Pub Date : 2024-03-01Epub Date: 2024-02-20DOI: 10.1107/S2053230X24000979
Anita Dornes, Christopher Nils Mais, Gert Bange
The GTPase FlhF, a signal recognition particle (SRP)-type enzyme, is pivotal for spatial-numerical control and bacterial flagella assembly across diverse species, including pathogens. This study presents the X-ray structure of FlhF in its GDP-bound state at a resolution of 2.28 Å. The structure exhibits the classical N- and G-domain fold, consistent with related SRP GTPases such as Ffh and FtsY. Comparative analysis with GTP-loaded FlhF elucidates the conformational changes associated with GTP hydrolysis. These topological reconfigurations are similarly evident in Ffh and FtsY, and play a pivotal role in regulating the functions of these hydrolases.
GTP酶FlhF是一种信号识别颗粒(SRP)型酶,在包括病原体在内的不同物种的空间-数量控制和细菌鞭毛组装中起着关键作用。本研究以 2.28 Å 的分辨率展示了 FlhF 在 GDP 结合态下的 X 射线结构。该结构表现出经典的 N 和 G 域折叠,与 Ffh 和 FtsY 等相关 SRP GTP 酶一致。与加载了 GTP 的 FlhF 的比较分析阐明了与 GTP 水解相关的构象变化。这些拓扑重组在 Ffh 和 FtsY 中同样明显,并在调节这些水解酶的功能方面发挥了关键作用。
{"title":"Structure of the GDP-bound state of the SRP GTPase FlhF.","authors":"Anita Dornes, Christopher Nils Mais, Gert Bange","doi":"10.1107/S2053230X24000979","DOIUrl":"10.1107/S2053230X24000979","url":null,"abstract":"<p><p>The GTPase FlhF, a signal recognition particle (SRP)-type enzyme, is pivotal for spatial-numerical control and bacterial flagella assembly across diverse species, including pathogens. This study presents the X-ray structure of FlhF in its GDP-bound state at a resolution of 2.28 Å. The structure exhibits the classical N- and G-domain fold, consistent with related SRP GTPases such as Ffh and FtsY. Comparative analysis with GTP-loaded FlhF elucidates the conformational changes associated with GTP hydrolysis. These topological reconfigurations are similarly evident in Ffh and FtsY, and play a pivotal role in regulating the functions of these hydrolases.</p>","PeriodicalId":7029,"journal":{"name":"Acta crystallographica. Section F, Structural biology communications","volume":null,"pages":null},"PeriodicalIF":0.9,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10910532/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139904727","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-01Epub Date: 2024-02-20DOI: 10.1107/S2053230X24000827
Sintayehu Manaye Shenkutie, Soshichiro Nagano, Jon Hughes
Sorghum, a short-day tropical plant, has been adapted for temperate grain production, in particular through the selection of variants at the MATURITY loci (Ma1-Ma6) that reduce photoperiod sensitivity. Ma3 encodes phytochrome B (phyB), a red/far-red photochromic biliprotein photoreceptor. The multi-domain gene product, comprising 1178 amino acids, autocatalytically binds the phytochromobilin chromophore to form the photoactive holophytochrome (Sb.phyB). This study describes the development of an efficient heterologous overproduction system which allows the production of large quantities of various holoprotein constructs, along with purification and crystallization procedures. Crystals of the Pr (red-light-absorbing) forms of NPGP, PGP and PG (residues 1-655, 114-655 and 114-458, respectively), each C-terminally tagged with His6, were successfully produced. While NPGP crystals did not diffract, those of PGP and PG diffracted to 6 and 2.1 Å resolution, respectively. Moving the tag to the N-terminus and replacing phytochromobilin with phycocyanobilin as the ligand produced PG crystals that diffracted to 1.8 Å resolution. These results demonstrate that the diffraction quality of challenging protein crystals can be improved by removing flexible regions, shifting fusion tags and altering small-molecule ligands.
{"title":"Expression, purification and crystallization of the photosensory module of phytochrome B (phyB) from Sorghum bicolor.","authors":"Sintayehu Manaye Shenkutie, Soshichiro Nagano, Jon Hughes","doi":"10.1107/S2053230X24000827","DOIUrl":"10.1107/S2053230X24000827","url":null,"abstract":"<p><p>Sorghum, a short-day tropical plant, has been adapted for temperate grain production, in particular through the selection of variants at the MATURITY loci (Ma1-Ma6) that reduce photoperiod sensitivity. Ma3 encodes phytochrome B (phyB), a red/far-red photochromic biliprotein photoreceptor. The multi-domain gene product, comprising 1178 amino acids, autocatalytically binds the phytochromobilin chromophore to form the photoactive holophytochrome (Sb.phyB). This study describes the development of an efficient heterologous overproduction system which allows the production of large quantities of various holoprotein constructs, along with purification and crystallization procedures. Crystals of the Pr (red-light-absorbing) forms of NPGP, PGP and PG (residues 1-655, 114-655 and 114-458, respectively), each C-terminally tagged with His<sub>6</sub>, were successfully produced. While NPGP crystals did not diffract, those of PGP and PG diffracted to 6 and 2.1 Å resolution, respectively. Moving the tag to the N-terminus and replacing phytochromobilin with phycocyanobilin as the ligand produced PG crystals that diffracted to 1.8 Å resolution. These results demonstrate that the diffraction quality of challenging protein crystals can be improved by removing flexible regions, shifting fusion tags and altering small-molecule ligands.</p>","PeriodicalId":7029,"journal":{"name":"Acta crystallographica. Section F, Structural biology communications","volume":null,"pages":null},"PeriodicalIF":0.9,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10910535/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139904726","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-02DOI: 10.1107/S2053230X2400102X
Mark J. van Raaij
Acta Cryst. F – Structural Biology Communications plans to introduce more video content to articles and Dialpuri et al. [(2024). Acta Cryst. F80, 30–35] provide an early example.
{"title":"Private glycan investigations and public video content","authors":"Mark J. van Raaij","doi":"10.1107/S2053230X2400102X","DOIUrl":"10.1107/S2053230X2400102X","url":null,"abstract":"<p><i>Acta Cryst. F – Structural Biology Communications</i> plans to introduce more video content to articles and Dialpuri <i>et al.</i> [(2024). <i>Acta Cryst.</i> F<b>80</b>, 30–35] provide an early example.</p>","PeriodicalId":7029,"journal":{"name":"Acta crystallographica. Section F, Structural biology communications","volume":null,"pages":null},"PeriodicalIF":0.9,"publicationDate":"2024-02-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139669670","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-02DOI: 10.1107/S2053230X24000621
Phillip G. Pierce, Brian E. Hartnett, Tosha M. Laughlin, Joy M. Blain, Stephen J. Mayclin, Madison J. Bolejack, Janette B. Myers, Tate W. Higgins, David M. Dranow, Amy Sullivan, Donald D. Lorimer, Thomas E. Edwards, Timothy J. Hagen, James R. Horn, Peter J. Myler
The methylerythritol phosphate (MEP) pathway is a metabolic pathway that produces the isoprenoids isopentyl pyrophosphate and dimethylallyl pyrophosphate. Notably, the MEP pathway is present in bacteria and not in mammals, which makes the enzymes of the MEP pathway attractive targets for discovering new anti-infective agents due to the reduced chances of off-target interactions leading to side effects. There are seven enzymes in the MEP pathway, the third of which is IspD. Two crystal structures of Burkholderia thailandensis IspD (BtIspD) were determined: an apo structure and that of a complex with cytidine triphosphate (CTP). Comparison of the CTP-bound BtIspD structure with the apo structure revealed that CTP binding stabilizes the loop composed of residues 13–19. The apo structure of Mycobacterium paratuberculosis IspD (MpIspD) is also reported. The melting temperatures of MpIspD and BtIspD were evaluated by circular dichroism. The moderate Tm values suggest that a thermal shift assay may be feasible for future inhibitor screening. Finally, the binding affinity of CTP for BtIspD was evaluated by isothermal titration calorimetry. These structural and biophysical data will aid in the discovery of IspD inhibitors.
{"title":"Crystal structure and biophysical characterization of IspD from Burkholderia thailandensis and Mycobacterium paratuberculosis","authors":"Phillip G. Pierce, Brian E. Hartnett, Tosha M. Laughlin, Joy M. Blain, Stephen J. Mayclin, Madison J. Bolejack, Janette B. Myers, Tate W. Higgins, David M. Dranow, Amy Sullivan, Donald D. Lorimer, Thomas E. Edwards, Timothy J. Hagen, James R. Horn, Peter J. Myler","doi":"10.1107/S2053230X24000621","DOIUrl":"10.1107/S2053230X24000621","url":null,"abstract":"<p>The methylerythritol phosphate (MEP) pathway is a metabolic pathway that produces the isoprenoids isopentyl pyrophosphate and dimethylallyl pyrophosphate. Notably, the MEP pathway is present in bacteria and not in mammals, which makes the enzymes of the MEP pathway attractive targets for discovering new anti-infective agents due to the reduced chances of off-target interactions leading to side effects. There are seven enzymes in the MEP pathway, the third of which is IspD. Two crystal structures of <i>Burkholderia thailandensis</i> IspD (<i>Bt</i>IspD) were determined: an apo structure and that of a complex with cytidine triphosphate (CTP). Comparison of the CTP-bound <i>Bt</i>IspD structure with the apo structure revealed that CTP binding stabilizes the loop composed of residues 13–19. The apo structure of <i>Mycobacterium paratuberculosis</i> IspD (<i>Mp</i>IspD) is also reported. The melting temperatures of <i>Mp</i>IspD and <i>Bt</i>IspD were evaluated by circular dichroism. The moderate <i>T</i><sub>m</sub> values suggest that a thermal shift assay may be feasible for future inhibitor screening. Finally, the binding affinity of CTP for <i>Bt</i>IspD was evaluated by isothermal titration calorimetry. These structural and biophysical data will aid in the discovery of IspD inhibitors.</p>","PeriodicalId":7029,"journal":{"name":"Acta crystallographica. Section F, Structural biology communications","volume":null,"pages":null},"PeriodicalIF":0.9,"publicationDate":"2024-02-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139669860","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-01Epub Date: 2024-01-25DOI: 10.1107/S2053230X24000645
Monika Witzenberger, Robert Janowski, Dierk Niessing
Human tRNA (uracil-5-)-methyltransferase 2 homolog A (TRMT2A) is the dedicated enzyme for the methylation of uridine 54 in transfer RNA (tRNA). Human TRMT2A has also been described as a modifier of polyglutamine (polyQ)-derived neuronal toxicity. The corresponding human polyQ pathologies include Huntington's disease and constitute a family of devastating neurodegenerative diseases. A polyQ tract in the corresponding disease-linked protein causes neuronal death and symptoms such as impaired motor function, as well as cognitive impairment. In polyQ disease models, silencing of TRMT2A reduced polyQ-associated cell death and polyQ protein aggregation, suggesting this protein as a valid drug target against this class of disorders. In this paper, the 1.6 Å resolution crystal structure of the RNA-recognition motif (RRM) from Drosophila melanogaster, which is a homolog of human TRMT2A, is described and analysed.
{"title":"Crystal structure of the RNA-recognition motif of Drosophila melanogaster tRNA (uracil-5-)-methyltransferase homolog A.","authors":"Monika Witzenberger, Robert Janowski, Dierk Niessing","doi":"10.1107/S2053230X24000645","DOIUrl":"10.1107/S2053230X24000645","url":null,"abstract":"<p><p>Human tRNA (uracil-5-)-methyltransferase 2 homolog A (TRMT2A) is the dedicated enzyme for the methylation of uridine 54 in transfer RNA (tRNA). Human TRMT2A has also been described as a modifier of polyglutamine (polyQ)-derived neuronal toxicity. The corresponding human polyQ pathologies include Huntington's disease and constitute a family of devastating neurodegenerative diseases. A polyQ tract in the corresponding disease-linked protein causes neuronal death and symptoms such as impaired motor function, as well as cognitive impairment. In polyQ disease models, silencing of TRMT2A reduced polyQ-associated cell death and polyQ protein aggregation, suggesting this protein as a valid drug target against this class of disorders. In this paper, the 1.6 Å resolution crystal structure of the RNA-recognition motif (RRM) from Drosophila melanogaster, which is a homolog of human TRMT2A, is described and analysed.</p>","PeriodicalId":7029,"journal":{"name":"Acta crystallographica. Section F, Structural biology communications","volume":null,"pages":null},"PeriodicalIF":0.9,"publicationDate":"2024-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10836426/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139545168","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-02-01Epub Date: 2024-01-24DOI: 10.1107/S2053230X24000359
Jordan S Dialpuri, Haroldas Bagdonas, Lucy C Schofield, Phuong Thao Pham, Lou Holland, Paul S Bond, Filomeno Sánchez Rodríguez, Stuart J McNicholas, Jon Agirre
Owing to the difficulties associated with working with carbohydrates, validating glycan 3D structures prior to deposition into the Protein Data Bank has become a staple of the structure-solution pipeline. The Privateer software provides integrative methods for the validation, analysis, refinement and graphical representation of 3D atomic structures of glycans, both as ligands and as protein modifiers. While Privateer is free software, it requires users to install any of the structural biology software suites that support it or to build it from source code. Here, the Privateer web app is presented, which is always up to date and available to be used online (https://privateer.york.ac.uk) without installation. This self-updating tool, which runs locally on the user's machine, will allow structural biologists to simply and quickly analyse carbohydrate ligands and protein glycosylation from a web browser whilst retaining all confidential information on their devices.
{"title":"Online carbohydrate 3D structure validation with the Privateer web app.","authors":"Jordan S Dialpuri, Haroldas Bagdonas, Lucy C Schofield, Phuong Thao Pham, Lou Holland, Paul S Bond, Filomeno Sánchez Rodríguez, Stuart J McNicholas, Jon Agirre","doi":"10.1107/S2053230X24000359","DOIUrl":"10.1107/S2053230X24000359","url":null,"abstract":"<p><p>Owing to the difficulties associated with working with carbohydrates, validating glycan 3D structures prior to deposition into the Protein Data Bank has become a staple of the structure-solution pipeline. The Privateer software provides integrative methods for the validation, analysis, refinement and graphical representation of 3D atomic structures of glycans, both as ligands and as protein modifiers. While Privateer is free software, it requires users to install any of the structural biology software suites that support it or to build it from source code. Here, the Privateer web app is presented, which is always up to date and available to be used online (https://privateer.york.ac.uk) without installation. This self-updating tool, which runs locally on the user's machine, will allow structural biologists to simply and quickly analyse carbohydrate ligands and protein glycosylation from a web browser whilst retaining all confidential information on their devices.</p>","PeriodicalId":7029,"journal":{"name":"Acta crystallographica. Section F, Structural biology communications","volume":null,"pages":null},"PeriodicalIF":0.9,"publicationDate":"2024-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10836424/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139541238","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}