Academic Journal
Reaction-based cryo-EM resolves the continuous conformational spectrum of CTP synthase catalysis.
| Τίτλος: | Reaction-based cryo-EM resolves the continuous conformational spectrum of CTP synthase catalysis. |
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| Συγγραφείς: | Guo CJ; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China., Wu YF; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China., Guo SY; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China., Fu Y; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China., Wang W; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China., Liu JL; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.; Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom. |
| Πηγή: | Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2026 Oct 20; Vol. 123 (42), pp. e2620271123. Date of Electronic Publication: 2026 Oct 09. |
| Τύπος έκδοσης: | Journal Article |
| Γλώσσα: | English |
| Στοιχεία περιοδικού: | Publisher: National Academy of Sciences Country of Publication: United States NLM ID: 7505876 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1091-6490 (Electronic) Linking ISSN: 00278424 NLM ISO Abbreviation: Proc Natl Acad Sci U S A Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: Washington, DC : National Academy of Sciences |
| Ιατρικοί όροι (MeSH): | Cryoelectron Microscopy*/methods , Carbon-Nitrogen Ligases*/chemistry , Carbon-Nitrogen Ligases*/metabolism , Carbon-Nitrogen Ligases*/ultrastructure , Drosophila melanogaster*/enzymology, Adenosine Triphosphate/metabolism ; Adenosine Triphosphate/chemistry ; Uridine Triphosphate/metabolism ; Uridine Triphosphate/analogs & derivatives ; Uridine Triphosphate/chemistry ; Cytidine Triphosphate/metabolism ; Cytidine Triphosphate/chemistry ; Animals ; Protein Conformation ; Models, Molecular ; Phosphorylation ; Catalysis ; Biocatalysis |
| Περίληψη: | Capturing enzymes under native turnover conditions remains a grand challenge in structural biology. Here, we develop a reaction-based cryo-electron microscopy (cryo-EM) strategy that directly samples Drosophila melanogaster cytidine triphosphate synthase (CTPS) from actively catalyzing mixtures containing only natural substrates and allosteric effectors. By integrating reaction-based sampling with three-dimensional variability analysis, we resolve a continuous conformational spectrum of CTPS during catalysis. This approach visualizes the chemical progression of 4-phosphoryl-uridine triphosphate (4Pi-UTP) formation and reveals a previously unobserved co-occupancy pattern of adenosine triphosphate (ATP) and CTP at the reaction end point. Cross-validation with nonhydrolyzable ATP analogs demonstrates that UTP phosphorylation by ATP, rather than mere ATP binding, shifts the conformational ensemble toward more closed states. Our findings establish reaction-based cryo-EM as a framework for resolving chemically annotated conformational ensembles without predefining a single trapped intermediate. |
| Competing Interests: | Competing interests statement:The authors declare no competing interest. |
| Grant Information: | 2021YFA0804700 Ministry of Science and Technology of the People's Republic of China (MOST); 32370744; 32350710195 MOST | National Natural Science Foundation of China (NSFC); MC_UU_12021/3; MC_U137788471 UKRI | Medical Research Council (MRC) |
| Contributed Indexing: | Keywords: 3D variability analysis; CTP synthase; conformational spectrum; enzyme catalysis; reaction-based cryo-EM |
| Substance Nomenclature: | EC 6.3.- (Carbon-Nitrogen Ligases) EC 6.3.4.2 (CTP synthetase) 8L70Q75FXE (Adenosine Triphosphate) UT0S826Z60 (Uridine Triphosphate) 65-47-4 (Cytidine Triphosphate) |
| Entry Date(s): | Date Created: 20261009 Date Completed: 20261009 Latest Revision: 20261023 |
| Update Code: | 20261023 |
| PubMed Central ID: | PMC13684251 |
| DOI: | 10.1073/pnas.2620271123 |
| PMID: | 42853686 |
| Βάση Δεδομένων: | MEDLINE |
| ISSN: | 1091-6490 |
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| DOI: | 10.1073/pnas.2620271123 |