dataesr/scientific-paragraphs-categorization huggingface dataset:
MM was supported by the  Czech Ministry of Education, Youth and Sports  (project  LTC17016 ). SGS acknowledges the financial support of  UEFISCDI  grant  PN-III-P1-1. 1-TE-2016-2147 (CORIMAG). VB and PPG acknowledge the  France-BioImaging  infrastructure supported by the  French National Research Agency  ( ANR-10-INBS-04 ).
Radiation damage limits the accuracy of macromolecular structures in X-ray crystallography. Cryogenic (cryo-) cooling reduces the global radiation damage rate and, therefore, became the method of choice over the past decades. The recent advent of serial crystallography, which spreads the absorbed energy over many crystals, thereby reducing damage, has rendered room temperature (RT) data collection more practical and also extendable to microcrystals, both enabling and requiring the study of specific and global radiation damage at RT. Here, we performed sequential serial raster-scanning crystallography using a microfocused synchrotron beam that allowed for the collection of two series of 40 and 90 full datasets at 2-and 1. 9-Å resolution at a dose rate of 40. 3 MGy/s on hen egg white lysozyme (HEWL) crystals at RT and cryotemperature, respectively.
The diffraction intensity halved its initial value at average doses (D 1/2 ) of 0. 57 and 15. 3 MGy at RT and 100 K, respectively. Specific radiation damage at RT was observed at disulfide bonds but not at acidic residues, increasing and then apparently reversing, a peculiar behavior that can be modeled by accounting for differential diffraction intensity decay due to the nonuniform illumination by the X-ray beam. Specific damage to disulfide bonds is evident early on at RT and proceeds at a fivefold higher rate than global damage. The decay modeling suggests it is advisable not to exceed a dose of 0. 38 MGy per dataset in static and time-resolved synchrotron crystallography experiments at RT. This rough yardstick might change for proteins other than HEWL and at resolutions other than 2 Å.
Since then, specific damage to disulfide bonds at RT has been observed in a traditional oscillation study performed on a rotating anode X-ray generator  (26) , in a serial synchrotron crystallography (SSX) experiment  (30)  and in two multicrystal investigations  (31, 32). Also classifying as specific damage, the iron-water bond in a metalloprotein has been shown to elongate as a consequence of RT radiation damage at a dose as low as 0. 033 MGy  (33). In contrast, another multicrystal RT study on insulin did not detect any specific damage to disulfide bonds (maximum dose was 0. 5 MGy)  (34). Likewise, multiple sequential RT datasets collected on single large crystals of the fluorescent protein Cerulean, AtPhot2-LOV2, and lysozyme, respectively, hardly showed signs of specific radiation damage in Fourier difference maps, although Raman spectroscopy did show spectral changes characteristic of damage to disulfide bonds  (35).
Inspired by SFX, serial helical line scans were carried out at a synchrotron source on microcrystals of cathepsin B at 100 K  (41) , paving the way for the implementation of SSX. Since then, various sample presentation methods have been developed, diversifying the means by which SSX data can be collected at RT. Briefly, crystals can be flowed through quartz capillaries  (42)  or a microfluidic flow-focusing device  (43) , or embedded in lipidic cubic phase  (44)  or other viscous media  (45) , and then slowly extruded across the X-ray beam. Alternatively, crystals can be presented on fixed targets, such as silicon nitride wafer sandwiches  (30) , silicon chips  (46, 47) , or Mylar sheet-on-sheet sandwiches  (48) , and X-ray data collected by raster scanning. As an in-between the injection and fixed-target approaches, tape drives carrying microcrystals are yet another way of delivering crystalline samples to the X-ray beam  (49, 50).
Together with the availability of fast readout detectors and of X-ray microbeams with increasing flux densities, these various SSX methods provide the potential to systematically study and compare both global and specific radiation damage at RT and at 100 K. Common to all of these SSX approaches is that the per-crystal X-ray radiation damage is reduced, but not eliminated, by distributing the absorbed energy per dataset over a multitude of crystals  (33) , as opposed to being accumulated in oscillation-based approaches. Here, we performed fixed-target raster-scanning SSX at one of the most brilliant microfocus synchrotron beamlines currently available to investigate specific and global radiation damage in HEWL microcrystals at both RT and 100 K. The questions we address include whether or not specific radiation damage can be observed at RT, and if yes, in which respect it differs from that observed at 100 K and at what rate it develops compared to global RT radiation damage.
The combination of a high photon flux density, the use of an EIGER-X 4M detector framing at close to 500 Hz, and distribution of the dose over ∼20,000 (RT) and ∼9,000 (100 K) microcrystals through a raster-scanning SSX approach  (30)  allowed data collection at high dose rates with 2. 01-ms exposure time per frame. At an average dose rate of 40. 3 MGy/s, 40 (RT) and 90 (100 K) datasets could be assembled sequentially and D 1/2 values (used here to define the global radiation damage limit of HEWL crystals diffracting to 2-Å resolution) were determined to be 0. 57 MGy at RT and 15. 3 MGy at 100 K. The signal in sequential Fourier difference maps, reflecting specific damage, increased up to 0. 5 MGy in the RT study before decreasing with increasing average dose, a phenomenon that can be explained by nonuniform illumination with a beam whose shape can be well approximated with a Voigt function (i.
e. , a convolution of a Gaussian and a Lorentzian beam, or alternatively, a Gaussian beam with significant tails). Furthermore, we collected RT data at 2. 4 MGy/s using the same methodology and provide evidence that the gain in crystal D 1/2 dose tolerance at 40. 3 MGy/s is at most 58% compared to the 2. 4 MGy/s data. Our study contributes to extending to RT the vast knowledge of radiation damage manifestations and implications gained over the past two decades at 100 K (1) and sets a framework for future SSX experiments.
Raster-Scanning Serial Crystallography Allows Radiation Damage Studies with Dose Fine Slicing. The combination of the highly brilliant microfocus X-ray beam available at the European Synchrotron Radiation Facility (ESRF) beamline ID13, a fast readout EIGER-X 4M detector  (51) , and a raster-scanning approach to serial crystallography  (30)  enabled the collection of up to 90 full datasets with minimum dose increments on HEWL crystals (Fig. 1 ). For RT (22 °C in our study) data collection, the crystals were sandwiched between two Si 3 N 4 membranes as reported earlier (30) while they were deposited, for the control cryo-data collection, on a microporous silicon chip (  52 ) that was flash-cooled after blotting of excess solvent, and maintained in the gaseous-nitrogen stream of a cryocooler operating at 100 K.
The membranes and chips were raster-scanned in lines from left to right across the micrometer-sized X-ray beam (3. 0 μm [hori-zontal (h)] × 1. 5 μm [vertical (v)] [full width at half-maximum (FWHM)]) of 2. 67 × 10 12 photons/s if unattenuated. Positions at which data were collected were spaced by 10 μm in both the horizontal and the vertical directions, and at each position, 40 to 90 consecutive still diffraction patterns (i. e. , without rotation during exposure) were acquired using about a 500-Hz data collection rate. For assessing a potential dose rate effect at RT, the flux of the X-ray beam was decreased 17-fold and a low-dose rate series also acquired. Patterns of equivalent dose collected at each spot were then assembled into equivalent-dose datasets by processing of still diffraction images using serial-crystallography protocols (see Materials and Methods section for details).
In this serial approach, the dose increment for each of the above equivalent-dose datasets was low (5 kGy for the RT low-dose rate series and 81 kGy for the RT high-dose rate and cryo-series) while the high-frame rate data collection in combination Fig. 1. Serial radiation damage raster-scanning approach. Protein crystals (cf. photo) are sandwiched between two Si 3 N 4 membranes and presented to the X-ray beam on a solid support  (30)  for RT data collection or deposited on a microporous silicon chip  (52)  for data collection at 100 K maintained by a stream of gaseous nitrogen. As an example, the scheme shows the RT high-dose rate series for which two Si 3 N 4 sandwiches were used. The support was raster scanned across the micrometer-sized X-ray beam from the upper left to the lower right with each line being scanned from left to right.
e. , corresponding to 19,818 indexed hits, from multiple membranes) are then assembled into 40 equivalent-dose serialcrystallography datasets from which protein structures at increasing dose are solved. with the brilliant X-ray beam minimized the total data collection time. Progression of global and specific radiation damage as a function of dose was then monitored by inspecting the decrease in diffraction intensities and by analyzing Fourier difference maps calculated with respect to the lowest-dose dataset, respectively. We refer to this experimental approach as sequential serial raster-scanning (SSRS) crystallography. Global Radiation Damage, Dose Limits, and Characteristic Decay Constants at RT and at 100 K. We recorded SSRS series at RT at two different dose rates (2. 4 and 40. 3 MGy/s) and a control series at 100 K at a dose rate of 40.
At this energy and for these crystals, the photoelectron median range is calculated to be 2 μm, so that taking their escape into account, the calculated average dose is reduced by 47% in the high-dose rate series (40. 3 instead of 76. 0 MGy/s). In order to monitor global radiation damage as it progresses from dataset to dataset, intensities of all predicted Bragg reflections on the detector, i. e. , including those that extended beyond the resolution limit of the highest-dose dataset in each series (2. 04 and 1. 9 Å for both RT series and the cryoseries, respectively), were summed for all indexed patterns of dataset n and added to yield a total intensity I n. Normalized by the total intensity of the first dataset (I 1 ) and plotted as a function of dose, the I n /I 1 ratio reflects the dose-dependent decrease in diffraction power for consecutive datasets (Fig.
3A ). The dose dependence of I n /I 1 at 100 K differs from that at RT (Fig. 2 ) and the decrease at 100 K can be well explained by a linear fit, but it can also be well fitted according to SI Appendix, Eq. S6 (SI Appendix, Fig. S3 ). Linear extrapolation indicates a half-dose D 1/2 of 15. 3 MGy for the cryo-dataset at 40. 3 MGy/s-a value that is similar to the D 1/2 reported earlier for lysozyme crystals assessed at 100 K at similar resolution [12. 5 MGy (57), 9. 2 MGy (35)] but 27 times larger than the corresponding value at RT at the same dose rate. In contrast to the cryotemperature (SI Appendix, Fig. S3 ) and RT high-dose rate series (Fig. 3A  and SI Appendix, Fig. S1 ), the intensity decay I n /I 1 of the RT low-dose rate series cannot be well fitted according to SI Appendix, Eq. S6 (SI Appendix, Fig.
S2 ), because the beam profile deviated considerably more from a Gaussian function for the collection of the RT low-dose rate than for the RT high-dose rate series. Specific Radiation Damage at Room Temperature and at 100 K. The progression of specific structural and chemical radiation damage is best visualized in Fourier difference maps calculated between datasets collected with different average doses  (2) (3) (4). Sequential difference Fourier maps were calculated for the RT series with respect to the lowest-dose dataset of the RT low-dose rate  (   (2. 4 MGy/s) series (i. e. , datasets RT-LDR2 to RT-LDR80), and datasets 1 to 40 for the RT high-dose rate (40. 3 MGy/s) series (i. e. , datasets RT-HDR1 to RT-HDR40). For the cryo-series, sequential-difference Fourier maps were calculated with respect to its lowest-dose dataset (CR1), i.
CR1 , with n being datasets 2 to 90 for the cryo-(40. 3 MGy/s) series (i. e. , datasets CR2 to CR90) (Fig. 4 ). As an example, Fig. 4A  shows difference electron density at the disulfide bond Cys6-Cys127 in the F o CR43 -F o CR1 map. The negative density highlights specific damage to this disulfide bond in the 43rd dataset of the cryo-series (3. 47 MGy), with indications of damage on Cys6 being larger than on Cys127. In order to follow specific radiation damage as a function of dose, difference peaks around each sulfur in a disulfide were integrated within a sphere of radius proportional to its isotropic atomic displacement parameter, i. e. , B factor (ref. 58 ; see Materials and Methods for details). Fig. 4B  shows the sum of the integrated difference density around both sulfur atoms within a disulfide bond as a function of dose.
Starting from zero, the sum monotonically decreases for all four disulfide bonds with increasing dose, reflecting previously well-described radiation-induced bond rupture at 100 K (2-4). The radiation sensitivity of the four disulfide bonds differ, with Cys6-Cys127 being the most sensitive and Cys64-Cys80 being the least sensitive, as already documented previously  (3, 59). Our SSRS approach also allows the observation of specific radiation damage at RT, owing both to the fine-slicing of the dose and to the collection of an ultralow-dose dataset (5 kGy, RT-LDR1) as a reference RT dataset for the calculation of F o RT-LDRN -F o RT-LDR1 difference maps. Fig. 4  C and E shows difference electron density at the disulfide bond Cys6-Cys127 in the F o RT-HDR6 -F o RT-LDR1 and the F o RT-LDR80 -F o RT-LDR1 maps, respectively.
S11); this fit yields a characteristic half-dose D 1/2,s of 0. 08 MGy for the Cys6-Cys127 disulfide bridge. The alternative per-voxel simulation (SI Appendix, Supplementary Text S2) reproduces the peculiar dose dependence equally well (Fig. 4D ). The characteristic half-dose of specific disulfide damage D 1/2,s (0. 08 MGy) is five times lower than the characteristic half-dose of global damage D 1/2,g (0. 38 MGy), suggesting that global and specific damage evolve at different rates at RT. Despite being composed of 80 datasets, our RT low-dose rate series does not extend in dose beyond 0. 4 MGy, so we cannot conclude that collection at a lower dose rate would result in a better preservation of structural differences. However, we note that peak heights in F o RT-HDRn -F o RT-LDR1 and F o RT-LDRn -F o RT-LDR1 Fourier difference maps are of similar heights at 0.
S6B ). It is possible this proline peak is due to noise accumulating on the symmetry axis. Concerning tryptophan residues, we observe no peaks in sequential difference Fourier maps in the cryo-and RT low-dose rate series, but in the RT high-dose rate series, a negative peak is already seen at the five-membered pyrrole ring of Trp108 and Trp111 in the F o RT-HDR1 -F o RT-LDR1 map (-0. 063 e -/A 3 [-3. 5 σ] and -0. 067 e -/A 3 [-3. 7 σ], respectively). A B Fig. 3. Modeling and simulation of the decrease in diffraction power for the RT series at 40. 3 MGy/s. (A) The sum of the intensities of all predicted reflections for each indexed pattern of a dataset n was averaged and normalized by the sum of the first (i. e. , lowest dose) dataset (triangles, same data as in Fig. 2 ). The fit to these data according to a three-beam model (hot, cold, and nondamaging beams; see SI Appendix, Supplementary Text S1 for details) approximating the Voigt-shaped beam is shown as a dashed line.
Discussion The SSRS approach described here is based on the combination of a highly brilliant microfocus X-ray beam, a fast readout detector (EIGER-X 4M)  (51) , and a raster-scanning approach to serial crystallography  (30). The protocol allowed SSX data to be collected at RT and 100 K with 2-ms exposure time per frame, and a total dose per dataset as low as 81 kGy for the high-dose rate series (RT and cryotemperature) and 5 kGy for the RT lowdose rate series. The serial approach used here inherently allows the dose to be distributed over a multitude of crystals (here, several thousand per dataset), minimizing the total dose per dataset. By collecting several (up to 90) consecutive diffraction images at each sample position, equivalent-dose datasets were assembled. This enabled a fine-sliced monitoring of global and specific radiation damage on HEWL crystals at both RT and 100 K, and allowed dose limits to be determined at both temperatures.
The dose at which the diffracted intensity of the doseequivalent datasets decreased to one-half of its initial value 0. 39 MGy (D Positions at which data were collected were spaced by 10 μm both in the horizontal and the vertical directions-a distance more than double that across which global radiation damage has been measured to spread (4 μm at 18. 5 keV) beyond a micrometer-sized beam at 100 K  (61) , and more than four times that across which photoelectrons are calculated to travel in this experiment [∼2 μm at 13. 45 keV  (53) ]. Since there have been no equivalent systematic studies at RT, we cannot exclude the possibility that the 10-μm spacing was insufficient and that global or specific radiation damage affected unexposed positions, in particular in the vertical direction where damaging species had more time to diffuse between probing neighboring spots than in the horizontal direction.
However, no indication of data degradation was visible during data collection time. Also, an indication that global radiation damage at RT might not spread beyond 10 μm has been reported in a study using a 2. 5 μm (FWHM, v) × 5. 1 μm (FWHM, h) beam  (23). However, we expect that the broad tails of the Voigt-shaped beam that was used in our study have preexposed neighboring positions on the crystals. Visualizing the buildup of specific radiation damage at RT was made possible by the dose fine-slicing inherent in the SSRS approach and by the assembly of full datasets, all constituted from data originating from the same crystals, for each sequential dose point. In the RT 40. 3 MGy/s series, sequential Fourier difference maps show the appearance and progression of specific damage to disulfide bonds up to a dose of about 0.
38 MGy and of specific disulfide damage D 1/2,s is 0. 08 MGy). This is somewhat at variance with a recent systematic study  (35)  that determined the decoupling factor between global and specific damage of lysozyme crystals at RT to be of only 1. 3, although in significantly different experimental conditions (e. g. , oscillation datasets obtained from unique large crystals). At 100 K, the decoupling factor has been determined to be 12. 4  (35). Whereas specific damage to disulfide bonds is easier to observe at 100 K than at RT  (35) , our study shows it can readily be observed at RT in dose fine-sliced SSRS using a highly intense microbeam. The experimentally determined dose limit of 0. 38 MGy (D 1/2,g ) provides guidance for the growing number of RT SSX experiments being conducted (see SI Appendix, Table  S2  for a summary of all D 1/2 values determined in this study).
Another possibility would be to report the dose averaged over the FWHM of the beam, which would be most appropriate for crystals that are the same size as the beam FWHM. Independently of which average dose is being reported, one should keep in mind that for crystals larger than the beam most of the illuminated crystal volume experiences a dose that is not the average dose, due to the high dose contrast and the time-dependent hole burning  (23). It is anticipated that time-resolved (TR) SSX experiments (65-68) will gain importance following the current upgrade to (or building of) fourth-generation synchrotron sources (e. g. , ESRF-EBS, NSLS II, and Max IV), and experimenters will be advised to respect the 0. 38-MGy limit. Indeed, structural changes in TR-SSX experiments are visualized in Fourier difference maps calculated between datasets obtained with and without reaction triggering.
The chip was then flash-cooled in a gaseous nitrogen stream of a cryocooler (Oxford Cryosystems) operating at 100 K. Data collection was performed on the microbranch of the ESRF "Microfocus Beamline" (ID13) using the raster-scanning serial crystallography approach  (30). The temperature in the experimental hutch was controlled by air conditioning and set to 22 °C and is referred to as RT in this study. In the focal plane, the shape of the X-ray beam can be accurately represented using a Voigt profile: similar to a Gaussian with weak but broad tails caused by parasitic scattering and aberrations of the X-ray optics. For the collection of high-dose rate series datasets at RT and at 100 K, an unattenuated beam with 2. 7 × 10 12 photons/s and a FWHM of 3. 0 μm (h) × 1. 5 μm (v) was used. In order to collect a RT low-dose rate series, the beam was attenuated by adjusting the gap of the beamline's u18 in-vacuum undulator (giving then 1.
This phenomenon, known as photoelectron escape, is significant for the microbeams used in this study and needs to be considered in dose calculations. The most recent version of RADDOSE-3D (53) takes photoelectron escape into account as well as the entry into the crystal of photoelectrons from irradiation of surrounding liquid. For the crystals and incident X-ray energy used, the median range of photoelectrons is predicted to be 2 μm. The high-dose rate series, both at RT and at 100 K, were collected at 40. 3 MGy/s, while the RT low-dose rate series was collected at 2. 4 MGy/s. If photoelectron escape had not been taken into account, apparent average dose rates would have been 76. 0 and 5. 0 MGy/s, respectively. Data Reduction and Processing and Structure Refinement. NanoPeakCell  (30)  was used for processing of SSRS data.
Selected hits were indexed and integrated with CrystFEL 0. 6. 2 (71). Indexing relied on MOSFLM (72), XDS  (73) , and Dirax  (74). Real-space refinement was carried out in Coot (75) and reciprocal space refinement in Phenix  (76). Details can be found in SI Appendix. Difference Map Calculation and Integration in Real Space. Structure factor amplitude Fourier difference maps were calculated for all three series with respect to the lowest-dose dataset of the RT low-dose rate and the cryoseries, i. e. , , with RT-LDRn, RT-HDRn, and CRn being the 80 RT low-dose rate, the 40 RT highdose rate, and the 90 cryo-datasets, respectively. Because of a high structure factor isomorphism between the two RT series, the lowest-dose dataset of the RT low-dose rate (RT-LDR1; corresponding to a dose of 5 kGy) could be used as the reference dataset for both RT low-and high-dose rate series.
Qweighted difference maps were calculated as described earlier (77), using custom-made scripts  (78)  implemented in CNS (79). Maps were calculated on an absolute scale (e -per cubic angstrom) at a resolution of 2. 04 and 1. 9 Å for both RT series and the cryo-series, respectively. The resolution cutoff of each series was set to that of the highest-dose dataset in each series. In order to quantify specific radiation damage, a custom-made Python script was used to integrate electron density in the difference maps around all atoms of the model  (58). The integration volume was a sphere with a radius r proportional to the square root of the refined B factor of the respective atom of the first dataset in each series used for the calculation of difference maps (LDR1 or cryo1): r = ð1=2πÞ ffiffiffiffiffiffiffiffi B=2 p , as described in ref.
80. For disulfide bonds, integration was performed over the two sulfur atoms and summed (Fig. 4 ). For Pro70, it was carried out over all modeled atoms (SI Appendix, Fig. S6 ). As a control for the RT high-dose rate series (Fig. 4D ), we integrated over a sphere with a radius proportional to the square root of the refined B factor of the respective atom of the nth dataset. In the resulting graph, the dose at which the integrated signal in Fourier difference maps was maximum (about 0. 5 MGy) remained unchanged. Code Availability. All Python scripts have been deposited on GitHub and are accessible at https://github. com/coquellen/RADDAM_2019 (58).