Methanosarcina mazei casposase can integrate varied forms of the casposon end in vitro, and recapitulates several properties of CRISPR-Cas integrases including site-specificity. The X-ray structure of the casposase bound to DNA representing the product of integration reveals a tetramer with target DNA bound snugly between two dimers in which single-stranded casposon end binding resembles that of spacer 3'-overhangs. The differences between transposase and CRISPR-Cas integrase are largely architectural, and it appears that evolutionary change involved changes in protein-protein interactions to favor Cas2 binding over tetramerization; this in turn led to preferred integration of single spacers over two transposon ends.Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that the  CRISPR-Cas systems, encoded in the genomes of many bacterial and archaeal species, function as defense systems against invading foreign nucleic acids such as bacteriophages and plasmids . They prcas2 gene 2(Cas1)2 heterohexamer in which two catalytic Cas1 monomers carry out metal-dependent transesterification to integrate protospacers into the target repeat sequence  in vitro , and tested its in vitro activity using an integration assay . This choice was motivated by the apparent preference of Cas1-Cas2 of some CRISPR-Cas systems to integrate spacers with short 3'-overhangs  of the linear plasmid products generated when pUC19+TSD+30 bp was used as the integration target . Assays M. mazei casposase is remarkably sequence-specific, and that the most important target sequence features directing integration are clustered around the 5'-CGCA motif detected in vivo , with sequences from all five samples revealing overlapping positive and negative strand insertions consistent with the 14 bp offset corresponding to the size of casposon TSDs . For the in vivo .To explore aspects of target recognition by casposases in more detail, we employed an integration assay using short target oligonucleotides  containiWe took advantage of this assay to more finely dissect the stimulatory effect of recessing the top strand of the casposon end . StartinM. mazei casposase towards integration substrates and by the report of direct integration of ssRNA by a type III reverse transcriptase-Cas1 fusion protein  and between R192 and G20 (the base opposite the second C of the 5'-CGCA motif)  . The remA motif) .To confirm the catalytic importance of R191 and R192, we expressed and purified a double mutant  is substantially smaller than the total Cas1-Cas2 buried surface in the heterohexamer of either E. coli  was cloned into a modified pBAD vector as previously described for the A. boonei casposase (E. coli strain B834(DE3). Expression was performed using the M9 minimal media kit and selenomethionine from Molecular Dimensions Inc  according to the manufacturer's instructions. Full selenomethionine substitution was confirmed by LC/MS . Purification and crystallization were identical to those of the unsubstituted protein. All purification steps were carried out at 4°C.The gene encoding the casposase from asposase . For optasposase . For selA C-terminally truncated casposase, residues 1-341 (ΔHTH), was generated by the deletion of residues 342-406 from the Trx-casposase fusion protein by PCR mutagenesis. Expression and purification was identical to that of the full-length version. Single and double point mutations were introduced by PCR, and sequences of the full coding frame confirmed. Purification was essentially as the wild-type with the exception of the Y206A/R280A double mutant, which yielded only ~10% of the level of soluble protein when compared to wild-type and was not completely cleaved by TEV, despite prolonged incubation. Due to the low level of recovered protein, it was assayed without performing the usual final size exclusion chromatography step.2 and incubated at room temperature for the times indicated. For each set of reactions, a "no end" control was performed in which the oligonucleotide was omitted. Reactions were initiated by the addition of 150 ng target plasmid and stopped at time points indicated by adding EDTA to a final concentration of 25 mM followed by proteinase K digestion for 30 min at 37◦C . Glycogen was then added, the reaction products ethanol-precipitated, and the tubes air-dried. Products were resuspended in 1X DNA loading buffer, run on a 1.5% agarose gel in 1X TAE at 100V, and visualized using ethidium bromide. Bands were quantified where indicated using ImageJ.Oligonucleotides (IDT) were resuspended in 10 mM Tris and, as needed, mixed in the appropriate molar ratio, heated to 95°C for 15 minutes, and allowed to slowly cool to room temperature. Modified pUC19 target plasmids were constructed using the QuikChange method by introducing target sequences between bp 575 and 576. For assays, purified protein (75 nM) and oligonucleotide (200 nM) were mixed in buffer consisting of 25 mM Tris pH 7.5, 150 mM KCl, 0.05 mg/ml BSA, and 5 mM MnCl2. Reactions at room temperature were initiated by the addition of the target oligonucleotide, and were stopped at the time points indicated by mixing 1:1 with formamide gel buffer. Reactions were run on 15% acrylamide TBE-urea gels (Novex) at 250V in 1X TBE and visualized using SYBR Gold (Invitrogen). Bands were quantified as indicated using the AlphaView band analysis software of an Alpha Innotech imager.Protein (1.4 μM) and casposon end oligonucleotides (1.2 μM) were mixed in buffer consisting of 25 mM Tris pH 7.5, 150 mM KCl, 0.05 mg/ml BSA, and 5 mM MnClTo prepare four of the DNA samples for sequencing, the in vitro integration reaction was carried out in quadruplicate for 60 min using dsLE31 and pUC19+TSD+30bp as described above. The resulting samples were run on 1.5% agarose and, for each, the band corresponding to the linear product of integration was isolated using the QIAquick Gel Extraction Kit (Qiagen). The resulting DNA was then used as the template for four independent PCR reactions using a LE31 primer to amplify the product. The reaction was carried out for either 17 or 30 cycles. Amplified products corresponding to the size of linearized pUC19 were again isolated from agarose prior to library preparation. For the fifth sample, the integration reaction was scaled up four-fold and carried out in triplicate for 2 hours using TR29 as the integration substrate. Bands corresponding to the linear integration product were isolated from agarose, subjected to one round of extension using Pfu Ultra II Fusion DNA polymerase (Agilent) for 30 sec at 72°C, and purified again using the QIAquick PCR purification kit (Qiagen). The five sequencing libraries were prepared from the supplied DNA using Nextera XT DNA sample prep kit  according to the manufacturer’s protocols. The libraries were then sequenced in a stranded 75 bases paired-end run on the MiSeq instrument and analyzed.R (V3.5.1) using custom scripts. Specifically, the reads were first collapsed by sequence, abundance of individual sequences was recorded, and results were stored in fasta format. Each fasta file was filtered, extracting only the unique sequences containing casposon end sequence (5`-GAGTTACCTATATCCC) for further analysis. These sequences were then trimmed, removing the casposon end fragment and retaining only downstream regions for aligned to the plasmid using Rsubread package (V1.34.7). Generated alignment files were used to visualize sequence alignments and coverages using Gviz package (V1.26.5). To generate sequence logos, the aligned sequences were first extracted from alignment files by strand, and each sequence was then duplicated based on the recorded abundance information obtained from the fastq files. The sequences were then aligned at the 5`end and alignment was used to calculate nucleotide frequency matrix extending from positions +1 up to +30 nucleotides downstream of integration of casposon end sequence. The logos were then visualized using ggseqlogo (V0.1) and negative strand logos flipped using Adobe Illustrator to reflect the complimentary base pairing. The alignment, conservation and upstream sequences of different target sites were performed and depicted using Jalview (V1.0) software. Additional packages used in the analysis include Biostrings (V2.50.2), Rsamtools (V1.34.1), ShortRead (V1.40.0), GenomicAlignments (V1.18.1). The scripts are available upon request. The fastq files have been deposited to GEO under the accession number: GSE139037.The read 1 fastq files for each sample were processed, aligned and analyzed in After samples were mixed at various protein:DNA ratios and dialyzed overnight into 35 mM CHES pH 9.0, 0.5 M KCl, 105 (w/v) glycerol, 0.2 mM TCEP, protein multimerization and DNA binding was assessed. Samples were injected onto a Superdex 200 Increase 3.2/300 column equilibrated in dialysis buffer and eluted at 0.05 ml/min at 4°C. A standard curve was generated using solutions at 1 mg/ml in dialysis buffer of blue dextran, thyroglobulin (669 kDa), apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa).Samples at ~1 mg/ml (~25 μM) were diluted to 25-50 nM in buffer containing 30 mM CHES pH 9.0, 2 mM EDTA, and either 1 M or 0.5 M KCl immediately prior to measurement using a G10 RefeynOne mass photometer. For each experiment, thousands of protein molecules were counted and their contrasts measured. Contrast values were converted to molar masses using a standard curve prepared using BSA (monomer+dimer), alcohol dehydrogenase (monomer+dimer), ovalbumin, and thyroglobulin in PBS buffer. Distributions were fitted using a Gaussian distribution model.Oligonucleotides were combined in several molar excess with purified casposase and dialyzed overnight into Complex Buffer 1 . This was followed by size exclusion chromatography in the same buffer on a 16/60 Superdex 200 column. Fractions containing the complex were subsequently combined and concentrated at room temperature in ThermoScientific 30K CO units. Glycerol was removed by extensive dialysis against Complex Buffer 1 without glycerol. Complexes were stored at 10°C to prevent cold-induced precipitation.Crystallization was performed in hanging drop format at room temperature. Briefly, protein-DNA complexes were mixed with equal volume of reservoir solution containing 0.15-0.185 M calcium acetate, 84 mM sodium acetate pH 4.4-3.8, 7.5-9% PEG 8000, and 0.5 M KCl. Crystals grew to final size within 48h, and addition of 0.5 M NaCl to the reservoir encouraged further growth. Crystals were cryoprotected by transfer to mother liquor and quick serial transfer in increasing concentrations of ethylene glycol to 15-17.5% (v/v) final. Data was collected at the Southeast Regional Collective Access Team (SERCAT) beamline ID22 of the Advanced Photon Source.Diffraction data collected at three energies around the Se K edge  were intThe resulting experimental electron density map was of excellent quality at 3.2 Å with clearly visible side chain densities at most places and clear density for the bound DNA. The molecular model was built using O  and refihttp://www.ebi.ac.uk/pdbe/prot_int/pistart.html). All structural figures were generated using PyMOL .Interfaces were analyzed using Protein interfaces, surfaces and assemblies' service PISA  at the E In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Methanosarsina mazei. They demonstrate that the MmCasposase integrates a wide variety of RNA and DNA substrates into a specific location. Integration requires substrates with a free 3'-OH and a target that contains specific sequence motifs. To understand the mechanism of integration the authors crystallize the casposase bound to DNA. The structure reveals a tetrameric architecture in which two casposase dimers sandwich the target DNA. The authors compare this structure to available crystal structures of Cas1 and Cas1-2 integration complexes. This analysis shows that the casposase tetramer is mutually exclusive with Cas2 binding and suggests how architectural changes in the Cas1-Cas2 complex may have led to mechanistic differences between the two types of integrases.Cas1 and Cas2 are characteristic of the CRISPR-Cas adaptive immune systems found in most archaeal and many bacterial genomes. It is hypothesized that Cas1 evolved from a rare class of transposases, called casposases. Here, Hickman et al. express and purify a casposase from Decision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled "Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas" for consideration by eLife, in its present form.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in In this manuscript, Hickman et al. present the first crystal structure of a transposon-encoded casposase bound to an integration product mimic. The structure reveals a tetrameric architecture in which two casposase dimers sandwich the target DNA. The authors compare this structure to available crystal structures of Cas1 and Cas1-2 complexes. This analysis shows that the casposase tetramer is mutually exclusive with Cas2 binding and suggests how architectural changes in the Cas1-Cas2 complex may have led to mechanistic differences between the two types of integrases.The reviewers all agree that the structure presented in this manuscript is potentially important. However, the structural data are not supported by biochemical experiments that advance our understanding of mechanism.eLife for further consideration, should the authors wish to do so.Some of the specific points raised during the review and in the discussion between reviewers and the editors are noted below, and the individual reviews are appended. We concluded that it could take substantial time to address all of these issues in a satisfactory manner. At this point, the authors may simply wish to submit this high quality paper elsewhere, without delay. If, however, these points can be addressed satisfactorily then the paper could be resubmitted to 1) Clarification on the DNA substrates used in the biochemical assay, and an explanation for how these substrates relate to the biology are required. Additional controls and additional time points are necessary. Important controls are missing from the results presented in Figure 1, most notably negative controls in which the plasmid is incubated with casposase in the presence of divalent metal ions but no transposon end substrates. Previous Cas1 studies have observed considerable nicking activity (or possibly nicking/cleavage from contaminating proteins), so it is crucial that the baseline activity of the casposase protein prep on plasmid DNA is evaluated. Furthermore, for the data presented in panels C and D, no lanes exist showing the starting plasmid substrate at time 0 (i.e. in the absence of protein and transposon end substrate); since any given plasmid prep will have variable ratios of supercoiled to open-circular plasmid, it is critical that the reader have this comparison – for the exact substrate used in the experiments – to fully evaluate the biochemical activity of casposase and discriminate this from the starting status of the substrate. Labeling the bands "target," "SE," and "DE," is misleading, since a plasmid will naturally purify from a bacterial culture with some fraction in the open circular form, which will migrate the same as a SE product but is certainly not a single-end integration product.2) Provide more experimental support and/or justification for the specific choice of the 8-nt overhang integration product. Why was 8 selected as the overhang length? Was a larger crystallization screen performed, and this was the only substrate geometry that resulted in diffracting crystals? Is there any further evidence that this is a physiologically relevant ssDNA end sequence? For at least one reviewer, it was unclear what the SEC data provided that was not show by the iSCAMS analysis. All reviewers commented on misplaced labels and inappropriate units (time rather than volume) in the SEC. There was some discussion about moving the SEC to the supplement but regardless of where it is discussed, the interpretation of these data must be clarified.3) The authors gloss over the interesting finding that the HTH domain is disordered in the crystals and fail to follow-up with further experiments to investigate the role of this domain on biochemical integration activity, and whether the domain may be important in the context of more physiologically relevant substrates.eLife, the authors should perform bioinforamtics analyses to determine whether related casposons are always present in genomes adjacent to similar target sites. Please mention, in the Discussion, the recent observation that the Type V-C Cas1 is much less sequence-specific in its integration site preference.4) A major take-away from this manuscript would be that this casposase integrates substrates primarily at a specific target site rather than randomly, as has been previously touched upon in the literature. However, apparent integration events in pUC19 alone and farther away from the target site have been detected in this and previous studies. No sequencing data for these events are shown, and the authors fail to perform additional analysis on integration patterns. The sequences should be shown and discuss. It has become routine in spacer acquisition studies with Cas1 to deep sequence half-site integration products in order to gain deeper insights into specificity. Similar NGS experiments are warranted here to enhance their biochemical results. While NGS experiments are not required for publication in 5) When describing the structure, the authors note several protein-DNA contacts, but it is unclear whether any of these contacts contribute to the specificity for the TSD target or LE substrate. From the data shown in Figure 1C, it appears that the casposase is somewhat specific for the LE sequence, especially for shorter single-stranded DNAs. Can this specificity be explained by the structure? Do R191 and R192 contribute to the specificity of the casposase for the TSD? The authors speculate about the importance of stacking interactions for ssDNA substrate binding (subsection “ssDNA casposon end binding resembles that of spacer 3' overhangs”). Did the authors perform any mutagenesis experiments to establish the importance of these contacts for target and substrate specificity? Experiments supporting structure-based hypotheses proposed by the authors are required.6) The order in which substrates are shown on the left gel in Figure 1C is confusing. If possible, it would be helpful if this gel could be re-run to place casposon-specific and random substrates of similar type (e.g. TR17 vs ranTR17) in adjacent lanes on the gel. Is a dsran17 substrate (i.e. a fully double-stranded random substrate of 17 bp) also integrated? In addition, ssran12 and ssLE11 could be omitted from this gel, as these substrates are also tested in the rightmost gel in the panel.7) In paragraph two of the Discussion section, the authors suggest a similarity in the lack of sequence specificity on the bottom strand of the casposase TSD with a lack of sequence specificity for spacer-side integration by Cas1-Cas2. However, it is important to note that Cas1-Cas2 does display specificity for the entire direct repeat sequence, likely through sequence-specific deformations of the repeat that occur during full-site integration . In that study, it was shown that mutations in the repeat affect spacer-side integration, but do not have an effect on leader-side integration. Thus, the conclusion stated by the authors that bottom strand integration is sequence-independent and instead based on a "molecular ruler" is not consistent with current models for spacer-side integration.Reviewer #1:eLife. However, they use this qualitative biochem to identify substrates that result in higher order assemblies and they determine the structure of the tetrameric complex bound to DNA. Overall the experiments appear technically sound, and the paper delivers the first structure of a casposase. These mechanistic insights and evolutionary connection of this system to CRISPR-Cas immune systems are important and warrant publication in eLife. However, the text and figure are a challenge to interpret  and considerable revision is required to make this work accessible to a broad readership.Cas1 and Cas2 are hallmarks of the CRISPR-Cas adaptive immune systems found in most archaeal and many bacterial genomes. Koonin and colleagues have previously identified homology between Cas1 and casposon family proteins (doi: 10.1186/1741-7007-12-36) and predicted that CRISPR repeats evolved from the target site duplications (doi: 10.1016/j.mib.2017.04.004). These predictions have been largely validated with in vitro biochemical studies . Here, Hickman et al., extend the previous work by defining essential features of the target site and testing a wide-variety of integration substrates. This on its own would not warrant publication in Methanosarcina mazei and determine sequence requirements for integration. The authors describe a sequencing method to determine the location of integration. 7 of 8 integration occurs at the TSD sequence with concomitant generation of a 14 bp TSD. "The other sequenced product arose from integration into a site ~110 bp away from the specific sequence and was also accompanied by a 14 bp TSD." Please include more information. Was there sequence similarity between these sites? If not, what does this say about the mechanism of recognition and how does this relate what the structures teaches us about recognition?The authors purify a casposases from Figure 1A. It is not clear what the authors are intending to teach the reader in panel A. Based on the figure legend title ("Casposase structure and function") the reader is expecting to see a structure, rather than a schematic of the genomic context. Most of the arrows in the schematic are different colors, which have no explanations, the sequence inserted below the schematic is too small to decipher, and the blue box inserted to the right is not labeled or sufficiently explained. To easily understand this figure, readers will need to be familiar with the papers by Koonin et al. I think this is an unfair expectation of readers.Seems like it would be worth showing a gel or at least referencing the gel in Figure 5—figure supplement 4, when the authors first claim to have "expressed and purified a representative casposase".Figure 1B. I was unable to understand how the TIR (introduced in font size ~2 in panel B) is different form the TSD, which is introduced in panel A. I understand that it's a tandem inverted repeat but where is the sequence, how long, how is it related to the biology (I'm lost)..? Why is the first gel white on black and the second is black on white? Are these different imaging methods of the same assay? If the 5'-CGCA motif is conserved, then why do the authors mutate the first 6 rather than the first 4 bps of the TSD sequence?Figure 1C. What are the red dots one end of the orange oligonucleotides?Figure 2A. In my opinion, seeing a gel of the purified protein is more important to me than seeing a series of SEC runs with increasing concentration of the DNA substrates. If the authors choose to show these in the main text, then a vertical line extending from the protein alone peak down though the other runs will help illustrate the shift. The authors disclose that "the complex did not produce the expected shift on SEC for a tetramer", but the peak does not look symmetrical to me and the left edge appears to be almost vertical. This would suggest that the complex may be eluting in the void, but the x-axis is in time rather than volume, so it is hard to know what this mean for a16/60 Superdex 200 column. Try a Sup 6 column and include a regression line fit with standers. It appears that the labels for the x-axis are misplaced for the last column ("integration products"). In my opinion this entire figure could go in the supplemental material and Figure 1 should be refined and split into two figures.The text for Table 1 overlaps the table itself making it a little hard to interpret. I suggest adding a sentence to the text that states how the structure was determined .Figure 3. Active site subunits "shown in pink sticks" are nearly impossible to see. I could not find an explanation for the green spheres in panel B.Figure 4. The authors state that "Each casposase monomer exhibits the fold expected of a Cas1 homolog" but none of the figures do a good job of showing this and I could not find an RMSD for a define set of equivalently positioned atoms.Discussion paragraph five: delete word "is". “It is an intriguing possibility is that some casposases may already possess the ability to integrate short CRISPR-like spacers,…..”Reviewer #2:In their manuscript, Hickman and colleagues present the first crystal structure of a transposon-encoded casposase bound to an integration product mimic, revealing a tetrameric architecture in which two Cas1 dimers sandwich the target DNA destined to be duplicated during downstream molecular steps within the pathway. Biochemical experiments are presented to argue that single-stranded transposon ends may be the natural substrate utilized during integration, and that specific sequence motifs in both the transposon end and the target site affect biochemical integration activity. By comparing their casposase-DNA structure to available crystal structures of Cas1 homologs in the context of the Cas1-Cas2 complexes that mediate spacer acquisition during adaptive immunity in CRISPR-Cas systems, the authors argue a plausible evolutionary scenario by which gaining Cas2 interactions could have directed casposases towards an integration mechanism that is more akin to spacer acquisition than transposition.The crystal structure is a notable advance that broadens our understanding of the Cas1/casposase protein family, and is undoubtedly a substantive contribution to the field. However, in this reviewer's opinion, the biochemical experiments that accompany this finding fall short of providing compelling evidence for a specific integration mechanism, and instead leave more to be desired in terms of what overhang geometries are optimal, how integration sites are chosen, and how casposons are excised. In addition, the authors gloss over the interesting finding that the HTH domain is disordered in the crystals and fail to follow-up with further experiments to investigate the role of this domain on biochemical integration activity, and whether the domain may be important in the context of more physiologically relevant substrates. I believe additional experiments are merited for this study to go beyond reporting a novel structure, to helping yield new mechanistic insights for the process of casposon transposition.1) Some important controls are missing from the results presented in Figure 1, most notably negative controls in which the plasmid is incubated with casposase in the presence of divalent metal ions but no transposon end substrates. Previous Cas1 studies have observed considerable nicking activity (or possibly nicking/cleavage from contaminating proteins), so it is crucial that the baseline activity of the casposase protein prep on plasmid DNA is evaluated. Furthermore, for the data presented in panels C and D, no lanes exist showing the starting plasmid substrate at time 0 (i.e. in the absence of protein and transposon end substrate); since any given plasmid prep will have variable ratios of supercoiled to open-circular plasmid, it is critical that the reader have this comparison – for the exact substrate used in the experiments – to fully evaluate the biochemical activity of casposase and discriminate this from the starting status of the substrate. I would even argue that naming the bands "target," "SE," and "DE," is misleading, since a plasmid will naturally purify from a bacterial culture with some fraction in the open circular form, which will migrate the same as a SE product but is certainly not a single-end integration product.2) A major question is left unaddressed (and mostly unmentioned) in the manuscript, namely, in what form the casposon is excised and integrated. I understand this is still unclear in the literature, that previous attempts to monitor excision by this group were unsuccessful , and that there is as yet no in vivo transposition data for casposons. Nevertheless, the reader should certainly be given more context on this topic, in the Introduction/Discussion, and in the Results section to better motivate the range of donor substrates tested. I would also like to see the authors test a larger panel of "Top Strand Recessed" substrates, and to more quantitatively compare integration activities amongst these substrates, and between these substrates and blunt dsDNA substrates. Similar experiments in the context of spacer acquisition by Cas1 complexes  have revealed the likely substrates that are used biologically, based on a clear optimum in terms of integration product formation kinetics. The authors have an opportunity to make similar discoveries with their casposase, but the use of just two timepoints, the lack of any product quantification, and the relatively small set of substrates tested, limits the potential of their biochemical assays.3) Related to #2, I would prefer more experimental support and/or justification for the specific choice of the 8-nt overhang integration product. Why was 8 selected as the overhang length? Was a larger crystallization screen performed, and this was the only substrate geometry that resulted in diffracting crystals? Is there any further evidence that this is a physiologically relevant ssDNA end sequence? What happens if the casposon end substrates are shorter, or partially double-stranded? For example, it seems that the iSCAMS analysis could be scaled up to test a wider range of model integration products, to investigate whether tetramer formation is more or less favored with certain substrates, which could help suggest what may or may not be more 'relevant.'4) The authors point out that capsposases have an additional C-terminal HTH domain that is absent in Cas1, and that this domain could not be modeled into the experimental electron density. In light of these interesting observations, the authors should test the biochemical activity casposase mutants in which this domain is truncated; does these mutants show behave similarly? Differently? Although it would be interesting to further investigate whether this domain becomes ordered with different DNA substrates, I understand that pursuing additional structures may be too much to ask. However, certainly some biochemical investigations are warranted here. I would also caution the authors against stating that the HTH domain "is not in a folded state" – although I am not a structural biologist, the absence of ordered density in a X-ray crystal structure is not equivalent to a domain being unfolded.5) A major take-away from this manuscript would be that this casposase integrates substrates primarily at a specific target site rather than randomly, as has been previously touched upon in the literature. However, apparent integration events in pUC19 alone and farther away from the target site have been detected in this and previous studies. No sequencing data for these events are shown, and the authors failed to perform additional analysis on integration patterns. It has not become routine in spacer acquisition studies with Cas1 to deep sequence half-site integration products in order to gain deeper insights into specificity, and the authors should perform similar NGS experiments here to enhance their biochemical results. These experiments could provide much more data on the target site requirements, but looking for conservation (or lack of conservation) in the motifs that are preferred adjacent to integration sites other than at the 3' end of tRNA-Leu.Reviewer #3:Methanosarcina mazei, and perform biochemical experiments to establish the target sequence specificity and substrate preferences for this casposase. They also report, for the first time, a structure of the casposase bound to an integration product mimic. The casposase forms a tetramer composed of two casposase dimers sandwiching the DNA. The structure provides insight into the mechanism of substrate and target recognition by the casposase. In addition, comparison of the casposase and Cas1-Cas2 complex structures show that the casposase tetramer is mutually exclusive with Cas2 binding, and suggests how architectural changes in the Cas1-Cas2 complex may have led to mechanistic differences between the two types of integrases. The structure of the casposase is an important first step in understanding casposon function and the evolution of CRISPR-Cas adaptation mechanisms, and the authors propose several interesting hypotheses based on the structure. However, these hypotheses are largely speculative and could greatly benefit from experimental evidence. In particular, the structural data is not supported by any biochemical experiments to establish a structure-function relationship. Thus, the structure is largely descriptive, although the authors speculate on the importance of a number of observed interactions within the structure. Several examples are provided in comment 1 below, in addition to few other concerns that should also be addressed by the authors.In this study, Hickman et al. describe the activity and structure of a casposase, the hypothesized evolutionary ancestor of the Cas1 integrase found in CRISPR-Cas adaptive immune systems. The authors demonstrate in vitro DNA integration by the casposase from 1) When describing the structure, the authors note several protein DNA contacts, but it is unclear whether any of these contacts contribute to the specificity for the TSD target or LE substrate. From the data shown in Figure 1C, it appears that the casposase is somewhat specific for the LE sequence, especially for shorter single-stranded DNAs. Can this specificity be explained by the structure? Do R191 and R192 contribute to the specificity of the casposase for the TSD? The authors speculate about the importance of stacking interactions for ssDNA substrate binding (subsection “ssDNA casposon end binding resembles that of spacer 3' overhangs”). Did the authors perform any mutagenesis experiments to establish the importance of these contacts for target and substrate specificity? In general, the study would be greatly improved by experiments supporting the structure-based hypotheses proposed by the authors when describing the structure.2) The order in which substrates are shown on the left gel in Figure 1C is confusing. If possible, it would be helpful if this gel could be re-run to place casposon-specific and random substrates of similar type (e.g. TR17 vs ranTR17) in adjacent lanes on the gel. Is a dsran17 substrate (i.e. a fully double-stranded random substrate of 17 bp) also integrated? In addition, ssran12 and ssLE11 could be omitted from this gel, as these substrates are also tested in the rightmost gel in the panel.3) In paragraph two of the Discussion section, the authors suggest a similarity in the lack of sequence specificity on the bottom strand of the casposase TSD with a lack of sequence specificity for spacer-side integration by Cas1-Cas2. However, it is important to note that Cas1-Cas2 does display specificity for the entire direct repeat sequence, likely through sequence-specific deformations of the repeat that occur during full-site integration . In that study, it was shown that mutations in the repeat affect spacer-side integration, but do not have an effect on leader-side integration. Thus, the conclusion stated by the authors that bottom strand integration is sequence-independent and instead based on a "molecular ruler" is not consistent with current models for spacer-side integration.eLife. Your revised article has been evaluated by John Kuriyan (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas" for further consideration by eLife. The reviewers offer a few additional suggestions, as outlined below:The reviewers agree that the manuscript has been dramatically improved and there is unanimous consensus that the paper should be published in Summary:Methanosarsina mazei. They demonstrate that the MmCasposase integrates a wide variety of RNA and DNA substrates into a specific location. Integration requires substrates with a free 3'-OH and a target that contains specific sequence motifs. To understand the mechanism of integration the authors attempt to crystallize the casposase bound to DNA oligonucleotides representing different stages of the integration reaction. Diffracting crystals of the casposase bound to a substrate that mimics the post integration step were used to determine a 3.1 Å resolution structure. The structure reveals a tetrameric architecture in which two casposase dimers sandwich the target DNA. The authors highlight several interactions with target and the substrate, and mutants are used to test the importance of these interactions. The authors compare this structure to available crystal structures of Cas1 and Cas1-2 complexes. This analysis shows that the casposase tetramer is mutually exclusive with Cas2 binding and suggests how architectural changes in the Cas1-Cas2 complex may have led to mechanistic differences between the two types of integrases. Collectively, the biochemistry and structure presented support a speculative discussion that connects architectural changes in the casposase that would accommodate Cas2 binding and the transition to a Cas1-Cas2 complex that is necessary for spacer integration. While clarity of presentation could be improved in places, the work is rigorously performed, and the conclusions are of interest to a broad readership.In this manuscript, Hickman et al. express and purify a casposase from Organization of the presentation:The initial characterization of casposon substrate and target specificities is much improved by the new experiments. However, the presentation is a bit disjointed, as it jumps back and forth between characterization of substrate and target requirements. For example, Figures 1C, 3A-B and 3D all investigate casposon end requirements, while Figures 2 and 3C are on target specificity. Indeed, in one case this requires that the reader read ahead to understand the experiment explained in Figure 2 . This section could flow better if the authors combine Figure 1C, 3A-B, and 3D into a new Figure, which could be Figure 2. This would also allow the authors to show a panel describing all substrates used in these three panels, as it is currently confusing that TR17 and ssLE17 are shown in Figure 1C but not mentioned until much later in the text. Figures 2 and 3C could then be combined to form a new Figure 3. With these changes, the authors could first define casposon end requirements, and then describe the characterization of the target sequence. [Editors’ note: what follows is the authors’ response to the first round of review]In this manuscript, Hickman et al. present the first crystal structure of a transposon-encoded casposase bound to an integration product mimic. The structure reveals a tetrameric architecture in which two casposase dimers sandwich the target DNA. The authors compare this structure to available crystal structures of Cas1 and Cas1-2 complexes. This analysis shows that the casposase tetramer is mutually exclusive with Cas2 binding and suggests how architectural changes in the Cas1-Cas2 complex may have led to mechanistic differences between the two types of integrases.The reviewers all agree that the structure presented in this manuscript is potentially important. However, the structural data are not supported by biochemical experiments that advance our understanding of mechanism.Some of the specific points raised during the review and in the discussion between reviewers and the editors are noted below, and the individual reviews are appended. We concluded that it could take substantial time to address all of these issues in a satisfactory manner. At this point, the authors may simply wish to submit this high quality paper elsewhere, without delay. If, however, these points can be addressed satisfactorily then the paper could be resubmitted to eLife for further consideration, should the authors wish to do so.1) Clarification on the DNA substrates used in the biochemical assay, and an explanation for how these substrates relate to the biology are required.M. mazei casposon 31mer TIR . In the text, we now reference the work on CRISPR Cas1-Cas2 proteins to explain our exploration of substrates with 3'-OH overhangs. The use of single-stranded substrates was the outcome of performing control reactions (or what we thought would be control reactions), and their activity was unexpected. Since then, Krupovic et al. (2019) have reported that a single-stranded casposon end is an effective substrate for the A. boonei casposase, and we have cited their work. Unfortunately, as there are many unknowns regarding the experimental biology for casposons, we can only speculate as to how these particular substrates relate to their biology.We have repeated all of the biochemical assays and incorporated more controls and time points (see below). In doing so, we took the opportunity to perform the biochemical assay first discussed in the text using an authentic Additional controls and additional time points are necessary. Important controls are missing from the results presented in Figure 1, most notably negative controls in which the plasmid is incubated with casposase in the presence of divalent metal ions but no transposon end substrates. Previous Cas1 studies have observed considerable nicking activity (or possibly nicking/cleavage from contaminating proteins), so it is crucial that the baseline activity of the casposase protein prep on plasmid DNA is evaluated. Furthermore, for the data presented in panels C and D, no lanes exist showing the starting plasmid substrate at time 0 (i.e. in the absence of protein and transposon end substrate); since any given plasmid prep will have variable ratios of supercoiled to open-circular plasmid, it is critical that the reader have this comparison – for the exact substrate used in the experiments – to fully evaluate the biochemical activity of casposase and discriminate this from the starting status of the substrate. Labeling the bands "target," "SE," and "DE," is misleading, since a plasmid will naturally purify from a bacterial culture with some fraction in the open circular form, which will migrate the same as a SE product but is certainly not a single-end integration product.We have repeated the experiments presented in the original manuscript  and included additional controls and time points. In our revised manuscript, for all experiments involving plasmid substrates, a negative control is shown in which plasmid is incubated with the casposase and metal ion in the absence of a transposon end substrate. We have re-labeled the product band positions as suggested by the reviewers, and these are now indicated as "relaxed", and "linear". The target plasmid is now labelled "sc". We have retained the SE and DE terminology in the schematic in Figure 1B, as these are the products expected by the model. As a reader can now judge, there is not much nonspecific nicking with our purified casposase under the assay conditions used.2) Provide more experimental support and/or justification for the specific choice of the 8-nt overhang integration product. Why was 8 selected as the overhang length? Was a larger crystallization screen performed, and this was the only substrate geometry that resulted in diffracting crystals? Is there any further evidence that this is a physiologically relevant ssDNA end sequence?We apologize, as in our original submission we failed to mention that we tried a wide range of DNA oligonucleotides for structural studies over the course of many months, and that – to date – the only substrate that has yielded crystals that diffract X-rays well enough to allow us to solve the structure is the 8-nt overhang integration product. We believe that such exploration (riddled with many failures) is typical in experimental structural biology. As shown in Figure 3B, an 8-nt ssDNA casposon sequence produces products in an integration assay that are specific for the target site . Thus, it has the specificity expected for a biologically relevant end sequence but we cannot say more than this in the absence of an in vivo assay for casposon mobility.For at least one reviewer, it was unclear what the SEC data provided that was not show by the iSCAMS analysis. All reviewers commented on misplaced labels and inappropriate units (time rather than volume) in the SEC. There was some discussion about moving the SEC to the supplement but regardless of where it is discussed, the interpretation of these data must be clarified.We agree with the reviewer that the SEC data are perhaps not as important in light of the iSCAMS analysis; therefore we have chosen to present it (and some new data) now as supplemental material . The supplemental figures now show the chromatograms with corrected labels and volume units. We have been able to clarify the apparent disconnect between the iSCAMS and SEC results with further SEC experiments in which we performed a dilution series with the protein alone and complexes with TR17 and the crystallized integration product. These revealed that the elution properties of the casposase alone and its complexes change as a function of protein concentration. We conclude that the lower concentration used for iSCAMS more accurately reflects the property of the casposase in vivo. These results are now discussed in the revised text.3) The authors gloss over the interesting finding that the HTH domain is disordered in the crystals and fail to follow-up with further experiments to investigate the role of this domain on biochemical integration activity, and whether the domain may be important in the context of more physiologically relevant substrates.We used the structure as a guide to produce a soluble version of the casposase missing the C-terminal domain  – solubility being a property that had frustratingly eluded us in the absence of structural information. We have therefore been able to follow up with integration experiments using a range of substrates, and these results are now presented in Figure 6 and associated text. Indeed, the C-terminal domain is crucial, although we do not know if it is recognizing the oligonucleotide end or the target. To date, we have not been able to confirm an interaction with either.4) A major take-away from this manuscript would be that this casposase integrates substrates primarily at a specific target site rather than randomly, as has been previously touched upon in the literature. However, apparent integration events in pUC19 alone and farther away from the target site have been detected in this and previous studies. No sequencing data for these events are shown, and the authors fail to perform additional analysis on integration patterns. The sequences should be shown and discuss. It has become routine in spacer acquisition studies with Cas1 to deep sequence half-site integration products in order to gain deeper insights into specificity. Similar NGS experiments are warranted here to enhance their biochemical results. While NGS experiments are not required for publication in eLife, the authors should perform bioinforamtics analyses to determine whether related casposons are always present in genomes adjacent to similar target sites. Please mention, in the Discussion, the recent observation that the Type V-C Cas1 is much less sequence-specific in its integration site preference.M. mazei casposase .”We agree with the reviewer's suggestion that the need for an NGS experiment was justified. We recruited the expertise of our colleagues, Dr. Pavel and Dr. Haase, who helped us to perform the NGS analysis on the linear product of targeted integration. They are now included as co-authors, and the new data are presented in Figure 2 and discussed in the text. As suggested, we have added a sentence regarding Type V-C Cas1 to the Discussion as follows: “Finally, the V-C Cas1 is less sequence-specific in its integration site preference relative to other characterized Cas1-Cas2 integrases , in marked contrast to the stringent sequence-specificity we observed here for the 5) When describing the structure, the authors note several protein-DNA contacts, but it is unclear whether any of these contacts contribute to the specificity for the TSD target or LE substrate. From the data shown in Figure 1C, it appears that the casposase is somewhat specific for the LE sequence, especially for shorter single-stranded DNAs. Can this specificity be explained by the structure? Do R191 and R192 contribute to the specificity of the casposase for the TSD? The authors speculate about the importance of stacking interactions for ssDNA substrate binding (subsection “ssDNA casposon end binding resembles that of spacer 3' overhangs”). Did the authors perform any mutagenesis experiments to establish the importance of these contacts for target and substrate specificity? Experiments supporting structure-based hypotheses proposed by the authors are required.As suggested, in order to explain the specificity for short ssDNAs, we generated several mutants to test the roles of the residues observed to form interactions with the ssDNA. These include three mutant versions of the casposase that the structure predicted should disrupt the stacking interactions, as well as the double-mutant R191A/R192A. We have compared their activity on the TSD target vs. pUC19 to evaluate target specificity, and have tested their integration activity using a range of substrates to probe the role of these residues in substrate specificity . These data are now discussed in the text.6) The order in which substrates are shown on the left gel in Figure 1C is confusing. If possible, it would be helpful if this gel could be re-run to place casposon-specific and random substrates of similar type (e.g. TR17 vs ranTR17) in adjacent lanes on the gel. Is a dsran17 substrate (i.e. a fully double-stranded random substrate of 17 bp) also integrated? In addition, ssran12 and ssLE11 could be omitted from this gel, as these substrates are also tested in the rightmost gel in the panel.We apologize for the haphazard organization of the gels presented in our original submission. As suggested, we have repeated the experiment to include a dsran17 substrate and the order of the lanes has been changed. These data are now in Figure 3A.7) In paragraph two of the Discussion section, the authors suggest a similarity in the lack of sequence specificity on the bottom strand of the casposase TSD with a lack of sequence specificity for spacer-side integration by Cas1-Cas2. However, it is important to note that Cas1-Cas2 does display specificity for the entire direct repeat sequence, likely through sequence-specific deformations of the repeat that occur during full-site integration . In that study, it was shown that mutations in the repeat affect spacer-side integration, but do not have an effect on leader-side integration. Thus, the conclusion stated by the authors that bottom strand integration is sequence-independent and instead based on a "molecular ruler" is not consistent with current models for spacer-side integration.Upon repeating our initial experiments with time courses, it has become clear that our original statement has to be revised. As shown in Figure 3C, when the activity of the "mutR" substrate is compared to that of "symTSD", two substrates that differ in sequence at the site of "bottom strand" integration, there is clearly a difference. We have removed the entire section from the revised text.Reviewer #1:Cas1 and Cas2 are hallmarks of the CRISPR-Cas adaptive immune systems found in most archaeal and many bacterial genomes. Koonin and colleagues have previously identified homology between Cas1 and casposon family proteins (doi: 10.1186/1741-7007-12-36) and predicted that CRISPR repeats evolved from the target site duplications (doi: 10.1016/j.mib.2017.04.004). These predictions have been largely validated with in vitro biochemical studies . Here, Hickman et al., extend the previous work by defining essential features of the target site and testing a wide-variety of integration substrates. This on its own would not warrant publication in eLife. However, they use this qualitative biochem to identify substrates that result in higher order assemblies and they determine the structure of the tetrameric complex bound to DNA. Overall the experiments appear technically sound, and the paper delivers the first structure of a casposase. These mechanistic insights and evolutionary connection of this system to CRISPR-Cas immune systems are important and warrant publication in eLife. However, the text and figure are a challenge to interpret  and considerable revision is required to make this work accessible to a broad readership.The authors purify a casposases from Methanosarcina mazei and determine sequence requirements for integration. The authors describe a sequencing method to determine the location of integration. 7 of 8 integration occurs at the TSD sequence with concomitant generation of a 14 bp TSD. "The other sequenced product arose from integration into a site ~110 bp away from the specific sequence and was also accompanied by a 14 bp TSD." Please include more information. Was there sequence similarity between these sites? If not, what does this say about the mechanism of recognition and how does this relate what the structures teaches us about recognition?We have now performed next-generation sequencing on the linear product of the plasmid integration reaction, and included these results in the revised manuscript. As shown in Figure 2C, there is indeed sequence similarity between the favored integration site and the very few other detected sites of integration.Figure 1A. It is not clear what the authors are intending to teach the reader in panel A. Based on the figure legend title ("Casposase structure and function") the reader is expecting to see a structure, rather than a schematic of the genomic context. Most of the arrows in the schematic are different colors, which have no explanations, the sequence inserted below the schematic is too small to decipher, and the blue box inserted to the right is not labeled or sufficiently explained. To easily understand this figure, readers will need to be familiar with the papers by Koonin et al. I think this is an unfair expectation of readers.Methanosarcina mazei casposon organization and initial biochemical characterization."In light of these comments, we have substantially revised Figure 1A, and have included more labeling, increased the font size for the TSD sequence, and have included the sequence of the 31mer TIR. We have also added titles to the two figure sections. The figure legend title has also been changed to: "Seems like it would be worth showing a gel or at least referencing the gel in Figure 5—figure supplement 4, when the authors first claim to have "expressed and purified a representative casposase".In the revised manuscript, in addition to the gel in the supplemental figure showing dissolved crystals and the protein control , we now show a gel for all of the purified proteins used in this study in Figure 6A.Figure 1B. I was unable to understand how the TIR (introduced in font size ~2 in panel B) is different form the TSD, which is introduced in panel A. I understand that it's a tandem inverted repeat but where is the sequence, how long, how is it related to the biology (I'm lost)..?We have now included a better schematic including the entire relevant genomic sequence in Figure 1A.Why is the first gel white on black and the second is black on white?We thank the reviewer for pointing this out and, in fact, there was no good reason. This has been corrected in the revised figures.Are these different imaging methods of the same assay?The gels have now been standardized to black-on-white throughout the manuscript.If the 5'-CGCA motif is conserved, then why do the authors mutate the first 6 rather than the first 4 bps of the TSD sequence?The experiment shown in the original submission was performed before we recognized that the first 4 bp are important, not 6. We have therefore removed this experiment panel from Figure 1B; instead, we have tested the effect of mutating only the first 4 bp of the TSD sequence in the experiment shown in Figure 3C.Figure 1C. What are the red dots one end of the orange oligonucleotides?The red dot indicates the 3'-OH that is the nucleophile for the integration reaction, and this is now stated in the figure legend for the schematic in Figure 1B. We have replaced the original schematic involving red dots with the explicit sequences of the oligonucleotides used .Figure 2A. In my opinion, seeing a gel of the purified protein is more important to me than seeing a series of SEC runs with increasing concentration of the DNA substrates.We agree with the reviewer, and an SDS-PAGE gel is now shown in Figure 6A , and the SEC data are now in supplementary material .If the authors choose to show these in the main text, then a vertical line extending from the protein alone peak down though the other runs will help illustrate the shift.We appreciate the suggestion, and vertical lines are now incorporated.The authors disclose that "the complex did not produce the expected shift on SEC for a tetramer", but the peak does not look symmetrical to me and the left edge appears to be almost vertical. This would suggest that the complex may be eluting in the void, but the x-axis is in time rather than volume, so it is hard to know what this mean for a16/60 Superdex 200 column.To resolve these issues, we performed further SEC experiments over a range of protein concentrations and generated a standard curve for the Superdex 200 Increase 3.2/300 column to allow us to estimate MWs. Although the peak shape is indeed asymmetric, at three different concentrations the integration product complex eluted with a peak position consistent with a tetramer bound to one DNA substrate.Try a Sup 6 column and include a regression line fit with standers.We believe we have resolved the basis for our original confusion (we had not recognized the effect of protein concentration on elution position), so have not repeated the experiment on a Superose 6 column. The regression line fit is now shown in Figure 4—figure supplement 2.It appears that the labels for the x-axis are misplaced for the last column ("integration products").Thank you for pointing this out, and the figure has been corrected.In my opinion this entire figure could go in the supplemental material and Figure 1 should be refined and split into two figures.We agree, and this has been done.The text for Table 1 overlaps the table itself making it a little hard to interpret. I suggest adding a sentence to the text that states how the structure was determined .As suggested, we have included the following when we first introduce the structure: “…was solved to 3.1 Å using experimental phases obtained from a selenomethionine derivative.” The text for the table has been corrected.Figure 3. Active site subunits "shown in pink sticks" are nearly impossible to see. I could not find an explanation for the green spheres in panel B.2+ ions from the crystallization buffer) has been included in the figure legend to Figure 5B.The active site is now shown in a close-up view , and an explanation for the green spheres  with which it shares 32% sequence identity, the N-terminal domains have an RMSD of 1.68 Å over 72 shared Cα positions and the catalytic domains are even more similar, with an RMSD of 1.28 Å over 208 shared Cα positions."We have added a figure  that shows the side-by-side structures of the casposase dimer and that of Cas1 from Discussion paragraph five: delete word "is". “It is an intriguing possibility is that some casposases may already possess the ability to integrate short CRISPR-like spacers,…..”Done – thank you.Reviewer #2:In their manuscript, Hickman and colleagues present the first crystal structure of a transposon-encoded casposase bound to an integration product mimic, revealing a tetrameric architecture in which two Cas1 dimers sandwich the target DNA destined to be duplicated during downstream molecular steps within the pathway. Biochemical experiments are presented to argue that single-stranded transposon ends may be the natural substrate utilized during integration, and that specific sequence motifs in both the transposon end and the target site affect biochemical integration activity. By comparing their casposase-DNA structure to available crystal structures of Cas1 homologs in the context of the Cas1-Cas2 complexes that mediate spacer acquisition during adaptive immunity in CRISPR-Cas systems, the authors argue a plausible evolutionary scenario by which gaining Cas2 interactions could have directed casposases towards an integration mechanism that is more akin to spacer acquisition than transposition.The crystal structure is a notable advance that broadens our understanding of the Cas1/casposase protein family, and is undoubtedly a substantive contribution to the field. However, in this reviewer's opinion, the biochemical experiments that accompany this finding fall short of providing compelling evidence for a specific integration mechanism, and instead leave more to be desired in terms of what overhang geometries are optimal, how integration sites are chosen, and how casposons are excised. In addition, the authors gloss over the interesting finding that the HTH domain is disordered in the crystals and fail to follow-up with further experiments to investigate the role of this domain on biochemical integration activity, and whether the domain may be important in the context of more physiologically relevant substrates. I believe additional experiments are merited for this study to go beyond reporting a novel structure, to helping yield new mechanistic insights for the process of casposon transposition.1) Some important controls are missing from the results presented in Figure 1, most notably negative controls in which the plasmid is incubated with casposase in the presence of divalent metal ions but no transposon end substrates. Previous Cas1 studies have observed considerable nicking activity (or possibly nicking/cleavage from contaminating proteins), so it is crucial that the baseline activity of the casposase protein prep on plasmid DNA is evaluated. Furthermore, for the data presented in panels C and D, no lanes exist showing the starting plasmid substrate at time 0 (i.e. in the absence of protein and transposon end substrate); since any given plasmid prep will have variable ratios of supercoiled to open-circular plasmid, it is critical that the reader have this comparison – for the exact substrate used in the experiments – to fully evaluate the biochemical activity of casposase and discriminate this from the starting status of the substrate. I would even argue that naming the bands "target," "SE," and "DE," is misleading, since a plasmid will naturally purify from a bacterial culture with some fraction in the open circular form, which will migrate the same as a SE product but is certainly not a single-end integration product.These issues have been addressed by repeating the experiments in Figure 1, as discussed above.2) A major question is left unaddressed (and mostly unmentioned) in the manuscript, namely, in what form the casposon is excised and integrated. I understand this is still unclear in the literature, that previous attempts to monitor excision by this group were unsuccessful , and that there is as yet no in vivo transposition data for casposons. Nevertheless, the reader should certainly be given more context on this topic, in the Introduction/Discussion, and in the Results section to better motivate the range of donor substrates tested.We agree with the reviewer that the excision of casposons is an open question at this point. However, one possible consequence of the model for self-synthesizing transposons is that excision may not in fact be occurring as the element might be synthesized at the donor site without the need for excision. However, this is pure speculation at this point. We have included a few sentences in the revised Introduction in which we discuss how free casposon ends might be generated.I would also like to see the authors test a larger panel of "Top Strand Recessed" substrates, and to more quantitatively compare integration activities amongst these substrates, and between these substrates and blunt dsDNA substrates. Similar experiments in the context of spacer acquisition by Cas1 complexes  have revealed the likely substrates that are used biologically, based on a clear optimum in terms of integration product formation kinetics. The authors have an opportunity to make similar discoveries with their casposase, but the use of just two timepoints, the lack of any product quantification, and the relatively small set of substrates tested, limits the potential of their biochemical assays.As suggested, we have performed the plasmid integration assay with a larger panel of "Top Strand Recessed" substrates, ranging from blunt-ended to 7-nt recessed . We have also performed the assay to compare blunt dsDNA, recessed, and ssDNA . We also repeated the assay in triplicate in order to provide a quantitative comparison of their integration activities. These results are plotted in Figure 3—figure supplement 1.3) Related to #2, I would prefer more experimental support and/or justification for the specific choice of the 8-nt overhang integration product. Why was 8 selected as the overhang length? Was a larger crystallization screen performed, and this was the only substrate geometry that resulted in diffracting crystals? Is there any further evidence that this is a physiologically relevant ssDNA end sequence? What happens if the casposon end substrates are shorter, or partially double-stranded?As discussed above, we have tried a wide range of substrates in crystallization experiments and with the exception of the 8-nt overhang, none has yielded crystals of sufficient diffraction quality. We have added the following so the reader can understand why this particular complex was studied: "In an effort to further characterize the casposase and its interactions with its substrates, we attempted to crystallize the casposase bound to DNA oligonucleotides representing different stages of the integration reaction. Many casposase-DNA complexes yielded poorly-diffracting crystals including the casposase bound to recessed dsDNA oligonucleotides and ssDNA oligonucleotides of varying length representing the casposon ends. This was also the case with casposase bound to mimics of the post-integration step using both symmetric and asymmetric complete integration target sites and casposon ends of varying length and dsDNA-ssDNA composition. We were eventually able to obtain diffracting crystals of the casposase…"For example, it seems that the iSCAMS analysis could be scaled up to test a wider range of model integration products, to investigate whether tetramer formation is more or less favored with certain substrates, which could help suggest what may or may not be more 'relevant.'We agree with the reviewer, and this line of inquiry is one of our future plans.4) The authors point out that capsposases have an additional C-terminal HTH domain that is absent in Cas1, and that this domain could not be modeled into the experimental electron density. In light of these interesting observations, the authors should test the biochemical activity casposase mutants in which this domain is truncated; does these mutants show behave similarly? Differently?As discussed above, we have performed these experiments with a 1-341 construct (ΔHTH) and they are shown in Figure 6B. We note that we have not been able to express a range of truncated casposase mutants, as all other deletion constructs we generated prior to the structure determination were insoluble. In our hands, only Δ341 and Δ343 have been soluble.Although it would be interesting to further investigate whether this domain becomes ordered with different DNA substrates, I understand that pursuing additional structures may be too much to ask.Our pursuit of additional structures by X-ray crystallography is ongoing. However, in light of our experience to date with the difficulties of obtaining diffraction-quality crystals with interesting DNA constructs, we are also pursuing single particle cryo-EM experiments.However, certainly some biochemical investigations are warranted here. I would also caution the authors against stating that the HTH domain "is not in a folded state" – although I am not a structural biologist, the absence of ordered density in a X-ray crystal structure is not equivalent to a domain being unfolded.The reviewer is correct, and we have reworded this to indicate that "the C-terminal residues do not appear to be folded into a compact domain."5) A major take-away from this manuscript would be that this casposase integrates substrates primarily at a specific target site rather than randomly, as has been previously touched upon in the literature. However, apparent integration events in pUC19 alone and farther away from the target site have been detected in this and previous studies. No sequencing data for these events are shown, and the authors failed to perform additional analysis on integration patterns. It has not become routine in spacer acquisition studies with Cas1 to deep sequence half-site integration products in order to gain deeper insights into specificity, and the authors should perform similar NGS experiments here to enhance their biochemical results. These experiments could provide much more data on the target site requirements, but looking for conservation (or lack of conservation) in the motifs that are preferred adjacent to integration sites other than at the 3' end of tRNA-Leu.As mentioned above, NGS experiments have been performed and the results discussed in the text and shown in Figure 2.Reviewer #3:In this study, Hickman et al. describe the activity and structure of a casposase, the hypothesized evolutionary ancestor of the Cas1 integrase found in CRISPR-Cas adaptive immune systems. The authors demonstrate in vitro DNA integration by the casposase from Methanosarcina mazei, and perform biochemical experiments to establish the target sequence specificity and substrate preferences for this casposase. They also report, for the first time, a structure of the casposase bound to an integration product mimic. The casposase forms a tetramer composed of two casposase dimers sandwiching the DNA. The structure provides insight into the mechanism of substrate and target recognition by the casposase. In addition, comparison of the casposase and Cas1-Cas2 complex structures show that the casposase tetramer is mutually exclusive with Cas2 binding, and suggests how architectural changes in the Cas1-Cas2 complex may have led to mechanistic differences between the two types of integrases. The structure of the casposase is an important first step in understanding casposon function and the evolution of CRISPR-Cas adaptation mechanisms, and the authors propose several interesting hypotheses based on the structure. However, these hypotheses are largely speculative and could greatly benefit from experimental evidence. In particular, the structural data is not supported by any biochemical experiments to establish a structure-function relationship. Thus, the structure is largely descriptive, although the authors speculate on the importance of a number of observed interactions within the structure. Several examples are provided in comment 1 below, in addition to few other concerns that should also be addressed by the authors.1) When describing the structure, the authors note several protein DNA contacts, but it is unclear whether any of these contacts contribute to the specificity for the TSD target or LE substrate. From the data shown in Figure 1C, it appears that the casposase is somewhat specific for the LE sequence, especially for shorter single-stranded DNAs. Can this specificity be explained by the structure? Do R191 and R192 contribute to the specificity of the casposase for the TSD? The authors speculate about the importance of stacking interactions for ssDNA substrate binding (subsection “ssDNA casposon end binding resembles that of spacer 3' overhangs”). Did the authors perform any mutagenesis experiments to establish the importance of these contacts for target and substrate specificity? In general, the study would be greatly improved by experiments supporting the structure-based hypotheses proposed by the authors when describing the structure.As discussed above, we have now performed these experiments and the results are shown in Figure 7F and Figure 7—figure supplement 1.2) The order in which substrates are shown on the left gel in Figure 1C is confusing. If possible, it would be helpful if this gel could be re-run to place casposon-specific and random substrates of similar type (e.g. TR17 vs ranTR17) in adjacent lanes on the gel. Is a dsran17 substrate (i.e. a fully double-stranded random substrate of 17 bp) also integrated? In addition, ssran12 and ssLE11 could be omitted from this gel, as these substrates are also tested in the rightmost gel in the panel.We have rerun the assay to both expand the range of substrates tested and to place them in a logical order. The results are shown in Figure 3A.3) In paragraph two of the Discussion section, the authors suggest a similarity in the lack of sequence specificity on the bottom strand of the casposase TSD with a lack of sequence specificity for spacer-side integration by Cas1-Cas2. However, it is important to note that Cas1-Cas2 does display specificity for the entire direct repeat sequence, likely through sequence-specific deformations of the repeat that occur during full-site integration . In that study, it was shown that mutations in the repeat affect spacer-side integration, but do not have an effect on leader-side integration. Thus, the conclusion stated by the authors that bottom strand integration is sequence-independent and instead based on a "molecular ruler" is not consistent with current models for spacer-side integration.We thank the reviewer for pointing this out. As discussed above, upon further examination of our data, we realize that our original statement was not correct. We have removed the entire section from the revised manuscript.[Editors’ note: what follows is the authors’ response to the second round of review.]Organization of the presentation:The initial characterization of casposon substrate and target specificities is much improved by the new experiments. However, the presentation is a bit disjointed, as it jumps back and forth between characterization of substrate and target requirements. For example, Figures 1C, 3A-B and 3D all investigate casposon end requirements, while Figures 2 and 3C are on target specificity. Indeed, in one case this requires that the reader read ahead to understand the experiment explained in Figure 2 . This section could flow better if the authors combine Figure 1C, 3A-B, and 3D into a new Figure, which could be Figure 2. This would also allow the authors to show a panel describing all substrates used in these three panels, as it is currently confusing that TR17 and ssLE17 are shown in Figure 1C but not mentioned until much later in the text. Figures 2 and 3C could then be combined to form a new Figure 3. With these changes, the authors could first define casposon end requirements, and then describe the characterization of the target sequence.We agree that most of these suggestions would help with the logic and the flow. However, a minor problem arises with Figure 3D which is certainly related to substrate specificity  but uses an assay that is introduced and validated only in Figure 3C . The solution we chose is to keep 3C and 3D together. We have therefore created a new Figure 2 with 1C, 3A-B, and combined old Figures 2 and 3C-3D into new Figure 3.


Sequence comparison is an essential part of modern molecular biology research. In this study, we estimated the parameters of Markov chain by considering the frequencies of occurrence of the all possible amino acid pairs from each alignment-free protein sequence. These estimated Markov chain parameters were used to calculate similarity between two protein sequences based on a fuzzy integral algorithm. For validation, our result was compared with both alignment-based  and alignment-free methods on six benchmark datasets. The results indicate that our developed algorithm has a better clustering performance for protein sequence comparison. Phylogenetic study and comparative analysis between taxa are an essential part of molecular biology and bioinformatics. These studies, traditionally depended on multiple or pairwise sequence alignments which are the well established classical approach and regarded as a standard method for sequence analysis. However, producing reliable multiple sequence alignments become extremely difficult when more dissimilar protein sequences are considered. The traditional alignment-based methods5 are much empirical to select and create a sequence alignment score matrix, and variation of which may affect the alignment results. Various alignment-free tools13 have been developed over the past two decades to overcome the alignment complexity for phylogenetic analysis. An alignment-free approach consist of two steps for comparing protein sequences. At the first step, the protein sequences are converted into a fixed-length feature vectors. Feature extraction is a series of process for extracting the required information from the query sequences, which is critical for the accuracy of an alignment-free method. At the second step, these extracted feature vectors are used as an input data in vectors similarity comparison algorithm to perform downstream analysis like phylogenetic analysis. Methods based on graphical representation, distance frequency matrix, numerical characterization, K-string dictionary etc., have been introduced to overcome the complication of the sequence alignment. Graphical representation15 of protein sequences provides a simple way of viewing, sorting and comparing various sequences. It also provides mathematical descriptor which help in identifying differences among similar protein sequences quantitatively. Distance frequency of amino acid pairs suggest a new numerical characterization of protein sequence, which converts protein sequence into a distance frequency matrix16. Numerical characterization directly extracted from protein sequence would capture the essence of the amino acid composition and their distribution on the protein sequence in a quantitative aspect. In this approach, each sequence is mapped into a vector or matrix based on the numerical characterization extracted from the protein sequence. Subsequently, a similarity score is calculated by following distance measure tools, such as, Euclidean distance, Cosine distance, Manhattan distance, etc., among their corresponding vectors or matrices. K-string dictionary17 approach permit users to use a much lower dimensional frequency or probability vector to represent a protein sequence. It also significantly reduces the space requirement for their implementation. Furthermore, after getting the lower dimensional frequency vectors, Singular Value Decomposition (SVD) is used to get a better protein vector representation which helps user to obtain a precise phylogenetic tree. However, these above mentioned methods are lagging behind in terms of accuracy. Thus, more discriminatory features are still needed to be developed. In addition to the accuracy, these method have another drawback and that is, computational complexity. Motivated by the aforementioned work, in this study, we proposed to use fuzzy integral algorithm19 for analysis of protein sequence based on Markov chain20. Fuzzy integral similarity22 method assigns similarity score within the closed interval  between two protein sequences. A protein sequence consists of twenty amino acids. By taking these 20 amino acids as a state space M = {A, I, L, M, F, P, W, V, D, E, N, C, Q, G, S, T, Y, R, H, K}, we have used kth−step transition probability matrix, fuzzy measure23, fuzzy integral to describe protein sequence. We have used fuzzy integral similarity for getting distance matrix, which is used in neighbor program in PHYLIP package24 for constructing a phylogenetic tree. The advantage of our method is, it do not require any prior knowledge of homologous relationship (common ancestry) among the sequences, which makes it fully automated and robust. For validation of our developed algorithm, we implemented our approach on NADH Dehydrogenase-5 protein sequences, NADH Dehydrogenase-6 protein sequences, xylanases protein sequences in the F10 and G11 datasets, transferrin protein sequences, coronavirus spike protein sequences and beta-globin protein sequences. We compared the tree generated by our method with the trees generated by both alignment-free method, and alignment-based ClustalW method using MEGA package25. In addition, we used few standard statistical tools such as correlation coefficient (CC), Robinson-Foulds distance (RF-distance)26 and receiver operating characteristic (ROC)29 curve to compare distance matrices generated by our method with the other alignment-free methods. The main purpose of this study is to compare the performance among alignment-based and alignment-free protein clustering methods and to identify their strengths and weakness from the practical perspectives of the users.With the advent of the advanced sequencing techniques, researchers are generating a large number of protein sequences. This brings in a new challengeP =  represent the transition probability matrix of a discrete-time Markov chain20. Transition probability pi,j can be defined as follows:Zn represent the actual state at time n, ai is the ith state within 20 distinct states. In the context of protein sequence, the number of states is M = 20, which corresponds to the twenty amino acids symbol set M = {A = a1, I = a2, L = a3, M = a4, F = a5, P = a6, W = a7, V = a8, D = a9, E = a10, N = a11, C = a12, Q = a13, G = a14, S = a15, T = a16, Y = a17, R = a18, H = a19, K = a20}. The state transition probabilities satisfy the following constraintsai, aj), then 1st−step transition probability matrix from the state ai to the state aj is given byst step Markov chain and the kth step Markov chain can be obtained through the 1st step Markov chain. Let i after k steps to end with state j. Each state transition probability U, V and W, the following condition holds: p[U ∩ V|W] = p[U|V ∩ W]p[V|W]. Interpreting U as Zn+k = aj, V as Zn+t = ar and W as Zn = ai, we haveLet Hence, the matrix with element kth-step transition probability matrix can be expressed as:kth-step transition probability matrix, we optimized the step k = h, which is least positive integer under satisfying following condition for each protein sequence:rmsd represent root mean square distance between two consecutive transition probability matrices. After optimizing the step of transition probability matrix, i.e, Ph. We noted that, all 20 rows in optimized transition probability matrix are approximately identical with each other. Therefore, we took a single row from the transition probability matrix Ph as a input for further investigation, which reduced our time complexity.In the context of protein sequence, entclass1pt{minimaquations . After tG = {(bai)h = xi|i ∈ {1, 2, 3, ..., 20}, b ∈ M} be the finite set of hth–step amino acids starting from amino acid b and ending with amino acid ai, estimated from protein sequence. The finite set G is termed as feature vector.Let ν, τ ⊆ G and R(G) be the power set of G. A fuzzy measure μ is a real valued function:Let μ: R(G) → , satisfy the following condition,(i)(ii)μ, let μ(xi) = μi ∀ xi ∈ G. The mapping xi → μi is known as fuzzy density function. The fuzzy density of single element xi ∈ G, μi can be interpreted as the importance of xi in determining the set G. Based on the fuzzy measure definition μ, the measure of a subset is not just only the summation of the measure of its elements but also included the measure of each combination. This information could be delivered by an expert or observed through the problem. However, when handing with larger set, this job may become computationally complex, difficult or even not feasible. λ-measures is the possible solution for solving this problem. λ-fuzzy measure30 fulfills the criteria of fuzzy measure plus some additional property: for all ν, τ ⊂ G, ν ∩ τ = ϕ andλ can be obtained by solving following equation:μi .For a fuzzy measure ρ:G →  represent a function that maps every element of G to its evidence. The function ρ must satisfy descending order, which is as follows: ρ(x1) ≥ ρ(x2) ≥ ρ(x3) ≥ ... ≥ ρ(x20). If suppose ρ function does not satisfy the above condition, then reorder G so that ρ function must satisfy descending order condition and we will proceed further calculation based on the modified descending order condition. Let μ:R(G) →  be a fuzzy measure. Then the fuzzy integral of ρ with respect to the fuzzy measure μ is given byLet 31 and classification.The fuzzy integral examine the fact supplied by each element of a given set, and the assessment of each subset of elements (using a fuzzy measure) in its decision-making process. The combination of the important significance of the source and the extracted information makes the fuzzy integral appropriate for information fusion. This theory has capability to tackle uncertainties associated with issue related to the processing procedures and data extraction. Therefore, this theory has been extensively applied in pattern recognitionhth–step amino acids frequencies between the feature vector of the two sequences. Let ν and τ are feature vectors of the two sequences. We define fuzzy integral function ρ, which is given as:xi ∈ G .The fuzzy integral similarity is based on the λ–fuzzy measure properties described above, we can formulate μ using the fuzzy density of the individual element μi.Using fuzzy measure, we can determine the relative importance of subsets of amino acids being considered. Taking benefit of the xi ∈ G . Using equation  and (ii) of the fuzzy measure. Once we have ρ and μ, it is a straight forward using equation is fuzzy integral similarity between ν and τNext we calculate difference between two feature vectors n number of protein sequences. Finally, a distance matrix was generated. This distance matrix contained the dissimilarity information related to n protein sequences. This distance matrix was used as an input data to the neighbor.exe program in PHYLIP package24 for phylogenetic tree construction.The above process is continued for all pairwise combinations taken from AlgorithmThis section explains an algorithmic view of the developed method. The complete algorithm consists three stages.Stage 1: Calculation of optimal-step transition probability matrix using Markov chain estimated from observed protein sequences:Stage 2: Fuzzy integral similarity between two feature vectors F1 and F2:Stage 3: Integrate stage(1) and stage(2) for phylogenetic tree construction:32 of developed algorithm, we assumed that all operations took the same unit of time. Our algorithm was partitioned into three stages. For time complexity calculation: in the initial stage, transition probability matrices were calculated from the raw protein sequences. Time complexity of stage (1) is n is the total number of protein sequences, m is the number of amino acids, l is the average length of protein sequences and hi is the optimal-step of feature vector. In the second stage, fuzzy integral similarity is calculated between two feature vectors. Therefore, time complexity of stage (2) is O(m2m). In the third stage, we integrated both the stages for generating distance matrix. Here, we used parallel computation for reducing the time complexity. Therefore, total time complexity for generating distance matrix is:t is the number of threads and εt is the extra time taken in job assigning to all t threads. We also calculated the computational speed of our method and ClustalW method on tested datasets, which is given below in conclusion section.For calculating computational complexity24 for generating the phylogenetic tree. On the benchmark data, result generated using both the approaches has minor differences between them. However, we chose optimal tree based on taxonomic classification and compare with existing tools. The six benchmark datasets used in this study are as follows:(i)NADH Dehydrogenase 5 (ND 5) protein sequences.(ii)NADH Dehydrogenase 6 (ND 6) protein sequences.(iii)xylanases protein sequences in the F10 and G11 datasets.(iv)transferrin protein sequences.(v)coronavirus spike protein sequences.(vi)beta-globin protein sequences.To test our developed algorithm, we applied it to six sets of benchmark data. Different model might result different phylogenetic tree, therefore it is important to choose the most appropriate method. Here, we used Fitch-Margoliash or UPGMA (UPGMA = Unweighted Pair Group Method with Arithmetic Mean) approaches in PHYLIP package33. Therefore ND5 has been widely used for the analysis of the phylogenetic studies and their evolution. The phylogenetic tree generated by our method shown in Fig. Hominidae includes human, pigmy chimpanzee, common chimpanzee and gorilla; (ii) Balaenopteridae includes fin whale and blue whale; (iii) Muridae includes mouse and rat; and (iv) Didelphidae include opossum. From Fig. 25  method shows that, even the CC is very high (0.9403) as compared to our method CC (0.7378) but their corresponding the RF-distance is 4, which is higher than our method RF-distance which is 2  is topologically less similar as compared to our tree to the reference tree). Similarly in Table et al.35 and Yao et al.36 having CC 0.7324 and 0.6908, respectively, which is nearer to CC of our method (CC = 0.7378). However, in terms of topological similarity, the RF- distance of Wen et al.35 and Yao et al.36 are 4 which is higher than RF-distance of our method. The above analysis shows that higher or closer CC does not always implies that the two phylogenetic trees are more similar or closer to each other.We used correlation coefficient (CC) and Robinson-Foulds distance (RF-distance)ds Table  against 41. 8 sequences of ND6 protein belong to mammals can be divided into following four categories based on their taxonomic family; (i) Hominidae includes human, common chimpanzee and gorilla; (ii) Phocidae includes harbor seal and gray seal; (iii) Muridae includes mouse and rat; and (iv) Macropodidae include wallaroo. As shown in the tree generated by our method  and  in separate clades. However, Fig. 42 and Fig. The other benchmark dataset used in this study was 8 protein sequences of NADH Dehydrogenase 6 with nearly 175 amino acids Table . All thehod Fig. , the proe25 Fig. . The tre42 with ClustalW. CC and RF-distance were also calculated between the our method and with ClustalW. In Table et al.38 method has lower CC (0.4609) than CC (0.5982) generated by our method compared with ClustalW method, and their corresponding RF-distance (RF = 6) is much higher than our method (RF = 2). Therefore, phylogenetic tree generated by our method  as compared to our method CC (0.5982) but both the methods has the same RF-distance = 2.We calculated CC and RF-distance from previous studiesree Fig. . However37. Phylogenetic tree generated by our method , actinopterygii(green square) and amphibians(black diomand). Only Japanese flounder transferrin sequence belong to actinopterygii class was clustered with Frog transferrin sequence belong to amphibians class. In Fig. oncorhynchus and salvelinus were clustered in separate clades, and sequences belong to genus salmo were placed close to each other.In this study the other benchmark dataset used was 24 protein sequence of transferrins (TFs) from vertebratesds Table . All the46 indicates improvement in our approach. In Fig. mammalia class were clustered in a separate clade which were not observed in46. Moreover, species belong to genera oncorhynchus and salvelinus were grouped into separate clades, which is lacking in46. While comparing our tree  among the sequences which makes it fully automated and robust. We implemented our method on six benchmark datasets as discussed in the result section.oncorhynchus and salvelinus in separate branches, and sequences belong to genus salmo were closest to each other. In coronavirus spike protein, phylogenetic tree generated by our approach  for distance matrices generated by our method and other alignment-free tools from Alfree repository54. The results of ROC and AUC analysis for all benchmark datasets are given in supplementary material. We are yet to attain an highly efficient alignment-free method for phylogenetic analysis. However, our method shows an improvement over the other existing alignment-free methods in terms of sequence clustering. Based on the observed progress, this method would be useful for the researcher to develop hypothesis that can be examined further in details. Before continuing our research work for further improvement, we would like to emphasize that this is a probabilistic approach in nature. It can later be modified by including more biological evidence. Overall, our goal in this study was to bring a new methodology or algorithm to the proteomics study. This proposed algorithm can be used to guide the development of more powerful measures for sequence analysis.Our programs were executed on a linux server with 24 dual core processor with 384 GB RAM. We enriched our programs by incorporating parallel computation, which can reduce the execution time of our program by increasing the number of threads, depending on the number of sequences. In our program, we implemented two threads as a default parameter. However, the user can manipulate the parameter to single thread or multiple threads. The execution time of our method with two threads is shown in Table Dataset 1Dataset 2ROC_supplementary material