3, Protoships Inc. ). For cryogenic grid preparation at ACSMB, samples were prepared using the Vitrobot Mark IV (Thermo Fisher Scientific) at 4 °C in 95% humidity, with a blot time of 3 s.
Grids were prepared using 2 mg/ml of CDC48A by applying 3 µl of sample to glow discharged Quantifoil Cu R1. 2/1. 3 grids (EMS). Cryogenic blot-free grids were prepared using the Chameleon instrument (SPT Labtech) at Rosalind Franklin Institute (RFI), United Kingdom  (Razinkov et al. , 2016; Dandey et al. , 2018; Wei et al. , 2018). Self-wicking nanowire Cu R1. 2/0. 8 holey carbon grids with a rectangular bar cross-section were glow discharged in a Pelco Easiglow at 12 mA, 0. 39 mbar air pressure. Approximately 6 nl of the samples at 5 mg/ml were applied to each grid at a relative humidity between 75% and 85% at ambient temperature. Details, and comparison of the sample preparation for each cryo-EM dataset can be found in Supplementary Table  S1.
EM Data Collection Negative stain samples were screened on a Titan 80-300 (Thermo Fisher Scientific) operated at an acceleration voltage of 300 kV. Images were taken at different magnifications with a US4000 CCD camera (Gatan). For the 2D analysis of the negative stain sample, 75 images were recorded on Titan ST (Thermo Fisher Scientific) with a OneView camera (Gatan) at a magnification of 69k, and a pixel size of 1. 7 Å. Cryo-EM data were recorded in multiple facilities. At KAUST, cryo-EM data were recorded on a Titan Krios G1 (ThermoFisher Scientific) operated at 300 kV, equipped with a K2 Summit direct detector, and GIF Quantum968 Imaging Filter (Gatan company). Movies were collected at 130k magnification in super-resolution mode yielding a pixel size of 0. 52 Å/pixel at specimen level.
The initial dataset had 1,242 movies, optimized dataset 1 had 1,073, dataset 2 had 797, dataset 3 had 2,118, and dataset 4 had 2,284. The total exposure time was 5. 6 s, dose-fractionated into 32 frames (0. 175 s per frame) with a total dose of 50 e/Å 2. At ACSMB, cryo-EM data were recorded on a Titan Krios G2 (Thermo Fisher Scientific) operated at 300 kV equipped with K2 summit direct detector, and GIF BioQuantum967 Imaging Filter (Gatan company). A total of 1,311 movies were collected at 130k magnification in electron counting mode at a pixel size of 1. 07 Å/pixel. The total exposure time was 10 s, dose-fractionated into 60 frames (0. 167 s per frame) with a total dose of 75 e/Å 2. At eBIC, Diamond Light Source, cryo-EM data were recorded on a Glacios operated at 200 kV equipped Falcon IV direct detector (Thermo Fisher Scientific).
A total of 1,472 movies were collected at 120k magnification in electron counting mode at a pixel size of 1. 192 Å/pixel. The total exposure time was 10 s, dose-fractionated into 50 frames (0. 200 s per frame), with a total dose of 48. 1 e/Å 2. A comparison of the data collection conditions across all cryo-EM datasets can be found in Supplementary Table  S1.
EM Data Processing Negative stain data were processed to compute 2D class averages. Briefly, the contrast transfer function (CTF) parameters were estimated by Gctf  (Zhang, 2016) , and particles picked automatically by Gautomatch v0. 56 (https://www. mrc-lmb. cam. ac. uk/kzhang/Gautomatch/). A total of 74k particles were picked from 75 images, and extracted with a box size of 180 pixels in Relion-4. 0-beta-1-commit-11e38ba (Kimanius et al. , 2021) , and 2D classification was performed using the VDAM algorithm. Data collected at KAUST were motion-corrected using Warp  (Tegunov and Cramer, 2019)  with a binning factor of 2 and subsequently imported into cryoSPARC v3. 2. 0  (Punjani et al. , 2017)  for further processing. CTF parameters for each image were estimated by patch CTF estimation (multi).
Particles were picked automatically using the Topaz wrapper in cryoSPARC  (Bepler et al. , 2019). Briefly, a Topaz model was trained on a curated subset of CDC48A particles selected using template picking, and subsequently used to pick the particles. Particles were extracted with a box size of 256 pixels in the initial dataset and 380 pixels in the optimized datasets. From the initial dataset, 177k particles were extracted, and subjected to further 2D classification, with no further selection of particles. From the optimized datasets, around 91k particles were extracted from dataset 1 (71k in good classes), 111k from dataset 2 (78k in good classes), 400k from dataset 3 (270k in good classes), and 550k from dataset 4 (430k in good classes). Data collected at eBIC was processed at ETH Zurich using a similar procedure as above using cryoSPARC v3.