Jasmonates(JAs)are essential phytohormones that coordinate plant defense and development in response to unpredictable environments.Recent advances have highlighted the SCF COI1-JAZ-MYC2-MED25 module as a central hub f...Jasmonates(JAs)are essential phytohormones that coordinate plant defense and development in response to unpredictable environments.Recent advances have highlighted the SCF COI1-JAZ-MYC2-MED25 module as a central hub for JA signaling,orchestrating transcriptional repression,derepression,activation,amplification,and feedback termination.This review summarizes current insights into the roles of JA in the regulation of biotic and abiotic stress responses and agronomic traits,including root development,regeneration,fertility,flowering,leaf senescence,and seed development,with a particular emphasis on the crosstalk between JA and a wound-induced peptide hormone,systemin,which mediates systemic wound responses.A deeper understanding of the JA regulatory mechanisms will provide valuable strategies for engineering crops with enhanced stress resilience and improved yields.We further propose JA-based strategies as a promising avenue for crop improvement.展开更多
The H3 bivalent modifications of trimethylationat Lys9 and acetylation at Lys18(H3-K9 Me3-K18 Ac) were identified to collectively recruit TRIM33 in the nodal signaling pathway.To understand the underlying mechanism of...The H3 bivalent modifications of trimethylationat Lys9 and acetylation at Lys18(H3-K9 Me3-K18 Ac) were identified to collectively recruit TRIM33 in the nodal signaling pathway.To understand the underlying mechanism of TRIM33 recruitment,the nucleosome core particles(NCPs) containing full-length H3-K9 Me3-K18 Ac were indispensable samples.Herein we developed a pseudo dipeptide strategy to efficiently prepare peptide segments,facilitating the chemical synthesis of H3-K9 Me3-K18 Ac at a tens of milligram scale.The synthetic H3-K9 Me3-K18 Ac was then examined by CD spectroscopy,which demonstrated a prominent shift compared to recombinant H3.Finally,bivalently modified NCPs were assembled and verified by gel mobility shift assay with good homogeneity.展开更多
Smart, real-time, low-cost, and distributed ecosystem monitoring is essential for understanding and managing rapidly changing ecosystems. However, new techniques in the big data era have rarely been introduced into op...Smart, real-time, low-cost, and distributed ecosystem monitoring is essential for understanding and managing rapidly changing ecosystems. However, new techniques in the big data era have rarely been introduced into operational ecosystem monitoring, particularly for fragile ecosystems in remote areas.We introduce the Internet of Things(IoT) techniques to establish a prototype ecosystem monitoring system by developing innovative smart devices and using IoT technologies for ecosystem monitoring in isolated environments. The developed smart devices include four categories: large-scale and nonintrusive instruments to measure evapotranspiration and soil moisture, in situ observing systems for CO2 and d13 C associated with soil respiration, portable and distributed devices for monitoring vegetation variables, and Bi-CMOS cameras and pressure trigger sensors for terrestrial vertebrate monitoring. These new devices outperform conventional devices and are connected to each other via wireless communication networks. The breakthroughs in the ecosystem monitoring IoT include new data loggers and longdistance wireless sensor network technology that supports the rapid transmission of data from devices to wireless networks. The applicability of this ecosystem monitoring IoT is verified in three fragile ecosystems, including a karst rocky desertification area, the National Park for Amur Tigers, and the oasis-desert ecotone in China. By integrating these devices and technologies with an ecosystem monitoring information system, a seamless data acquisition, transmission, processing, and application IoT is created. The establishment of this ecosystem monitoring IoT will serve as a new paradigm for ecosystem monitoring and therefore provide a platform for ecosystem management and decision making in the era of big data.展开更多
The plant hormone jasmonate(JA)regulates plant immunity and adaptive growth by orchestrating a genome-wide transcriptional program.Key regulators of JA-responsive gene expression include the master transcription facto...The plant hormone jasmonate(JA)regulates plant immunity and adaptive growth by orchestrating a genome-wide transcriptional program.Key regulators of JA-responsive gene expression include the master transcription factor MYc2,which is repressed by the conserved Groucho/Tup1-like corepressor TOPLESS(TPL)in the resting state.However,the mechanisms underlying TPL-mediated transcriptional repression of MYc2 activity and hormone-dependent switching between repression and de-repression remainenigmatic.Here,we report the regulation of TPLactivity and JAsignaling byreversibleacetylation of TPL.We found that the histone acetyltransferase GCN5 could mediate TPL acetylation,which enhances its interaction with the NOVEL-INTERACTOR-OF-JAZ(NINJA)adaptor and promotes its recruitment to MYc2 target promoters,facilitating transcriptional repression.Conversely,TPL deacetylation by the histonedeacetylase HDA6 weakens TPL-NINJA interaction and inhibitsTPL recruitmentto MYC2 target promoters,facilitating transcriptional activation.In the resting state,the opposing activities of GCN5 and HDA6 maintain TPL acetylation homeostasis,promoting transcriptional repression activity of TPL.In response to JA elicitation,HDA6 expression is transiently induced,resulted in decreased TPL acetylation and repressor activity,thereby transcriptional activation of MYC2 target genes.Thus,the GCN5-TPL-HDA6 module main tains the homeostasis of acetylated TPL,thereby determining the transcriptional state of JA-responsive genes.Our findings uncovered a mechanism by which the TPL corepressor activity in JA signaling is activelytuned inarapidandreversiblemanner.展开更多
MED25 has been implicated as a negative regulator of the abscisic acid(ABA)signaling pathway.However,it is unclear whether other Mediator subunits could associate with MED25 to participate in the ABA response.Here,we ...MED25 has been implicated as a negative regulator of the abscisic acid(ABA)signaling pathway.However,it is unclear whether other Mediator subunits could associate with MED25 to participate in the ABA response.Here,we used affinity purification followed by mass spectrometry to uncover Mediator subunits that associate with MED25 in transgenic plants.We found that at least26 Mediator subunits,belonging to the head,middle,tail,and CDK8 kinase modules,were copurified with MED25 in vivo.Interestingly,the tail module subunit MED16 was identified to associate with MED25 under both mock and ABA treatments.We further showed that the disruption of MED16 led to reduced ABA sensitivity compared to the wild type.Transcriptomic analysis revealedthattheexpressionofseveral ABA-responsive genes was significantly lower in med16 than those in wild type.Furthermore,we discovered that MED16 may possibly compete with MED25 to interact with the key transcription factor ABA INSENSITIVE 5(ABI5)to positively regulate ABA signaling.Consistently,med16 and med25 mutants displayed opposite phenotypes in ABA response,cuticle permeability,and differential ABI5-mediated EM1 and EM6 expression.Together,our data indicate that MED16 and MED25 differentially regulate ABA signaling byantagonisticallyaffectingABI5-mediated transcription in Arabidopsis.展开更多
Dear Editor,Fruit rot caused by necrotrophic pathogens results in substantial reductions in fruit yield and revenues worldwide(Petrasch et al.,2019).A widespread phenomenon in fleshy fruit species is the susceptibilit...Dear Editor,Fruit rot caused by necrotrophic pathogens results in substantial reductions in fruit yield and revenues worldwide(Petrasch et al.,2019).A widespread phenomenon in fleshy fruit species is the susceptibility of ripe fruits to necrotrophs(Silva et al.,2021),which facilitates seed dispersal(Forlani et al.,2019)but causes severe post-harvest losses in production.As most of the nutritional and sensory qualities of fruits are elaborated at the ripening stage(Liu et al.,2015),balancing fruit ripening and pathogen resistance to maintain fruit quality has proven to be challenging.A deeper understanding of the mechanisms underlying the increased susceptibility of fruits to necrotrophs during ripening could lead to new strategies for producing necrotrophy-resistant fruits without compromising ripening-relatedquality.展开更多
SKIP is a conserved protein from yeasts to plants and humans. In plant cells, SKIP is a bifunctional regulator that works in the nucleus as a splicing factor by integrating into the spliceosome and as a transcriptiona...SKIP is a conserved protein from yeasts to plants and humans. In plant cells, SKIP is a bifunctional regulator that works in the nucleus as a splicing factor by integrating into the spliceosome and as a transcriptional activator by interacting with the Pall complex. In this study, we identified two nuclear localization signals in SKIP and confirmed that each is sufficient to target SKIP to the nucleus. The SNW domain of SKIP is required for both its function as a splicing factor by promoting integration into the spliceosome in response to stress, and its function as a transcriptional activator by controlling its interaction with the Pall complex to participate in flowering. Truncated proteins that included the SNW domain and the N- or C-terminus of SKIP were still able to carry out the functions of the full-length protein in gene splicing and transcriptional activation in Arabidopsis. In addition, we found that SKIP undergoes 26S proteasome-mediated degrada- tion, and that the C-terminus of SKIP is required to maintain the stability of the protein in plant cells. Together, our findings demonstrate the structural domain organization of SKIP and reveal the core domains and motifs underlying SKIP function in plants.展开更多
Grain number is a flexible trait and contributes significantly to grain yield.In rice,the zinc finger transcription factor DROUGHT AND SALT TOLERANCE(DST)controls grain number by directly regulating cytokinin oxidase!...Grain number is a flexible trait and contributes significantly to grain yield.In rice,the zinc finger transcription factor DROUGHT AND SALT TOLERANCE(DST)controls grain number by directly regulating cytokinin oxidase!dehydrogenase 2(OsCKX2)expression.Although specific upstream regulators of the DST-OsCKX2 module have been identified,the mechanism employed by DST to regulate the expression of OsCKX2 remains unclear.Here,we demonstrate that DST-interacting protein 1(DIP1),known as Mediator subunit OsMED25,acts as an interacting coactivator of DST.Phenotypic analyses revealed that OsMED25-RNAi and the osmed25 mutant plants exhibited enlarged panicles,with enhanced branching and spikelet number,similar to the dst mutant.Genetic analysis indicated that OsMED25 acts in the same pathway as the DST-OsCKX2 module to regulate spikelet number per panicle.Further biochemical analysis showed that OsMED25 physically interacts with DST at the promoter region of OsCKX2,and then recruits RNA polymerase II(Pol II)to activate OsCKX2 transcription.Thus,OsMED25 was involved in the communication between DST and Pol II general transcriptional machinery to regulate spikelet number.In general,our findings reveal a novel function of OsMED25 in DST-OsCKX2 modulated transcriptional regulation,thus enhancing our un derstanding of the regulatory mechanism underlying DST-OsCKX2-mediated spikelet number.展开更多
Mycosin-1 protease(MycP1)is a serine protease anchored to the inner membrane of Mycobacterium tuberculosis,and is essential in virulence factor secretion through the ESX-1 type VII secretion system(T7SS).Bacterial phy...Mycosin-1 protease(MycP1)is a serine protease anchored to the inner membrane of Mycobacterium tuberculosis,and is essential in virulence factor secretion through the ESX-1 type VII secretion system(T7SS).Bacterial physiology studies demonstrated that MycP1 plays a dual role in the regulation of ESX-1 secretion and virulence,primarily through cleavage of its secretion substrate EspB.MycP1 contains a putative N-terminal inhibitory propeptide and a catalytic triad of Asp-His-Ser,classic hallmarks of a sub-tilase family serine protease.The MycP1 propeptide was previously reported to be initially inactive and activated after prolonged incubation.In this study,we have deter-mined crystal structures of MycP1 with(MycP124-422)and without(MycP1^(63-422))the propeptide,and conducted EspB cleavage assays using the two proteins.Very high struc-tural similarity was observed in the two crystal structures.Interestingly,protease assays demonstrated positive EspB cleavage for both proteins,indicating that the putative propeptide does not inhibit protease activity.Molecu-lar dynamic simulations showed higher rigidity in regions guarding the entrance to the catalytic site in MycP124-422 than in MycP1^(63-422),suggesting that the putative propeptide might contribute to the conformational stability of the active site cleft and surrounding regions.展开更多
Dear Editor,The functional diversity of proteins is related to the cooperation of multiple domains.Independent globular domains are typically joined by a fl exible length of polypeptide chain,which makes the structura...Dear Editor,The functional diversity of proteins is related to the cooperation of multiple domains.Independent globular domains are typically joined by a fl exible length of polypeptide chain,which makes the structural analysis of multi-domain proteins diffi cult.Here,we describe the combined use of solution NMR(nuclear magnetic resonance)and EPR(elec-tron paramagnetic resonance)for the structural analysis of a protein with two separate domains.The structure of each domain was determined independently using conventional NMR restraints,and the relative orientation of the two separate domains was confi ned using long-distance restraints obtained by NMR-PRE(paramagnetic relaxation enhancement)and EPR-DEER(double electron-electron resonance,also called PELDOR:pulsed electron double reso-nance.展开更多
基金supported by the National Natural Science Foundation of China(32370332 and 32202481)the Natural Science Foundation of Hainan Province(325RC839).
文摘Jasmonates(JAs)are essential phytohormones that coordinate plant defense and development in response to unpredictable environments.Recent advances have highlighted the SCF COI1-JAZ-MYC2-MED25 module as a central hub for JA signaling,orchestrating transcriptional repression,derepression,activation,amplification,and feedback termination.This review summarizes current insights into the roles of JA in the regulation of biotic and abiotic stress responses and agronomic traits,including root development,regeneration,fertility,flowering,leaf senescence,and seed development,with a particular emphasis on the crosstalk between JA and a wound-induced peptide hormone,systemin,which mediates systemic wound responses.A deeper understanding of the JA regulatory mechanisms will provide valuable strategies for engineering crops with enhanced stress resilience and improved yields.We further propose JA-based strategies as a promising avenue for crop improvement.
基金supported by the National Natural Science Foundation of China(Nos.21708036,31470740,U1732161)Anhui Provincial Natural Science Foundation (No.1808085QC63)。
文摘The H3 bivalent modifications of trimethylationat Lys9 and acetylation at Lys18(H3-K9 Me3-K18 Ac) were identified to collectively recruit TRIM33 in the nodal signaling pathway.To understand the underlying mechanism of TRIM33 recruitment,the nucleosome core particles(NCPs) containing full-length H3-K9 Me3-K18 Ac were indispensable samples.Herein we developed a pseudo dipeptide strategy to efficiently prepare peptide segments,facilitating the chemical synthesis of H3-K9 Me3-K18 Ac at a tens of milligram scale.The synthetic H3-K9 Me3-K18 Ac was then examined by CD spectroscopy,which demonstrated a prominent shift compared to recombinant H3.Finally,bivalently modified NCPs were assembled and verified by gel mobility shift assay with good homogeneity.
基金supported by the National Key Research & Development Program of China (2016YFC0500106)the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA20100104)the 13th Five-year Informatization Plan of the Chinese Academy of Sciences (XXH13505-06)
文摘Smart, real-time, low-cost, and distributed ecosystem monitoring is essential for understanding and managing rapidly changing ecosystems. However, new techniques in the big data era have rarely been introduced into operational ecosystem monitoring, particularly for fragile ecosystems in remote areas.We introduce the Internet of Things(IoT) techniques to establish a prototype ecosystem monitoring system by developing innovative smart devices and using IoT technologies for ecosystem monitoring in isolated environments. The developed smart devices include four categories: large-scale and nonintrusive instruments to measure evapotranspiration and soil moisture, in situ observing systems for CO2 and d13 C associated with soil respiration, portable and distributed devices for monitoring vegetation variables, and Bi-CMOS cameras and pressure trigger sensors for terrestrial vertebrate monitoring. These new devices outperform conventional devices and are connected to each other via wireless communication networks. The breakthroughs in the ecosystem monitoring IoT include new data loggers and longdistance wireless sensor network technology that supports the rapid transmission of data from devices to wireless networks. The applicability of this ecosystem monitoring IoT is verified in three fragile ecosystems, including a karst rocky desertification area, the National Park for Amur Tigers, and the oasis-desert ecotone in China. By integrating these devices and technologies with an ecosystem monitoring information system, a seamless data acquisition, transmission, processing, and application IoT is created. The establishment of this ecosystem monitoring IoT will serve as a new paradigm for ecosystem monitoring and therefore provide a platform for ecosystem management and decision making in the era of big data.
基金This work was supported by the founding from National Natural Science Foundationof China(32161133018,31730010,31991183,31900243)the Strategic Priority Research Program of theCAS(XDPB16)+1 种基金C.A.was supported by the National Postdoctoral Program for Innovative Talents(BX20180355)the postdoctoral fellowship from China PostdoctoralScienceFoundation.
文摘The plant hormone jasmonate(JA)regulates plant immunity and adaptive growth by orchestrating a genome-wide transcriptional program.Key regulators of JA-responsive gene expression include the master transcription factor MYc2,which is repressed by the conserved Groucho/Tup1-like corepressor TOPLESS(TPL)in the resting state.However,the mechanisms underlying TPL-mediated transcriptional repression of MYc2 activity and hormone-dependent switching between repression and de-repression remainenigmatic.Here,we report the regulation of TPLactivity and JAsignaling byreversibleacetylation of TPL.We found that the histone acetyltransferase GCN5 could mediate TPL acetylation,which enhances its interaction with the NOVEL-INTERACTOR-OF-JAZ(NINJA)adaptor and promotes its recruitment to MYc2 target promoters,facilitating transcriptional repression.Conversely,TPL deacetylation by the histonedeacetylase HDA6 weakens TPL-NINJA interaction and inhibitsTPL recruitmentto MYC2 target promoters,facilitating transcriptional activation.In the resting state,the opposing activities of GCN5 and HDA6 maintain TPL acetylation homeostasis,promoting transcriptional repression activity of TPL.In response to JA elicitation,HDA6 expression is transiently induced,resulted in decreased TPL acetylation and repressor activity,thereby transcriptional activation of MYC2 target genes.Thus,the GCN5-TPL-HDA6 module main tains the homeostasis of acetylated TPL,thereby determining the transcriptional state of JA-responsive genes.Our findings uncovered a mechanism by which the TPL corepressor activity in JA signaling is activelytuned inarapidandreversiblemanner.
基金supported by the National Natural Science Foundation of China(NSFC 31900238 and NSFC 32070307)to Y.Z.
文摘MED25 has been implicated as a negative regulator of the abscisic acid(ABA)signaling pathway.However,it is unclear whether other Mediator subunits could associate with MED25 to participate in the ABA response.Here,we used affinity purification followed by mass spectrometry to uncover Mediator subunits that associate with MED25 in transgenic plants.We found that at least26 Mediator subunits,belonging to the head,middle,tail,and CDK8 kinase modules,were copurified with MED25 in vivo.Interestingly,the tail module subunit MED16 was identified to associate with MED25 under both mock and ABA treatments.We further showed that the disruption of MED16 led to reduced ABA sensitivity compared to the wild type.Transcriptomic analysis revealedthattheexpressionofseveral ABA-responsive genes was significantly lower in med16 than those in wild type.Furthermore,we discovered that MED16 may possibly compete with MED25 to interact with the key transcription factor ABA INSENSITIVE 5(ABI5)to positively regulate ABA signaling.Consistently,med16 and med25 mutants displayed opposite phenotypes in ABA response,cuticle permeability,and differential ABI5-mediated EM1 and EM6 expression.Together,our data indicate that MED16 and MED25 differentially regulate ABA signaling byantagonisticallyaffectingABI5-mediated transcription in Arabidopsis.
基金supported by the National Natural Science Foundation of China(31991183,32161133018,and U22A20459)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDPB16)the Beijing Joint Research Program for Germplasm Innovation and New Variety Breeding(G20220628003).
文摘Dear Editor,Fruit rot caused by necrotrophic pathogens results in substantial reductions in fruit yield and revenues worldwide(Petrasch et al.,2019).A widespread phenomenon in fleshy fruit species is the susceptibility of ripe fruits to necrotrophs(Silva et al.,2021),which facilitates seed dispersal(Forlani et al.,2019)but causes severe post-harvest losses in production.As most of the nutritional and sensory qualities of fruits are elaborated at the ripening stage(Liu et al.,2015),balancing fruit ripening and pathogen resistance to maintain fruit quality has proven to be challenging.A deeper understanding of the mechanisms underlying the increased susceptibility of fruits to necrotrophs during ripening could lead to new strategies for producing necrotrophy-resistant fruits without compromising ripening-relatedquality.
文摘SKIP is a conserved protein from yeasts to plants and humans. In plant cells, SKIP is a bifunctional regulator that works in the nucleus as a splicing factor by integrating into the spliceosome and as a transcriptional activator by interacting with the Pall complex. In this study, we identified two nuclear localization signals in SKIP and confirmed that each is sufficient to target SKIP to the nucleus. The SNW domain of SKIP is required for both its function as a splicing factor by promoting integration into the spliceosome in response to stress, and its function as a transcriptional activator by controlling its interaction with the Pall complex to participate in flowering. Truncated proteins that included the SNW domain and the N- or C-terminus of SKIP were still able to carry out the functions of the full-length protein in gene splicing and transcriptional activation in Arabidopsis. In addition, we found that SKIP undergoes 26S proteasome-mediated degrada- tion, and that the C-terminus of SKIP is required to maintain the stability of the protein in plant cells. Together, our findings demonstrate the structural domain organization of SKIP and reveal the core domains and motifs underlying SKIP function in plants.
基金supported by the National Key Research and Development Program of China (Grant No. 2019YFD1000300)Agricultural Seed Project of Shandong Province (Grant Nos 2020LZGC005, 2021LZGC0017)+1 种基金the Tai-Shan Scholar Program from Shandong Province (Grant No. tsxk20150901)the K. C. Wong Education Foundation.
文摘Grain number is a flexible trait and contributes significantly to grain yield.In rice,the zinc finger transcription factor DROUGHT AND SALT TOLERANCE(DST)controls grain number by directly regulating cytokinin oxidase!dehydrogenase 2(OsCKX2)expression.Although specific upstream regulators of the DST-OsCKX2 module have been identified,the mechanism employed by DST to regulate the expression of OsCKX2 remains unclear.Here,we demonstrate that DST-interacting protein 1(DIP1),known as Mediator subunit OsMED25,acts as an interacting coactivator of DST.Phenotypic analyses revealed that OsMED25-RNAi and the osmed25 mutant plants exhibited enlarged panicles,with enhanced branching and spikelet number,similar to the dst mutant.Genetic analysis indicated that OsMED25 acts in the same pathway as the DST-OsCKX2 module to regulate spikelet number per panicle.Further biochemical analysis showed that OsMED25 physically interacts with DST at the promoter region of OsCKX2,and then recruits RNA polymerase II(Pol II)to activate OsCKX2 transcription.Thus,OsMED25 was involved in the communication between DST and Pol II general transcriptional machinery to regulate spikelet number.In general,our findings reveal a novel function of OsMED25 in DST-OsCKX2 modulated transcriptional regulation,thus enhancing our un derstanding of the regulatory mechanism underlying DST-OsCKX2-mediated spikelet number.
基金This work was supported by funds from the National Basic Research Program(973 Program)(Nos.2011CB911104 and 2012CB917202)the National Natural Science Foundation of China(Grant No.31100538)to F.W.,(Grant No.31170817)to C.T.
文摘Mycosin-1 protease(MycP1)is a serine protease anchored to the inner membrane of Mycobacterium tuberculosis,and is essential in virulence factor secretion through the ESX-1 type VII secretion system(T7SS).Bacterial physiology studies demonstrated that MycP1 plays a dual role in the regulation of ESX-1 secretion and virulence,primarily through cleavage of its secretion substrate EspB.MycP1 contains a putative N-terminal inhibitory propeptide and a catalytic triad of Asp-His-Ser,classic hallmarks of a sub-tilase family serine protease.The MycP1 propeptide was previously reported to be initially inactive and activated after prolonged incubation.In this study,we have deter-mined crystal structures of MycP1 with(MycP124-422)and without(MycP1^(63-422))the propeptide,and conducted EspB cleavage assays using the two proteins.Very high struc-tural similarity was observed in the two crystal structures.Interestingly,protease assays demonstrated positive EspB cleavage for both proteins,indicating that the putative propeptide does not inhibit protease activity.Molecu-lar dynamic simulations showed higher rigidity in regions guarding the entrance to the catalytic site in MycP124-422 than in MycP1^(63-422),suggesting that the putative propeptide might contribute to the conformational stability of the active site cleft and surrounding regions.
基金This research was supported by the National Basic Research Program(973 Program)(Nos.2011CB911104 and 2013CB910200)the Chinese Natural Science Foundation of China(Grant No.31100563)to Y.Xiong and(Grant No.31170817)to C.Tian.
文摘Dear Editor,The functional diversity of proteins is related to the cooperation of multiple domains.Independent globular domains are typically joined by a fl exible length of polypeptide chain,which makes the structural analysis of multi-domain proteins diffi cult.Here,we describe the combined use of solution NMR(nuclear magnetic resonance)and EPR(elec-tron paramagnetic resonance)for the structural analysis of a protein with two separate domains.The structure of each domain was determined independently using conventional NMR restraints,and the relative orientation of the two separate domains was confi ned using long-distance restraints obtained by NMR-PRE(paramagnetic relaxation enhancement)and EPR-DEER(double electron-electron resonance,also called PELDOR:pulsed electron double reso-nance.
