Activity modulation in anaerobic ribonucleotide reductases: nucleotide binding to the ATP-cone mediates long-range order-disorder transitions in the active site

Reviewer #1 (Public Review):

The goal of this study is to understand the allosteric mechanism of overall activity regulation in an anaerobic ribonucleotide reductase (RNR) that contains an ATP-cone domain. Through cryo-EM structural analysis of various nucleotide-bound states of the RNR, the mechanism of dATP inhibition is found to involve order-disorder transitions in the active site. These effects appear to prevent substrate binding and a radical transfer needed to initiate the reaction.

Strengths of the manuscript include the comprehensive nature of the work - including numerous structures of different forms of the RNR and detailed characterization of enzyme activity to establish the parameters of dATP inhibition. The manuscript could be improved, however, by performing additional experiments to establish that the mechanism of inhibition can be observed in other contexts and it is not an artifact of the structural approach. Additionally, some of the presentations of biochemical data could be improved to comply with standard best practices.

The work is impactful because it reports initial observations about a potentially new mode of allosteric inhibition in this enzyme class. It also sets the stage for future work to understand the molecular basis for this phenomenon in more detail.

General comments:

1. It would be ideal to perform an additional experiment of some type to confirm the order-disorder phenomena observed in the cryo-EM structures to rule out the possibility that it is an artifact of the structure determination approach. Circular dichroism might be a possibility?

2. Does the disordering phenomenon of one subunit in the ATP-bound structures have any significance - could it be related to half-of-sites activity? Does this RNR exhibit half-of-sites activity?

3. Does the disordering of the GRD with dATP bound have any long-term impact on the stability of the Gly radical? I realize that the authors tested the ability to form the Gly radical in the presence of dATP in Fig. 4 of the manuscript. But it looks like they only analyzed the samples after 20 min of incubation. Were longer time points analyzed?

4. Did the authors establish whether the effect of dATP inhibition on substrate binding is reversible? If dATP is removed, can substrates rebind?

5. In some figures (Fig. 6e, for example), the cryo-EM density map for the nucleotide component of the model is not continuous over the entire molecule. Can the authors comment on the significance of this phenomenon? Were the ligands validated in any way to ensure that the assignments were made correctly?

Reviewer #2 (Public Review):

This manuscript describes the functional and structural characterization of an anaerobic (Class III) ribonucleotide reductase (RNR) with an ATP cone domain from Prevotella copri (PcNrdD). Most significantly, the cryo-EM structural characterization revealed the presence of a flap domain that connects the ATP cone domain and the active site and provides structural insights about how nucleotides and deoxynucleotides bind to this enzyme. The authors also demonstrated the catalytic functions and the oligomeric states. However, many of the biochemical characterizations are incomplete, and it is difficult to make mechanistic conclusions from the reported structures. The reported nucleotide-binding constants may not be accurate because of the design of the assays, which complicates the interpretation of the effects of ATP and dATP on PcNrdD oligomeric states. Importantly, statistical information was missing in most of the biochemical data. Also, while the authors concluded that the dATP binding makes the GRD flexible based on the absence of cryo-EM density for GRD in the dATP-bound PcNrdD, no other supports were provided. There was also a concern about the relevance of the proposed GRD flexibility and the stability of Gly radical. Overall, the manuscript provides structural insights about Class III RNR with ATP cone domain and how it binds ATP and dATP allosteric effectors. However, ambiguity remains about the molecular mechanism by which the dATP binding to the ATP cone domain inhibits the Class III RNR activity.

Strengths:
1. The manuscript reports the first near-atomic resolution of the structures of Class III RNR with ATP domain in complex with ATP and dATP. These structures revealed the NxN flap domain proposed to form an interaction network between the substrate, the linker to the ATP cone domain, the GRD, and loop 2 important for substrate specificity. The structures also provided insights into how ATP and dATP bind to the ATP cone domain of Class III RNR. Also, the structures suggested that the ATP cone domain is directly involved in the tetramer formation by forming an interaction with the core domain in the presence of dATP. These observations serve as an important basis for future study on the mechanism of Allosteric regulation of Class III RNR.

2. The authors used a wide range of methodologies including activity assays, nucleotide binding assays, oligomeric state determination, and cryo-EM structural characterization, which were impressive and necessary to understand the complex allosteric regulation of RNR.

3. The activity assays demonstrated the catalytic function of PcNrdD and its ability to be activated by ATP and low-concentration dATP and inhibited by high-concentration dATP.

4. ITC and MST were used to show the ability of PcNrdD to bind NTP and dATP.

5. GEMMA was used successfully to determine the oligomeric state of PcNrdD, which suggested that PcNrdD exists in dimeric and tetrameric forms, whose ratio is affected by ATP and/or dATP.

Weaknesses:
1. Activity assays.
The activity assays were performed under conditions that may not represent the nucleotide reduction activity. The authors initiated the Gly radical formation and nucleotide reduction simultaneously. The authors also showed that the amount of Gly radical formation was different in the presence of ATP vs dATP. Therefore, it is possible that the observed Vmax is affected by the amount of Gly radical. In fact, some of the data fit poorly into the kinetic model. Also, the number of biological and technical replicates was not described, and no statistical information was provided for the curve fitting.

2. Binding assays.
The interpretation of the binding assays is complicated by the fact that dATP binds both a- and s-sites and ATP binds a- and active sites. dATP may also bind the active site as the product. It is unknown if ATP binds s-site in PcNrdD. Despite this complexity, the binding assays were performed under the condition that all the binding sites were available. Therefore, it is not clear which event these assays are reporting.

3. Oligomeric states.
Due to the ambiguity in the kinetic parameters and the binding constants determined above, the effects of ATP and dATP on the oligomeric states are difficult to interpret. The concentrations of ATP used in these experiments (50 and 100 uM) were significantly lower than KL determined by the activity assays (780 uM), while it is close to the Kd values determined by ITC or MST (~25 uM). Since it is unclear what binding events ITC and MST are reporting, the data in Figure 3 does not provide support for the claimed effects of ATP binding. For the effects of dATP, the authors did not observe a significant difference in oligomeric states between 50 or 100 uM dATP alone vs 50 uM dATP and 100 uM CTP. The former condition has dATP ~ 2x higher than the Kd and KL (Figure 1b) and therefore could be considered as "inhibited". On the other hand, NrdD should be fully active under the latter condition. Therefore, these observations show no correlation between the oligomeric state and the catalytic activity.

4. Effects of dATP binding on GRD structure
One of the key conclusions of this manuscript is that dATP binding induces the dissociation of GRD from the active site. However, the structures did not provide an explanation for how the dATP binding affects the conformation of GRD or whether the dissociation of GRD is a direct consequence of dATP binding or it is due to the absence of nucleotide substrate. Also, Gly radical is unlikely to be stable when it is not protected from the bulk solvent. Therefore, it is unlikely that the GRD dissociates from the active site unless the inhibition by dATP is irreversible. Further evidence is needed to support the proposed mechanism of inhibition by dATP.

5. Functional support for the observed structures.
Evidence for connecting structural observations and mechanistic conclusions is largely missing. For example, the authors proposed that the interactions between the ATP cone domain and the core domain are responsible for tetramer formation. However, no biochemical evidence was provided to support this proposal. Similarly, the functional significance of the interaction through the NxN flap domain was not proved by mutagenesis experiments.

Reviewer #3 (Public Review):

The manuscript by Bimai et al describes a structural and functional characterization of an anaerobic ribonucleotide reductase (RNR) enzyme from the human microbe, P. copri. More specifically, the authors aimed to characterize the mechanism by how (d)ATP modulates nucleotide reduction in this anaerobic RNR, using a combination of enzyme kinetics, binding thermodynamics, and cryo-EM structural determination. One of the principal findings of this paper is the ordering of a NxN 'flap' in the presence of ATP that promotes RNR catalysis and the disordering of both this flap and the glycyl radical domain (GRD) when the inhibitory effector, dATP, binds. The latter is correlated with a loss of substrate binding, which is the likely mechanism for dATP inhibition. It is important to note that the GRD is remote (>30 Ang) from the binding site of the dATP molecule, suggesting long-range communication of the structural (dis)ordering. The authors also present evidence for a shift in oligomerization in the presence of dATP. The work does provide evidence for new insights/views into the subtle differences of nucleotide modulation (allostery) of RNR through long-range interactions.

The strengths of the work are the impressive, in-depth structural analysis of the various regulated forms of PcRNR by (d)ATP using cryo-EM. The authors present seven different models in total, with striking differences in oligomerization and (dis)ordering of select structural features, including the GRD that is integral to catalysis. The authors present several, complementary biochemical experiments (ITC, MST, EPR, kinetics) aimed at resolving the binding and regulatory mechanism of the enzyme by various nucleotides. The authors present a good breadth of the literature in which the focus of allosteric regulation of RNRs has been on the aerobic orthologues.

Given the resolution of some of the structures in the remote regions that appear to be of importance, the rigor of the work could have been improved by complementing this experimental studies with molecular dynamics (MD) simulations to reveal the dynamics of the GRD and loops/flaps at the active site. The biochemical data supporting the loss of substrate binding with dATP association is compelling, but the binding studies of the (d)ATP regulatory molecules are not; the authors noted less-than-unity binding stoichiometries for the effectors. Also, the work would benefit from additional support for oligomerization changes using an additional biochemical/biophysical approach.

Overall, the authors have mostly achieved their overall aims of the manuscript. With focused modifications, including additional control experiments, the manuscript should be a welcomed addition to the RNR field.

Author Response

Reviewer #1 (Public Review):

The goal of this study is to understand the allosteric mechanism of overall activity regulation in an anaerobic ribonucleotide reductase (RNR) that contains an ATP-cone domain. Through cryo-EM structural analysis of various nucleotide-bound states of the RNR, the mechanism of dATP inhibition is found to involve order-disorder transitions in the active site. These effects appear to prevent substrate binding and a radical transfer needed to initiate the reaction.

Strengths of the manuscript include the comprehensive nature of the work - including numerous structures of different forms of the RNR and detailed characterization of enzyme activity to establish the parameters of dATP inhibition. The manuscript could be improved, however, by performing additional experiments to establish that the mechanism of inhibition can be observed in other contexts and it is not an artifact of the structural approach. Additionally, some of the presentations of biochemical data could be improved to comply with standard best practices.

The work is impactful because it reports initial observations about a potentially new mode of allosteric inhibition in this enzyme class. It also sets the stage for future work to understand the molecular basis for this phenomenon in more detail.

We thank the editor and reviewers for their positive evaluation of the potential impact of our work. We completely agree that hypotheses based on structural data require orthogonal experimental verification. However, the number and consistency of the cryo-EM structures speak in favour of the data being representative of conditions in solution. We feel that in particular cryo-EM data should be relatively free of artefacts, e.g. biased or incorrect relative domain orientations or artificially reduced mobility, compared to crystallography, where crystal packing effects can affect these parameters. As we write in response to Reviewer #2, it has been difficult to propose a direct structural mechanism for transmission of the allosteric signal from the a-site in the ATP-cone to the active site and GRD given that the ATP-cones and linker are disordered in the dATP-bound dimers and only partly ordered in the dATP-bound tetramers. Further verification experiments will be performed in future but are outside the scope of the present article.

We will improve the presentation of the biochemical data in a revised version.

General comments:

1. It would be ideal to perform an additional experiment of some type to confirm the order-disorder phenomena observed in the cryo-EM structures to rule out the possibility that it is an artifact of the structure determination approach. Circular dichroism might be a possibility?

Circular dichroism reports only on the approximate relative proportions of helix, sheet and loop structure in a protein; thus we believe that it would not be a sensitive enough tool to distinguish between ordered and disordered states of the GRD. We are considering what alternative methods might be appropriate.

1. Does the disordering phenomenon of one subunit in the ATP-bound structures have any significance - could it be related to half-of-sites activity? Does this RNR exhibit half-of-sites activity?

Half-of-sites activity has not been biochemically proven in any ribonucleotide reductase although it was first suggested in 1987 (PMID: 3298261). However, a strong structural indication was recently published in the form of the holo-complex of the class Ia ribonucleotide reductase from Escherichia coli, which is highly asymmetrical and in which productive contacts forming an intact proton-coupled electron transfer pathway are only formed between one of two pairs of monomers (PMID: 32217749). We have not been able to prove half-of-sites activity for PcNrdD due to low overall radical content, but the structural results are indeed consistent with such an activity.

1. Does the disordering of the GRD with dATP bound have any long-term impact on the stability of the Gly radical? I realize that the authors tested the ability to form the Gly radical in the presence of dATP in Fig. 4 of the manuscript. But it looks like they only analyzed the samples after 20 min of incubation. Were longer time points analyzed?

Radical content was measured after 5 min and 20 min incubation; 5 min incubations (not included in the manuscript) consistently gave higher radical content compared to 20 min incubation. Longer time points were not analysed, as we assumed that the radical content would be even lower after 20 min.

1. Did the authors establish whether the effect of dATP inhibition on substrate binding is reversible? If dATP is removed, can substrates rebind?

This is an interesting question. We measured KDs for dATP in the micromolar range and are hence confident that dATP binding is reversible. Our measurements do not, however, directly prove that inhibition of the enzyme is reversible. Nevertheless, it is worth noting that the protein as purified contained significant amounts of dATP and purification conditions had to be optimised to remove dATP. This is evidence that PcNrdD that has “seen” dATP can subsequently bind substrate in the presence of ATP. We will describe the purification more clearly in a revision.

1. In some figures (Fig. 6e, for example), the cryo-EM density map for the nucleotide component of the model is not continuous over the entire molecule. Can the authors comment on the significance of this phenomenon? Were the ligands validated in any way to ensure that the assignments were made correctly?

Indeed, we sometimes saw discontinuous density for the nucleotides, both in the active site and in the specificity site. However, the break was almost always near the C5’ carbon atom, which is common to all nucleotides. While we cannot readily explain this phenomenon, the nucleotides refined well with full occupancy, giving B-factors similar to those of the surrounding protein atoms. The identity of the nucleotide could always be inferred from a) the size of the base (purine or pyrimidine); b) the known nucleotide combinations added to the protein before grid preparation; c) prior knowledge on the combinations of effector and substrate that have been found valid for all RNRs since the first studies of allosteric specificity regulation.

Reviewer #2 (Public Review):

This manuscript describes the functional and structural characterization of an anaerobic (Class III) ribonucleotide reductase (RNR) with an ATP cone domain from Prevotella copri (PcNrdD). Most significantly, the cryo-EM structural characterization revealed the presence of a flap domain that connects the ATP cone domain and the active site and provides structural insights about how nucleotides and deoxynucleotides bind to this enzyme. The authors also demonstrated the catalytic functions and the oligomeric states. However, many of the biochemical characterizations are incomplete, and it is difficult to make mechanistic conclusions from the reported structures. The reported nucleotide-binding constants may not be accurate because of the design of the assays, which complicates the interpretation of the effects of ATP and dATP on PcNrdD oligomeric states. Importantly, statistical information was missing in most of the biochemical data. Also, while the authors concluded that the dATP binding makes the GRD flexible based on the absence of cryo-EM density for GRD in the dATP-bound PcNrdD, no other supports were provided. There was also a concern about the relevance of the proposed GRD flexibility and the stability of Gly radical. Overall, the manuscript provides structural insights about Class III RNR with ATP cone domain and how it binds ATP and dATP allosteric effectors. However, ambiguity remains about the molecular mechanism by which the dATP binding to the ATP cone domain inhibits the Class III RNR activity.

Strengths:

1. The manuscript reports the first near-atomic resolution of the structures of Class III RNR with ATP domain in complex with ATP and dATP. These structures revealed the NxN flap domain proposed to form an interaction network between the substrate, the linker to the ATP cone domain, the GRD, and loop 2 important for substrate specificity. The structures also provided insights into how ATP and dATP bind to the ATP cone domain of Class III RNR. Also, the structures suggested that the ATP cone domain is directly involved in the tetramer formation by forming an interaction with the core domain in the presence of dATP. These observations serve as an important basis for future study on the mechanism of Allosteric regulation of Class III RNR.

1. The authors used a wide range of methodologies including activity assays, nucleotide binding assays, oligomeric state determination, and cryo-EM structural characterization, which were impressive and necessary to understand the complex allosteric regulation of RNR.

1. The activity assays demonstrated the catalytic function of PcNrdD and its ability to be activated by ATP and low-concentration dATP and inhibited by high-concentration dATP.

1. ITC and MST were used to show the ability of PcNrdD to bind NTP and dATP.

1. GEMMA was used successfully to determine the oligomeric state of PcNrdD, which suggested that PcNrdD exists in dimeric and tetrameric forms, whose ratio is affected by ATP and/or dATP.

Weaknesses:

1. Activity assays.

The activity assays were performed under conditions that may not represent the nucleotide reduction activity. The authors initiated the Gly radical formation and nucleotide reduction simultaneously. The authors also showed that the amount of Gly radical formation was different in the presence of ATP vs dATP. Therefore, it is possible that the observed Vmax is affected by the amount of Gly radical. In fact, some of the data fit poorly into the kinetic model. Also, the number of biological and technical replicates was not described, and no statistical information was provided for the curve fitting.

The highest turnover activity of PcNrdD measured in presence of ATP was 1.3 s-1 (470 nmol/min/mg), a kcat comparable to recently reported values for anaerobic and aerobic RNRs from Neisseria bacilliformis, Leeuwenhoekiella blandensis, Facklamia ignava, Thermus virus P74-23, and Aquifex aeolicus (PMID: 25157154, PMID: 29388911, PMID: 30166338, PMID: 34314684, PMID: 34941255). The general trend illustrated in Figure 1 is that ATP has an activating effect, whereas high concentrations of dATP have an inactivating effect, which cannot be explained by suboptimal assay conditions since our EPR results consistently show that more radical is formed in incubations with dATP compared to incubations with ATP. Curve fitting methods used are listed in Materials and Methods (as specified in the Figure 1 legend), and standard errors for all specified curve fitting results (from triplicate experiments) are shown in Figure 1.

1. Binding assays.

The interpretation of the binding assays is complicated by the fact that dATP binds both a- and s-sites and ATP binds a- and active sites. dATP may also bind the active site as the product. It is unknown if ATP binds s-site in PcNrdD. Despite this complexity, the binding assays were performed under the condition that all the binding sites were available. Therefore, it is not clear which event these assays are reporting.

Both ITC and MST experiments involving ATP and dATP binding to the a-site were performed in the presence of at least 1 mM GTP substrate (5 mM in MST) to fill the active site, and 1 mM dTTP effector to fill the s-site (specified in the legend to Figure 2). These conditions enable binding of ATP or dATP only to the a-site in the ATP-cone.

1. Oligomeric states.

Due to the ambiguity in the kinetic parameters and the binding constants determined above, the effects of ATP and dATP on the oligomeric states are difficult to interpret. The concentrations of ATP used in these experiments (50 and 100 uM) were significantly lower than KL determined by the activity assays (780 uM), while it is close to the Kd values determined by ITC or MST (~25 uM). Since it is unclear what binding events ITC and MST are reporting, the data in Figure 3 does not provide support for the claimed effects of ATP binding. For the effects of dATP, the authors did not observe a significant difference in oligomeric states between 50 or 100 uM dATP alone vs 50 uM dATP and 100 uM CTP. The former condition has dATP ~ 2x higher than the Kd and KL (Figure 1b) and therefore could be considered as "inhibited". On the other hand, NrdD should be fully active under the latter condition. Therefore, these observations show no correlation between the oligomeric state and the catalytic activity.

The results in Figure 3 show that at in presence of 100 µM ATP plus 100 µM CTP the oligomeric equilibrium is 64% dimers plus 36% tetramers, and in presence of 50-100 µM dATP the oligomeric equilibrium is 32% dimers and 68% tetramers. We agree that there is no clear and strong correlation between oligomeric state and inhibition. We will also try to make it clearer in a revised version. Meanwhile, to add some further clarity, SEC experiments at higher nucleotide concentrations will be included in the revision.

1. Effects of dATP binding on GRD structure

One of the key conclusions of this manuscript is that dATP binding induces the dissociation of GRD from the active site. However, the structures did not provide an explanation for how the dATP binding affects the conformation of GRD or whether the dissociation of GRD is a direct consequence of dATP binding or it is due to the absence of nucleotide substrate. Also, Gly radical is unlikely to be stable when it is not protected from the bulk solvent. Therefore, it is unlikely that the GRD dissociates from the active site unless the inhibition by dATP is irreversible. Further evidence is needed to support the proposed mechanism of inhibition by dATP.

We admit that it has been difficult to propose a direct structural mechanism for transmission of the allosteric signal from the a-site in the ATP-cone to the active site and GRD given that the ATP-cones and linker are disordered in the dATP-bound dimers and that the linker can only be partly modelled in the dATP-bound tetramers. Most likely dATP binding causes a change in the dynamics of the linker region and NxN flap that directly affects substrate binding and simultaneously causes disorder of the GRD, given that all are part of a connected system (described as “nexus” in the manuscript). The structures determined in the presence of dATP and CTP show that CTP cannot bind in the absence of an ordered NxN flap.

In any case a major conclusion of the work is that dATP does not inhibit the anaerobic RNR by prevention of glycyl radical formation but by prevention of its subsequent transfer. We agree that further evidence is required to support the proposed mechanism but, given the extent of the data already presented in the manuscript, we feel that such studies should be the subject of a future publication.

1. Functional support for the observed structures.

Evidence for connecting structural observations and mechanistic conclusions is largely missing. For example, the authors proposed that the interactions between the ATP cone domain and the core domain are responsible for tetramer formation. However, no biochemical evidence was provided to support this proposal. Similarly, the functional significance of the interaction through the NxN flap domain was not proved by mutagenesis experiments.

We did actually make mutants to verify the observed interactions in the tetramer, but several of them did not behave well in our hands, e.g. with regard to protein stability. Since we have no evidence that oligomerisation is coupled to inhibition, and since we did not observe any conservation between protein sequences in the interaction area, we chose not to pursue this point further. The main merit of the tetramer structures is that they allowed a high-resolution view of dATP binding to the ATP-cone and a comparison to previously observed ATP-cones. Nevertheless, mutation experiments, also including the NxN flap, could be the subject of future work.

Reviewer #3 (Public Review):

The manuscript by Bimai et al describes a structural and functional characterization of an anaerobic ribonucleotide reductase (RNR) enzyme from the human microbe, P. copri. More specifically, the authors aimed to characterize the mechanism by how (d)ATP modulates nucleotide reduction in this anaerobic RNR, using a combination of enzyme kinetics, binding thermodynamics, and cryo-EM structural determination. One of the principal findings of this paper is the ordering of a NxN 'flap' in the presence of ATP that promotes RNR catalysis and the disordering of both this flap and the glycyl radical domain (GRD) when the inhibitory effector, dATP, binds. The latter is correlated with a loss of substrate binding, which is the likely mechanism for dATP inhibition. It is important to note that the GRD is remote (>30 Ang) from the binding site of the dATP molecule, suggesting long-range communication of the structural (dis)ordering. The authors also present evidence for a shift in oligomerization in the presence of dATP. The work does provide evidence for new insights/views into the subtle differences of nucleotide modulation (allostery) of RNR through long-range interactions.

The strengths of the work are the impressive, in-depth structural analysis of the various regulated forms of PcRNR by (d)ATP using cryo-EM. The authors present seven different models in total, with striking differences in oligomerization and (dis)ordering of select structural features, including the GRD that is integral to catalysis. The authors present several, complementary biochemical experiments (ITC, MST, EPR, kinetics) aimed at resolving the binding and regulatory mechanism of the enzyme by various nucleotides. The authors present a good breadth of the literature in which the focus of allosteric regulation of RNRs has been on the aerobic orthologues.

Given the resolution of some of the structures in the remote regions that appear to be of importance, the rigor of the work could have been improved by complementing this experimental studies with molecular dynamics (MD) simulations to reveal the dynamics of the GRD and loops/flaps at the active site.

We will discuss this option with expert colleagues.

The biochemical data supporting the loss of substrate binding with dATP association is compelling, but the binding studies of the (d)ATP regulatory molecules are not; the authors noted less-than-unity binding stoichiometries for the effectors.

Most of the methods used measure only binding strength, not the number of binding sites (N), whereas ITC also measures number of sites. N is dependent on the integrity of the protein, i.e. the number of protein molecules in a preparation that are involved in binding, and quite often gives lower values than the theoretical number of binding sites.

Also, the work would benefit from additional support for oligomerization changes using an additional biochemical/biophysical approach.

SEC (chromatography), GEMMA (mass spectrometry) and cryo-EM were used to study oligomerization. Since each method has restrictions on nucleotide concentrations as well as protein concentrations that can be used, the results are not directly comparable, but all three methods indicate nucleotide dependent oligomerization changes. The SEC results will be included in a revised version.

Overall, the authors have mostly achieved their overall aims of the manuscript. With focused modifications, including additional control experiments, the manuscript should be a welcomed addition to the RNR field.