dataesr/scientific-paragraphs-categorization huggingface dataset:
Molecular modelling of zSuFu zSuFu was modelled by homology using the Phyre2 server  (Kelley et al. , 2015). The best-ranked model was based on hSuFu structure PDB code 4KMH. Then missing residues in the IDR2 loop (73 residues) were added with Modeller, generating twenty models, amongst which five were chosen to have loops structures with different shapes. The chi 2 of the calculated scattering curve with respect to the experimental curve ranged between 1. 91 and 40 with an average of 12. 6 and a median of 5. 4. The five models were processed with Dadimodo, with residues 18 -255 for the N-terminal and residues 259 -268 and 356 -472 for the Cterminal domain being kept rigid while residues 1-19, 256-258 and 279-355 were left flexible. Five models were generated for each starting model and ranked according to the chi 2 value.
Synchrotron radiation circular dichroism (SRCD) shows that human SuFu is more stable than zebrafish and drosophila SuFu We measured SRCD spectra between 10 and 90 • C for dSuFu, hSuFu and zSuFu in buffer HEPES HCl 50 mM pH 7. 5, NaCl 200 mM, glycerol 10%, Dithiothreitol 2 mM. All proteins showed spectra typical of structured proteins at low temperature, and the spectra became shallower when the temperature was increased, (Fig. 2 A-C ). The spectra cross at an isobestic point characteristic of a two-state transition. Plotting the minimum CD value at 208 nm as a function of temperature results in sigmoid-like curves (Supplementary Figure  S2 ). The transition temperatures were calculated using a sigmoidal fit in Kaleidagraph software with the following equation: CD208(T) = CDmin + (CDmax -CDmin) /( 1 + (T/Tm) k ) where CDmin and CDmax are the minimum and maximum values of CD208; T is the temperature; Tm is the transition temperature (corresponding to the inflexion point of the curve); k is the slope variable.
The average envelope resulting from ab initio calculations encloses most of the crystal structure of dSuFu 4KMA with the exception of the 274-282 loop, which is mobile in the crystal structure, as shown by the thinner radius of the ribbon representation corresponding to a higher Bfactor (Fig. 4A ). This figure highlights the IDR2 region that lines the Cterminal domain (residues 308-340 on the bottom left of the figure). It is ordered but it has no regular secondary structure. The scattering curve calculated from the 4KMA crystal structure  (Zhang et al. , 2013 ) fits rather well with the SAXS spectrum, however the chi 2 value is 53. 3 and there is some disagreement between 0. 15 and 0. 25 Å -1 q value (Fig. 4B , blue experimental curve, compare with 4KMA green curve). We performed a modelling study in order to better characterise possible movements in dSuFu and find a model that fits the SAXS data better.
Sec-SAXS shows that the IDR2 disordered region spans a large volume for hSuFu and zSuFu We chose to compare proteins from vertebrates that are far from each other in evolution: zebra fish and human. hSuFu shows 36% identity with dSuFu and 81% identity with zSuFu, while zSuFu has 35% identity with dSuFu (Fig. 1 ). As for dSuFu, we measured SEC-MALS on purified hSuFu and SEC-SAXS data on purified hSuFu and zSuFu. A reducing agent was necessary to remove a dimer-like species at pH7. 5, coherent with the neutral pKa of cysteine protonation. We suspect that, above this pKa, a disulphide bridge is formed involving Cysteine 292 located in the IDR2 (data not shown). The mass derived from SEC-MALS measurements of hSuFu (supplementary figure  S3B ) is of the order of 55 kDa, consistent with a Fig. 4.
We went on to generate molecular models of SuFu whose calculated scattering curves fit the experimental curve well. We used the Dadimodo software  (Rudenko et al. , 2019)  to find models that fit the experimental curve better. For hSuFu, we ran Dadimodo from three sets of starting models. A) As explained in the Methods section, we started from the free hSuFu crystal structure (pdb id 4KM9  (Zhang et al. , 2013) ) and built the missing regions using Modeller. We thus generated twenty structures, amongst which we chose five with the most different loops as starting points for Dadimodo. Fig. 5C  shows the superposition of the five best output models. The fit between the model and the experimental scattering curves is very good with an average chi 2 value for the Dadimodo output models is 1. 27 ± 0.
Alphafold2 added ordered secondary structures to the IDR2 (colored yellow): a fourth beta strand added to the second sheet of the C-terminal domain and an alpha helix between the two domains. This makes the IDR2 much more compact, while the IDR1 is more extended than in our models. This conformation, although it looks realistic, does not fit our solution SAXS curve well, in particular its Rg is 30. 6 Å, significantly lower than our experimental Rg of 36 Å. C) In addition, we generated a starting model with closed domains similar to those of dSuFu whose domains are more closed with respect to each other even in the absence of the Gli peptide ligand. We thus generated a closed starting model using the Gli peptidebound conformation of hSuFu (pdb id 4KMD) on which the missing regions were attached similarly to the extended model.
We produced twenty models by adding the missing residues corresponding to IDR1 and IDR2 using Modeller and selected the five most different ones as starting points for Dadimodo. The five Dadimodo refined models have extended IDR2 loops similarly to hSuFu models (Fig. 5E ) and have low chi 2 values of 1. 18 ± 0. 06 (Fig. 5F  and Table  3 ). Thus, our SAXS data show that zSuFu has a solution structure similar to those of hSuFu. In summary, our SAXS measurements provide structural data that the crystal structures do not supply. They show that drosophila SuFu has a very different shape in solution than vertebrate SuFu. This difference is mainly due to the presence of an extended IDR2 similar in both human and zebrafish species.
, 2013). They find some increase in the protection of the C-terminal domain, suggesting that the IDR2 can fold back onto the main body of hSuFu. The Alpha Fold2 model of hSuFu is compatible with this interaction. Our models with extended loops fit our SAXS data much better than the Alpha Fold2 model; however, it is possible that the IDR2 undergoes transient interactions with the main body that can be further stabilized by partner proteins. The elongation of the IDR2 loop in vertebrates might be associated with an increased complexity in the Hh pathway from flies to vertebrates. This is exemplified by the transcription factor Ci that is unique in drosophila, while three Gli proteins are simultaneously present in mammals: Gli1, Gli2 and Gli3  (Ruppert et al. , 1988) , allowing finetuning of the gene expression pattern via other partner proteins.
Acknowledgements We are very grateful to  Marie-Hélène Le Du ,  Dominique Durand  and  Patrice Vachette  ( Institut de Biologie Intégrative de la Cellule I2BC, CEA, CNRS, Université Paris-Saclay, France ) for help with data processing software, the SOLEIL synchrotron for access to beamlines,  Javier Perez  at Swing beamline, and  Frank Wien  and  Matthieu Réfrégiers  at Disco beamline. We thank  Valérie Campanacci  ( I2BC ) and  Alexandre Pozza  ( UMR7099 ,  CNRS ) for SEC-MALS analyses. Molecular graphics figures and analyses were performed with  UCSF Chimera , developed by the  Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco , with support from  NIH   P41-GM103311.
Funding AJ was financed by a thesis grant from  Ministère de l'Enseignement Supérieur et de la Recherche  and by a fourth year thesis grant from  Association pour la Recherche contre le Cancer (ARC). SM was financed by a thesis grant from  Ministère de l'Enseignement Supérieur et de la Recherche -IDEX Paris Sciences Lettres. This work was supported by the  Centre National de la Recherche Scientifique , and by the " Initiative d'Excellence" program  from the  French State  (Grant " DYNAMO ",  ANR-11-LABEX-0011-01 ) and  Fondation ARC pour la recherche sur le cancer  (grant  1112 ) to APl and MS.
Research in context Evidence before this study The seminal meta-analysis of the Collaborative Trials in Neoadjuvant Breast Cancer working group published in 2014 showed that on an individual level, achieving a pathological complete response (pCR) following neoadjuvant chemotherapy was associated with improved long-term survival outcomes. On Sept 13, 2021, a search of the PubMed database using the term "pathological complete response and breast cancer prognosis" yielded 1531 published articles between Jan 1, 2014, and Dec 31, 2019. However, pCR does not provide distinction among patients with residual disease. The residual cancer burden (RCB) method was proposed in 2007 as a standardised methodological approach to evaluate and quantitate the extent of residual disease in breast and axillary lymph nodes following neoadjuvant chemotherapy.
Data on Eastern Cooperative Oncology Group performance status was not available in all patients and thus was not collected. Investigators from institutions or trials who were known to have assessed and reported RCB in a predefined cohort were invited to participate between October, 2018, and April, 2019 (and all accepted); pooled data was finalised in November, 2019. Participating investigators representing 12 groups (four trials and eight clinical cohorts; appendix pp 2-3) from Europe and the USA provided individual patient data. We provide references or registration numbers for trials and cohorts when available. The following trials were included: the ISPY 1 trial, 17 the ISPY 2 trial,  18, 20  the ARTemis trial,  16  and a trial led by the Instituto de Investigación Sanitaria Gregorio Marañón (IISGM; Madrid, Spain).
After neoadjuvant treatment and surgery, patients in each trial or cohort received adjuvant endocrine therapy, HER2 therapy, and locoregional radiotherapy, per their institution's standard of care. For the remainder of this Article, we refer to all these trials and clinical cohorts as cohorts. Details on the cohorts, including eligibility criteria, type of consent, ethical approval, enrolment period, and patient characteristics, are provided in the appendix (pp 2-3). All patient identifiers were removed from data before the data were transferred and collated into a single dataset for the present analysis.
In other cohorts, hormone receptor status was defined by the percentage of cells that stained positive on immunohistochemistry at either a 1% or 10% threshold, depending on the institution. HER2 (ERBB2) status was determined according to international guide lines at all institutions. 22 Hormone receptor and HER2 status were used to define four phenotypic subtypes (hormone receptornegative, HER2negative; hormone receptornegative, HER2positive; hormone receptor positive, HER2positive; and hormone receptorpositive, HER2negative) for analysis. Treatment information, such as neoadjuvant HER2targeted therapy use for HER2positive patients, and histologicaltype data were also collected from the cohorts. Data collected in each cohort for the purposes of this study are summarised in the appendix (pp 2-3).
Results of the HER2targeted subset are preferentially presented over results for the entire set of HER2positive subtypes due to clinical relevance. In addition, to evaluate the nonlinear effect of RCB on survival, we used Bsplines with two degrees of freedom in our mixedeffects models and constructed relative event rate plots (with an RCB score of 0 as the reference) as a function of increasing RCB. Mixedeffects analysis was conducted with the coxme package in R (version 3. 4. 3). KaplanMeier plots of eventfree survival and distant relapsefree survival by RCB class, overall and within breast cancer subtypes, were constructed with survival times truncated at 12 years (a time at which around 10% of the smallest patient group [RCB1] remained at risk for an event); survival estimates at 3, 5, and 10 years were computed.
SLT has received consulting fees from  AstraZeneca ,  Novartis ,  Roche ,  Pfizer ,  Celgene ,  PierreFabre ,  Eisai , and  Eli Lilly ; reports honoraria from  Eli Lilly ; and reports travel support from  Novartis ,  Celgene ,  Merck Sharp & Dohme ,  Roche , and  Pfizer. SBE reports institutional research funding from  Pfizer. All other authors declare no competing interests. Acknowledgments The project described was supported by grant number  P01CA210961  from the  National Cancer Institute at the US National Institutes of Health. Additional support was provided from grants RP# 180712  from the  Cancer Prevention and Research Institute of Texas  and  BCRF158  from the  Breast Cancer Research Foundation. The trial led by IISGM was supported by research grants from the  Carlos III Health Institute  ( PI15/00117 ,  PI18/01775 ), cofunded by  El Fondo Europeo de Desarrollo Regional.
, 2000). Short intense X-ray pulses of the duration of only a few femtoseconds are produced by an X-ray free electron laser (XFEL), illuminating one crystal and giving rise to one diffraction pattern at a time. The crystal is generally destroyed in the process and needs to be replaced by a new one for the next image. This method allows to record the scattering information before atomic displacement has time to occur resulting in a dataset effectively free from the effects of radiation damage  (Spence, 2017). Here we present the first SFX structures of R2b in complex with NrdI in the oxidized and hydroquinone state. The datasets reveal redox-dependent structural rearrangements both in the FMN binding pocket of NrdI and at the protein interface. Despite the significant rearrangement in the direct vicinity of the cofactor, FMN itself changes surprisingly little between redox states.
Results SFX crystal structures of Bacillus cereus R2b in complex with oxidized and hydroquinone NrdI collected at ambient temperature An initial crystallization condition for the R2b-NrdI complex was optimized to yield a sufficient amount of crystals smaller than 100 µm in the longest axis (Fig. S1 ), the maximum acceptable crystal size for the experimental setup. Crystals were initially tested at SACLA (SPring-8 Angstrom Compact free electron LAser, Japan) under aerobic conditions with a grease extruder setup (data not shown)  (Sugahara et al. , 2015; Tono et al. , 2013). The crystals diffracted to 2 Å and proved to be stable under room temperature for several days and sturdy enough to handle the physical stress of being manipulated for the experiment. At LCLS (Linac Coherent Light Source at SLAC National Accelerator Laboratory, USA), we obtained two structures of the Bacillus cereus R2b-NrdI (BcR2b-NrdI) complex in different defined redox states by SFX.
The datasets were collected at room temperature under anaerobic conditions using the drop-on-demand sample delivery method  (Fuller et al. , 2017)  (see Materials and Methods for details). BcR2b protein was produced metal-free to allow full control over metal loading during complex reconstitution. The metal content of the protein was determined by total-reflection X-ray fluorescence (TXRF) and only trace amounts of metals could be detected. The iron and manganese content per R2b monomer corresponded to metal-to-protein molar ratios of 0. 27 ± 0. 04%, and 0. 07 ± 0. 04%, respectively. The BcR2b-NrdI complex was reconstituted in vitro by mixing both proteins in a molar ratio of 1:1 and set up for batch crystallization. Manganese was present both during the complex reconstitution and in the crystallization condition.
Crystals for two different datasets were prepared to investigate different oxidation states of NrdI. The crystals for the first dataset were grown under aerobic conditions, yielding bright orange crystals, indicating that NrdI was fully oxidized (Fig. S1 ). The structure from these crystals is later referred to as BcR2bMnMn-NrdIox (PDB ID: 7Z3D). The second dataset was obtained by reducing BcR2bMnMn-NrdIox crystals chemically with sodium dithionite in an anaerobic environment. Consequently, the crystal colour changed from bright orange to faint yellow, indicating that NrdI underwent a two-electron reduction to the hydroquinone state (Fig. S1 ). The crystals were subsequently kept under anaerobic conditions, preventing reoxidation of NrdI. The corresponding structure is denoted BcR2bMnMn-NrdIhq (PDB ID: 7Z3E).
16/0. 20 for BcR2bMnMn-NrdIox and 0. 15/0. 18 for BcR2bMnMn-NrdIhq (Table  1 ). Hammerstad et al. previously  reported two crystal structures of the BcR2b-NrdI complex  (Hammerstad et al. , 2014) , obtained by single-crystal X-ray diffraction at a synchrotron source and under cryogenic conditions. Here, however, R2b harbours a di-iron active site. These structures will be referred to as BcR2bFeFe-NrdI-1 and BcR2bFeFe-NrdI-2 (PDB ID: 4BMO and 4BMP, respectively). Structural alignment between the published synchrotron and our XFEL structures shows that the overall fold of the complex is similar with C⍺-RMSD values between 0. 42 and 0. 47 Å (Fig. 1 B ). Compared to the BcR2bFeFe-NrdI structures we observe a 7-8 residue extended ordered C-terminus in our structures. The C-terminus in BcR2bMnMn-NrdIox continues in the same orientation as the C-terminal ⍺-helix without keeping a helical conformation.
R2b complex formation prevents butterfly bend of FMN in NrdI Three redox states of FMN are physiologically relevant for NrdI: oxidized (FMNox), neutral semiquinone (FMNsq) and anionic hydroquinone (FMNhq)  (Cotruvo et al. , 2013; Røhr et al. , 2010)  (Scheme 2). Reduction of free FMN causes the isoalloxazine to bend along the N5-N10 axis leading to a "bufferfly bend" where the isoalloxazine moiety deviates from planarity (Fig. 2 A)  (Zheng & Ornstein, 1996). 1. 2 Å resolution) structure was produced from protein in the semiquinone state and the butterfly bend was 11° after data collection, similar to the calculated FMNhq angle (Fig. 2 A ). The authors could confirm the reducing effect of the synchrotron radiation on FMN by comparing Raman spectra of the crystals before and after data collection.
These structures will be referred to as BcNrdIox+e and BcNrdIsq+e to emphasize the photoreduction. Johansson et al. observed the same discrepancy between the butterfly bend for a synchrotron structure of initially oxidized NrdI from Bacillus anthracis (BaNrdI), a protein with 99% sequence identity to BcNrdI and identical FMN protein environment  (Johansson et al. , 2010). BaNrdIox+e (PDB ID: 2XOD, 1. 0 Å resolution) is even more bent than BcNrdIox+e with 5. 7°, indicating significant photoreduction of FMN during data collection. Figure  2  A lists the FMN angles of the calculated and measured structures. The use of SFX allowed us, for the first time, to investigate the conformation of oxidized FMN in NrdI. In BcR2bMnMn-NrdIox, the isoalloxazine moiety of FMNox is almost planar, with a butterfly bend of -1°.
This value is comparable to the angle of -3. 1° for FMNox in NrdI calculated by QM/MM  (Røhr et al. , 2010). Unexpectedly, the bend of FMNhq in the BcR2bMnMn-NrdIhq structure is minimal with only 1. 5 ° and thus similar to FMNox in the BcR2bMnMn-NrdIox structure (Fig. 2 A ). Additionally, we calculated an isomorphous difference (Fo(ox)-Fo(hq)) map of BcR2bMnMn-NrdIox and BcR2bMnMn-NrdIhq using the phases of the oxidized dataset. These maps reduce model bias by directly comparing the experimental data and are very sensitive to subtle changes of atom positions in different data sets  (Rould & Carter, 2003). The Fo(ox)-Fo(hq) map shows a slight movement of the oxygen on C4 of FMN indicating a small twist of the pyrimidine ring between both structures but no further bending (Fig. 3 ). This angle of BcR2bMnMn-NrdIhq does not correspond to either the calculated bend of FMN of 14.
3 C ). This redox-dependent conformational change of residues 43 to 45 was previously also observed in crystal structures of BcNrdI and BaNrdI in absence of the R2b subunit (PDB ID: 2X2O, 2X2P, 2XOD, 2XOE)  (Johansson et al. , 2010; Røhr et al. , 2010). Redox-induced changes are also visible on the side of the isoalloxazine ring facing R2b. An unknown molecule is bound within 5 Å from the reactive C4a of FMN in BcR2bMnMn-NrdIox, which binds O2 to reduce it to O2 •-  (Ghisla & Massey, 1989 ). The molecule is 3-coordinated to Trp74 (NrdI), Ser196 (R2b) and a water (p-ox in Fig. 3 D  and E ). A similar density was previously observed in the di-iron BcR2b-NrdI synchrotron structures where it was modelled as a chloride ion. In our structure, placing a water in this position does not sufficiently explain the density while modelling it as a chloride ion leaves no residual density (Fig.
S3 ). Chloride is abundant in the crystallization condition; however, serine and tryptophane are not typical chloride ligands  (Carugo, 2014). The exact nature of the molecule could not be determined  Notably, the reduction of FMN within NrdI occurs at about 16 Å from the metal site and does not affect the metal coordination in BcR2b-NrdI (Fig. 3 A ). The Fo(ox)-Fo(hq) map shows slightly lower occupancy for both manganese ions in BcR2bMnMn-NrdIhq  (Fig S5). The loss of metals can be explained by the reduction treatment of the crystals for this dataset and Mn1 was modelled with 80%, Mn2 with 90% occupancy in the reduced structure. Both structures show the same metal-metal distance of 3. 8-3. 9 Å within experimental error for this resolution (Fig. 4 B ). The metal-ligand Glu161 is likely a key player in the activation of the di-manganese centre as it is proposed to gate the access to the metal site for the oxidant produced by NrdI  (Boal et al.
Discussion In this study we present two structures of the manganese containing R2b-NrdI complex of Bacillus cereus with NrdI in two different oxidation states. These structures were obtained by room temperature SFX data collection using XFEL radiation in contrast to all previously studied R2b or NrdI structures, which were obtained by classical single-crystal synchrotron data collection at cryogenic temperatures. Determination of the oxidation state of redox active enzymes via synchrotron radiation is problematic because exposure to X-rays exerts photoreduction on redox centres. Investigation of artifact-free oxidized enzymes is therefore exceedingly challenging with synchrotron radiation. In the context of class Ib RNRs this affects both the redox active metal centre of R2b and FMN cofactor of NrdI.
SFX eliminates the effects of photoreduction observed during synchrotron-based data collection. We describe SFX structures of the R2b-NrdI complex with NrdI in the oxidized and hydroquinone oxidation state. Both 2. 0 Å structures are of similar quality as the previously published synchrotron structures of the same complex with a di-iron centre  (Hammerstad et al. , 2014)  showing that the change of methodology does not affect the quality. This allowed detailed examination of structural reorganization induced by changes in FMN redox state. Notably, the FMN conformation per se changes little between the oxidized and hydroquinone oxidation state in R2b-NrdI. This is surprising since it was previously shown that FMN is bent in reduced NrdI when not bound to R2b. The complexation of R2b and NrdI thus prevents FMN bending and exerts strain on the isoalloxazine ring in the hydroquinone state.
Data collection The BcR2b-NrdI crystals were initially tested for stability and diffraction quality at SACLA in Japan during experiment 2017B8085. The sample was delivered with a grease extruder setup installed on site  (Sugahara et al. , 2015; Tono et al. , 2013). Hydroxyethyl cellulose matrix  (Sugahara et al. , 2017)  and the protein crystals were mixed together in a volume ratio matrix to crystal pellet of 9:1 and the mixture ejected with a HPLC pump through a 150 µm nozzle with a flow rate of 1-1. 5 µl/min, delivering the sample into the XFEL beam. X-rays were delivered as <10 fs long pulses at 10. 9 keV with 30 Hz repetition rate and a typical pulse energy of around 0. 32 mJ with a beam size of 2 x 2 µm 2 (fwhm). The diffraction of the sample was recorded 100 mm downstream of the interaction point on an Octal MPCCD detector.