NADPH-dependent thioredoxin reductase C (NTRC) is ubiquitously localized across all plastid types, including both chloroplasts and nonphotosynthetic plastids, serving as a central regulator in redox homeostasis. It plays pivotal roles in peroxidation resistance, redox signaling, tetrapyrrole biosynthesis, starch metabolism and photoperiod regulation. The NTRC/2-Cys peroxiredoxin (2CP)-mediated antioxidant defense system is also critically involved in counteracting biotic stress from pathogens. Relative studies demonstrate that the interaction between NTRC and 2CP is essential for modulating and integrating redox functions in chloroplasts. However, the molecular mechanism underlying the NTRC-2CP interaction remains elusive. In this study, we characterized the three-dimensional structure of Nicotiana benthamiana NTRC (NbNTRC) and resolved the crystal structure of its NTR domain by X-ray crystallography. Furthermore, we investigated the molecular basis of NbNTRC-Nb2CP interaction and stability using in vitro assays, including pull-down, size-exclusion chromatography, microscale thermophoresis and thermal stability assays. In summary, our work identifies two cysteine residues crucial for the intrinsic stability of the complex between NbNTRC and Nb2CP and the intermolecular interaction between them, determines the structure of the NbNTRC NTR domain and provides novel mechanistic insights into the NbNTRC-Nb2CP regulatory complex.
{"title":"Structural study of Nicotiana benthamiana NADPH-dependent thioredoxin reductase C and its molecular mechanism of interaction with 2-Cys peroxiredoxin.","authors":"Jiatai Zhang,Xuehui Bai,Shaodong Dai,Zhongzhou Chen","doi":"10.1107/s2059798326000495","DOIUrl":"https://doi.org/10.1107/s2059798326000495","url":null,"abstract":"NADPH-dependent thioredoxin reductase C (NTRC) is ubiquitously localized across all plastid types, including both chloroplasts and nonphotosynthetic plastids, serving as a central regulator in redox homeostasis. It plays pivotal roles in peroxidation resistance, redox signaling, tetrapyrrole biosynthesis, starch metabolism and photoperiod regulation. The NTRC/2-Cys peroxiredoxin (2CP)-mediated antioxidant defense system is also critically involved in counteracting biotic stress from pathogens. Relative studies demonstrate that the interaction between NTRC and 2CP is essential for modulating and integrating redox functions in chloroplasts. However, the molecular mechanism underlying the NTRC-2CP interaction remains elusive. In this study, we characterized the three-dimensional structure of Nicotiana benthamiana NTRC (NbNTRC) and resolved the crystal structure of its NTR domain by X-ray crystallography. Furthermore, we investigated the molecular basis of NbNTRC-Nb2CP interaction and stability using in vitro assays, including pull-down, size-exclusion chromatography, microscale thermophoresis and thermal stability assays. In summary, our work identifies two cysteine residues crucial for the intrinsic stability of the complex between NbNTRC and Nb2CP and the intermolecular interaction between them, determines the structure of the NbNTRC NTR domain and provides novel mechanistic insights into the NbNTRC-Nb2CP regulatory complex.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"13 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146044686","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-02-01DOI: 10.1107/s2059798326000422
Kelly L Parker,John D Clarke,Xiaojiao Liu,Barbara F Gomes,Lauren E A Eyssen,Nicholas Furnham,Floriano Paes Silva-Jr,Raymond J Owens
Schistosoma mansoni cathepsin D1 (SmCD1) has been shown to be an essential enzyme for helminth metabolism due to its role in haemoglobin degradation: a key amino-acid source for the developing parasite. Therefore, the enzyme is a potential target for the development of antischistosomal inhibitors. SmCD1 has significant sequence identity to cathepsin D-like proteases found in other schistosome species and homology to mammalian aspartic proteases. Here, we report the first crystal structures of a helminth cathepsin D, SmCD1, and have identified a single-domain antibody (nanobody) that specifically binds to SmCD1 with nanomolar affinity but does not recognize human cathepsin D. We have mapped the epitope of the nanobody by determining the crystal structure of the enzyme-nanobody complex, revealing the conformation of SmCD1 in the propeptide-bound state.
{"title":"Crystal structure of Schistosoma mansoni cathepsin D1 in complex with a nanobody reveals the conformation of the propeptide-bound state.","authors":"Kelly L Parker,John D Clarke,Xiaojiao Liu,Barbara F Gomes,Lauren E A Eyssen,Nicholas Furnham,Floriano Paes Silva-Jr,Raymond J Owens","doi":"10.1107/s2059798326000422","DOIUrl":"https://doi.org/10.1107/s2059798326000422","url":null,"abstract":"Schistosoma mansoni cathepsin D1 (SmCD1) has been shown to be an essential enzyme for helminth metabolism due to its role in haemoglobin degradation: a key amino-acid source for the developing parasite. Therefore, the enzyme is a potential target for the development of antischistosomal inhibitors. SmCD1 has significant sequence identity to cathepsin D-like proteases found in other schistosome species and homology to mammalian aspartic proteases. Here, we report the first crystal structures of a helminth cathepsin D, SmCD1, and have identified a single-domain antibody (nanobody) that specifically binds to SmCD1 with nanomolar affinity but does not recognize human cathepsin D. We have mapped the epitope of the nanobody by determining the crystal structure of the enzyme-nanobody complex, revealing the conformation of SmCD1 in the propeptide-bound state.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"77 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146056912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-02-01DOI: 10.1107/s2059798326000306
Charles Barchet,Ottilie von Loeffelholz,Roberto Bahena-Ceron,Bruno P Klaholz,Alexandre G Urzhumtsev
The quality of three-dimensional macromolecular image reconstruction by cryo electron microscopy (cryo-EM) strongly depends on the number and the quality of the respective two-dimensional projections and on their angular distribution in space. Distributions with one or a few strongly preferred particle orientations may result in maps that are deformed in certain directions. A simple removal of overrepresented views may improve the quality of the reconstructed maps when the level of noise in the two-dimensional (2D) projections is low and the data-set size can afford this removal, but is counterproductive otherwise. Complementarily, giving an increased weight to underrepresented views, or taking multiple copies of them during the reconstruction, may improve the results, naturally, depending on how non-uniform the view distribution is. This work describes the results of three-dimensional (3D) reconstructions using an explicit correction of the number of overrepresented and underrepresented projections for non-uniformly distributed sets. Such correction can be considered as a potential preprocessing, fast and simple, during 3D reconstruction in the image-processing and cryo-EM structure-determination workflow.
{"title":"Explicit correction of severely non-uniform distributions of cryo-EM views.","authors":"Charles Barchet,Ottilie von Loeffelholz,Roberto Bahena-Ceron,Bruno P Klaholz,Alexandre G Urzhumtsev","doi":"10.1107/s2059798326000306","DOIUrl":"https://doi.org/10.1107/s2059798326000306","url":null,"abstract":"The quality of three-dimensional macromolecular image reconstruction by cryo electron microscopy (cryo-EM) strongly depends on the number and the quality of the respective two-dimensional projections and on their angular distribution in space. Distributions with one or a few strongly preferred particle orientations may result in maps that are deformed in certain directions. A simple removal of overrepresented views may improve the quality of the reconstructed maps when the level of noise in the two-dimensional (2D) projections is low and the data-set size can afford this removal, but is counterproductive otherwise. Complementarily, giving an increased weight to underrepresented views, or taking multiple copies of them during the reconstruction, may improve the results, naturally, depending on how non-uniform the view distribution is. This work describes the results of three-dimensional (3D) reconstructions using an explicit correction of the number of overrepresented and underrepresented projections for non-uniformly distributed sets. Such correction can be considered as a potential preprocessing, fast and simple, during 3D reconstruction in the image-processing and cryo-EM structure-determination workflow.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"276 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146005285","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-01DOI: 10.1107/s2059798325010290
Mario Milani,Michela Visintin,Ivet Krastanova,Marco Visentini,Elisa Margotti,Gabriele Ugolini,Martino Bolognesi,Lucio C Rovati,Eloise Mastrangelo
The ADAMTS (a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motifs) family of secreted metalloproteinases plays essential roles in extracellular matrix remodeling. ADAMTS-5 contributes to cartilage degradation, cleaving proteoglycans such as aggrecan and versican, and being involved in both physiological tissue turnover and pathological processes such as osteoarthritis and atherosclerosis. Although structural insights into its catalytic domain have informed inhibitor development, the role of ancillary domains, particularly the spacer domain, in substrate recognition and specificity remains underexplored. Here, we report the crystal structure of a segment of human ADAMTS-5 encompassing the C-terminal portion of the cysteine-rich domain and the spacer domain (residues 694-876). This structure reveals critical features of the spacer domain, including the hypervariable loops that function as exosites essential for the binding of aggrecan and versican. Our findings provide new structural insights into the molecular determinants of the substrate specificity of ADAMTS-5 and underscore the spacer domain as a promising target for the development of selective inhibitors.
{"title":"Structure, substrate recognition and therapeutic targeting of the human ADAMTS-5 spacer domain.","authors":"Mario Milani,Michela Visintin,Ivet Krastanova,Marco Visentini,Elisa Margotti,Gabriele Ugolini,Martino Bolognesi,Lucio C Rovati,Eloise Mastrangelo","doi":"10.1107/s2059798325010290","DOIUrl":"https://doi.org/10.1107/s2059798325010290","url":null,"abstract":"The ADAMTS (a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motifs) family of secreted metalloproteinases plays essential roles in extracellular matrix remodeling. ADAMTS-5 contributes to cartilage degradation, cleaving proteoglycans such as aggrecan and versican, and being involved in both physiological tissue turnover and pathological processes such as osteoarthritis and atherosclerosis. Although structural insights into its catalytic domain have informed inhibitor development, the role of ancillary domains, particularly the spacer domain, in substrate recognition and specificity remains underexplored. Here, we report the crystal structure of a segment of human ADAMTS-5 encompassing the C-terminal portion of the cysteine-rich domain and the spacer domain (residues 694-876). This structure reveals critical features of the spacer domain, including the hypervariable loops that function as exosites essential for the binding of aggrecan and versican. Our findings provide new structural insights into the molecular determinants of the substrate specificity of ADAMTS-5 and underscore the spacer domain as a promising target for the development of selective inhibitors.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"28 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145664256","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Since the fiscal year 2022, `Support Program for Life Science and Drug Discovery Research (BINDS Phase II)', funded by the Japan Agency for Medical Research and Development (AMED), has been supporting structural analysis research in Japan, promoting the further development of various advanced structural analysis technologies. The BINDS project aims to offer more timely and cutting-edge support by establishing a new macromolecular crystallography endstation (MX-ES) at NanoTerasu, Japan's first fourth-generation synchrotron-radiation facility. MX-ES has been equipped with microbeam focusing optics and a state-of-the-art measurement system for fully automated, high-throughput data collection from cooled crystals. Commissioning and initial experiments have been completed. User operations are expected to begin in the second half of the fiscal year 2025.
{"title":"A new macromolecular crystallography endstation at NanoTerasu for accelerating structural biology and drug discovery.","authors":"Yusuke Yamada,Keisuke Sakurai,Akiya Fukuda,Hiroyuki Yamane,Tadashi Hatano,Tetsuya Nakamura,Go Ueno,Takuya Masunaga,Masaki Yamamoto,Eriko Nango","doi":"10.1107/s2059798325011234","DOIUrl":"https://doi.org/10.1107/s2059798325011234","url":null,"abstract":"Since the fiscal year 2022, `Support Program for Life Science and Drug Discovery Research (BINDS Phase II)', funded by the Japan Agency for Medical Research and Development (AMED), has been supporting structural analysis research in Japan, promoting the further development of various advanced structural analysis technologies. The BINDS project aims to offer more timely and cutting-edge support by establishing a new macromolecular crystallography endstation (MX-ES) at NanoTerasu, Japan's first fourth-generation synchrotron-radiation facility. MX-ES has been equipped with microbeam focusing optics and a state-of-the-art measurement system for fully automated, high-throughput data collection from cooled crystals. Commissioning and initial experiments have been completed. User operations are expected to begin in the second half of the fiscal year 2025.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145786403","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-01DOI: 10.1107/s2059798325010617
Alice Brink,John R Helliwell,Francois J F Jacobs
The aim of our structures for eventual clinical application is to be relevant. Regulation of pharmaceutical lead compounds, however, does not yet involve the need for patient-relevant macromolecular structures determined at 37°C, as it is not yet known whether crystal growth and diffraction at 37°C versus standard cryo-condition practices will reveal significant binding variations applicable for drug development or, in the case of extremophiles, provide insight into their function. However, for select examples in the literature interesting changes occurred, and support the initiative that data collection at high temperatures should be considered. This topical review considers a Protein Data Bank (PDB) and Cambridge Structural Database (CSD) data survey of crystal structures that have been determined at elevated temperatures, i.e. neither under cryogenic conditions nor at typical room-temperature conditions of 20-25°C, and reveals a few hurdles as well as many successes in reaching such patient-relevant structures. It highlights key methodology that appears in the literature which could benefit those considering related research. Since it is possible for crystallographic structure-determination methods to be adapted to 37°C, amid some challenges, we encourage the initiative that many more could be determined at 37°C. Included in the studies deposited in the PDB are some that have been performed at temperatures in excess of >37°C, and surprisingly several at even higher temperatures (i.e. 50-90°C). The overall aim of determining the 3D structure of a biological macromolecule at its natural body temperature has in principle to include crystallization and diffraction data collection. In the survey we find very few crystallizations performed at 37°C followed by data collection at the same temperature, and few have conducted a systematic study of comparing the changes occurring at 100 K versus 37°C. It is of course assumed that some key drug binding in proteins may occur over a narrow temperature range appropriate for mesophilic organisms, whereas for thermophilic organisms the protein may well exist over a wide temperature range reflecting that in which the organism is able to thrive. For the higher temperature structure solutions, those in the range which is more appropriate for thermophiles or hyperthermophiles, no crystallizations at these extreme temperatures have yet been conducted. The ability to conduct crystallization at 37°C and obtain acceptable high-resolution data at the same temperature is surely encouraging to the crystallographic community to build on these achievements for this and the full temperature range. We describe aspects of crystallization, mounting and transfer of crystals, data collection, reporting of metadata within databases etc. that have been notable during the survey of the data and highlight them here for the benefit of the community which may be considering 37°C data analysis from pre-crystal growth to re-refinement o
{"title":"Towards patient-relevant structures: reviewing body-temperature biological macromolecules and their ligands for pharmaceutical applications.","authors":"Alice Brink,John R Helliwell,Francois J F Jacobs","doi":"10.1107/s2059798325010617","DOIUrl":"https://doi.org/10.1107/s2059798325010617","url":null,"abstract":"The aim of our structures for eventual clinical application is to be relevant. Regulation of pharmaceutical lead compounds, however, does not yet involve the need for patient-relevant macromolecular structures determined at 37°C, as it is not yet known whether crystal growth and diffraction at 37°C versus standard cryo-condition practices will reveal significant binding variations applicable for drug development or, in the case of extremophiles, provide insight into their function. However, for select examples in the literature interesting changes occurred, and support the initiative that data collection at high temperatures should be considered. This topical review considers a Protein Data Bank (PDB) and Cambridge Structural Database (CSD) data survey of crystal structures that have been determined at elevated temperatures, i.e. neither under cryogenic conditions nor at typical room-temperature conditions of 20-25°C, and reveals a few hurdles as well as many successes in reaching such patient-relevant structures. It highlights key methodology that appears in the literature which could benefit those considering related research. Since it is possible for crystallographic structure-determination methods to be adapted to 37°C, amid some challenges, we encourage the initiative that many more could be determined at 37°C. Included in the studies deposited in the PDB are some that have been performed at temperatures in excess of >37°C, and surprisingly several at even higher temperatures (i.e. 50-90°C). The overall aim of determining the 3D structure of a biological macromolecule at its natural body temperature has in principle to include crystallization and diffraction data collection. In the survey we find very few crystallizations performed at 37°C followed by data collection at the same temperature, and few have conducted a systematic study of comparing the changes occurring at 100 K versus 37°C. It is of course assumed that some key drug binding in proteins may occur over a narrow temperature range appropriate for mesophilic organisms, whereas for thermophilic organisms the protein may well exist over a wide temperature range reflecting that in which the organism is able to thrive. For the higher temperature structure solutions, those in the range which is more appropriate for thermophiles or hyperthermophiles, no crystallizations at these extreme temperatures have yet been conducted. The ability to conduct crystallization at 37°C and obtain acceptable high-resolution data at the same temperature is surely encouraging to the crystallographic community to build on these achievements for this and the full temperature range. We describe aspects of crystallization, mounting and transfer of crystals, data collection, reporting of metadata within databases etc. that have been notable during the survey of the data and highlight them here for the benefit of the community which may be considering 37°C data analysis from pre-crystal growth to re-refinement o","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"4 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145704659","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-01DOI: 10.1107/s2059798325009982
Matthew D Giammar,Joshua L Dickerson,Laina N Hall,Bronwyn A Lucas
The ability to generate high-resolution views of cells with cryogenic electron microscopy (cryo-EM) can reveal the molecular mechanisms of biological processes in their native cellular context. The revolutionary impact of this strategy is limited by the difficulty of accurately annotating structures within these images. 2D template matching (2DTM), in which high-resolution structural models are used as computational probes to locate and orient molecular complexes with high precision, has shown initial promise in annotating single molecules in cellular cryo-EM images. While the scientific community works to identify best practices for applying 2DTM to specific biological questions and to maximize sensitivity and throughput, a modular and extensible software architecture would support the rapid development of novel methodological approaches, thus accelerating innovation within the field. To achieve this, we developed Leopard-EM (Location and orientation of particles found using two-dimensional tEmplate Matching), a modular Python-based 2DTM implementation built to be easily customizable. We implemented an automated pixel-size refinement procedure and find that 2DTM is sensitive to pixel-size errors of ∼0.2%. To demonstrate the flexibility of the Leopard-EM architecture, we developed a constrained search protocol that improved small ribosomal subunit detection by approximately eightfold by using initial locations and orientations determined for the large ribosomal subunit. Using this strategy, we captured a distribution of ribosome rotation states within the living cell at single-molecule resolution. We envision that Leopard-EM can be used as a platform for the development of in situ cryo-EM data-processing workflows, facilitating the rapid development of this field. Leopard-EM is available at https://github.com/Lucaslab-Berkeley/Leopard-EMgithub.com/Lucaslab-Berkeley/Leopard-EM.
{"title":"Leopard-EM: an extensible 2D template-matching package to accelerate in situ structural biology.","authors":"Matthew D Giammar,Joshua L Dickerson,Laina N Hall,Bronwyn A Lucas","doi":"10.1107/s2059798325009982","DOIUrl":"https://doi.org/10.1107/s2059798325009982","url":null,"abstract":"The ability to generate high-resolution views of cells with cryogenic electron microscopy (cryo-EM) can reveal the molecular mechanisms of biological processes in their native cellular context. The revolutionary impact of this strategy is limited by the difficulty of accurately annotating structures within these images. 2D template matching (2DTM), in which high-resolution structural models are used as computational probes to locate and orient molecular complexes with high precision, has shown initial promise in annotating single molecules in cellular cryo-EM images. While the scientific community works to identify best practices for applying 2DTM to specific biological questions and to maximize sensitivity and throughput, a modular and extensible software architecture would support the rapid development of novel methodological approaches, thus accelerating innovation within the field. To achieve this, we developed Leopard-EM (Location and orientation of particles found using two-dimensional tEmplate Matching), a modular Python-based 2DTM implementation built to be easily customizable. We implemented an automated pixel-size refinement procedure and find that 2DTM is sensitive to pixel-size errors of ∼0.2%. To demonstrate the flexibility of the Leopard-EM architecture, we developed a constrained search protocol that improved small ribosomal subunit detection by approximately eightfold by using initial locations and orientations determined for the large ribosomal subunit. Using this strategy, we captured a distribution of ribosome rotation states within the living cell at single-molecule resolution. We envision that Leopard-EM can be used as a platform for the development of in situ cryo-EM data-processing workflows, facilitating the rapid development of this field. Leopard-EM is available at https://github.com/Lucaslab-Berkeley/Leopard-EMgithub.com/Lucaslab-Berkeley/Leopard-EM.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"115 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145696906","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01DOI: 10.1107/s2059798325008782
Ryan Griffiths,Hans Pfalzgraf,Dimitris Latousakis,Gareth Ashworth,Changjiang Dong,Andrew Hemmings,Nathalie Juge
The accessory secretion (aSec) system is a protein export pathway that is uniquely present in Gram-positive bacteria and is dedicated to the secretion of large, glycosylated cell wall-anchored adhesins called serine-rich repeat proteins (SRRPs). Strain-specific glycosylation of SRRPs has previously been reported in Limosilactobacillus reuteri and attributed to GtfC, a glycosyltransferase belonging to family 113, with LrGtfC100-23 from L. reuteri rat strain 100-23C showing specificity for UDP-Glc, while LrGtfC53608 from L. reuteri pig strain ATCC 53608, which differs at only ten amino-acid positions, shows a preference for UDP-GlcNAc. However, the structural basis underpinning GtfC sugar-donor specificity remains unclear. Here, we report X-ray crystal structures of the tetrameric LrGtfC100-23 in the apo form and its complexes with UDP and with the noncognate sugar donor UDP-N-acetylglucosamine (UDP-GlcNAc). Analysis of the LrGtfC100-23 structures identified candidate residues implicated in donor-sugar substrate specificity, which were supported by site-directed mutagenesis. Reciprocal swaps of candidate residues combined with thermal shift assays revealed that the W240C variant of LrGtfC100-23 could bind both UDP-sugar donors, while the P243S variant of LrGtfC53608 became specific for UDP-Glc, opening the door for glycoengineering approaches in bacteria.
辅助分泌（aSec）系统是一种在革兰氏阳性细菌中独特存在的蛋白质输出途径，专门用于分泌被称为富丝氨酸重复蛋白（srrp）的大的、糖基化的细胞壁锚定黏附蛋白。此前在罗伊氏乳酸杆菌中曾报道过srrp的菌株特异性糖基化，并将其归因于属于113家族的糖基转移酶GtfC，其中来自罗伊氏乳杆菌大鼠菌株100-23C的LrGtfC100-23对UDP-Glc具有特异性，而来自罗伊氏乳杆菌猪菌株ATCC 53608的LrGtfC53608仅在10个氨基酸位置上存在差异，表现出对UDP-GlcNAc的偏好。然而，支持GtfC糖供体特异性的结构基础仍不清楚。在这里，我们报道了四聚体LrGtfC100-23载子形式的x射线晶体结构及其与UDP和非同源糖供体UDP- n -乙酰氨基葡萄糖（UDP- glcnac）的配合物。对LrGtfC100-23结构的分析发现了与供体糖底物特异性有关的候选残基，这得到了定点突变的支持。候选残基的互反交换结合热移实验表明，LrGtfC100-23的W240C变体可以结合两种udp -糖供体，而LrGtfC53608的P243S变体对UDP-Glc具有特异性，为糖工程方法在细菌中的应用打开了大门。
{"title":"A structural basis for the strain-dependent UDP-sugar specificity of glycosyltransferase C from the Limosilactobacillus reuteri accessory secretion system.","authors":"Ryan Griffiths,Hans Pfalzgraf,Dimitris Latousakis,Gareth Ashworth,Changjiang Dong,Andrew Hemmings,Nathalie Juge","doi":"10.1107/s2059798325008782","DOIUrl":"https://doi.org/10.1107/s2059798325008782","url":null,"abstract":"The accessory secretion (aSec) system is a protein export pathway that is uniquely present in Gram-positive bacteria and is dedicated to the secretion of large, glycosylated cell wall-anchored adhesins called serine-rich repeat proteins (SRRPs). Strain-specific glycosylation of SRRPs has previously been reported in Limosilactobacillus reuteri and attributed to GtfC, a glycosyltransferase belonging to family 113, with LrGtfC100-23 from L. reuteri rat strain 100-23C showing specificity for UDP-Glc, while LrGtfC53608 from L. reuteri pig strain ATCC 53608, which differs at only ten amino-acid positions, shows a preference for UDP-GlcNAc. However, the structural basis underpinning GtfC sugar-donor specificity remains unclear. Here, we report X-ray crystal structures of the tetrameric LrGtfC100-23 in the apo form and its complexes with UDP and with the noncognate sugar donor UDP-N-acetylglucosamine (UDP-GlcNAc). Analysis of the LrGtfC100-23 structures identified candidate residues implicated in donor-sugar substrate specificity, which were supported by site-directed mutagenesis. Reciprocal swaps of candidate residues combined with thermal shift assays revealed that the W240C variant of LrGtfC100-23 could bind both UDP-sugar donors, while the P243S variant of LrGtfC53608 became specific for UDP-Glc, opening the door for glycoengineering approaches in bacteria.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145440833","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01DOI: 10.1107/s205979832500912x
Henri Colyn Bwanika,Jingjing Zhao,Gerhard Hofer,Uwe H Sauer,Hongyi Xu
Microcrystal electron diffraction (MicroED), also known as three-dimensional electron diffraction (3D ED), allows the collection of diffraction data from submicrometre-sized crystals under low electron-dose conditions. Despite having several advantages over conventional X-ray crystallographic techniques, susceptibility to radiation damage is a great challenge that remains to be solved in MicroED. Similar to X-ray crystallography, radiation damage to the macromolecular crystal structures in MicroED manifests in two forms: global damage that affects the overall order of the crystal lattice and site-specific damage that affects highly sensitive residues and moieties in macromolecules. Traditionally, the unit e- Å-2 has been used for electron-dose estimations, which does not consider the interaction between the incident electron beam and the sample. In this study, we clarify the terminology for describing `dose' in electron crystallography, including the procedure for converting values from e- Å-2 to grays (Gy). Furthermore, we investigated data-processing strategies that could be used to limit the effects of radiation damage to the crystal. During MicroED data collection, radiation damage increases with the number of acquired ED frames because the accumulated electron dose increases. Data collected from several crystals and processed in this way can be merged to increase the completeness and subsequently be used for structure refinement. According to our results, this approach improves the resolution of the data, the data statistics, the structure determination and the quality of the final structure.
{"title":"Limiting the effects of radiation damage in MicroED through dose selection during data processing.","authors":"Henri Colyn Bwanika,Jingjing Zhao,Gerhard Hofer,Uwe H Sauer,Hongyi Xu","doi":"10.1107/s205979832500912x","DOIUrl":"https://doi.org/10.1107/s205979832500912x","url":null,"abstract":"Microcrystal electron diffraction (MicroED), also known as three-dimensional electron diffraction (3D ED), allows the collection of diffraction data from submicrometre-sized crystals under low electron-dose conditions. Despite having several advantages over conventional X-ray crystallographic techniques, susceptibility to radiation damage is a great challenge that remains to be solved in MicroED. Similar to X-ray crystallography, radiation damage to the macromolecular crystal structures in MicroED manifests in two forms: global damage that affects the overall order of the crystal lattice and site-specific damage that affects highly sensitive residues and moieties in macromolecules. Traditionally, the unit e- Å-2 has been used for electron-dose estimations, which does not consider the interaction between the incident electron beam and the sample. In this study, we clarify the terminology for describing `dose' in electron crystallography, including the procedure for converting values from e- Å-2 to grays (Gy). Furthermore, we investigated data-processing strategies that could be used to limit the effects of radiation damage to the crystal. During MicroED data collection, radiation damage increases with the number of acquired ED frames because the accumulated electron dose increases. Data collected from several crystals and processed in this way can be merged to increase the completeness and subsequently be used for structure refinement. According to our results, this approach improves the resolution of the data, the data statistics, the structure determination and the quality of the final structure.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"31 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145499612","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-28DOI: 10.1107/s2059798325010496
Lauren Giles,Tom Burnley
Here, we describe the first ten years of the CCP-EM Spring Symposium, an annual conference to bring together the cryogenic-sample electron microscopy (cryoEM) community to present and discuss the latest methodological advances and applications. We quantify the growth of the event and provide a detailed breakdown of the demographics of the tenth edition.
{"title":"Ten years of the CCP-EM Spring Symposium.","authors":"Lauren Giles,Tom Burnley","doi":"10.1107/s2059798325010496","DOIUrl":"https://doi.org/10.1107/s2059798325010496","url":null,"abstract":"Here, we describe the first ten years of the CCP-EM Spring Symposium, an annual conference to bring together the cryogenic-sample electron microscopy (cryoEM) community to present and discuss the latest methodological advances and applications. We quantify the growth of the event and provide a detailed breakdown of the demographics of the tenth edition.","PeriodicalId":501686,"journal":{"name":"Acta Crystallographica Section D","volume":"21 1","pages":"656-659"},"PeriodicalIF":0.0,"publicationDate":"2025-11-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145644916","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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