Technical approaches of 3D reconstruction from protein complex using the mixture of differently stained images: providing suggestive evidence for improving its resolution
Abstract Negative staining electron microscopy remains a valuable tool for structural biology, particularly for initial characterization of large protein complexes. However, the limitations of single staining methods often result in incomplete structural information. Here, we present a novel multi-s...
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SpringerOpen
2025-05-01
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| Series: | Applied Microscopy |
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| Online Access: | https://doi.org/10.1186/s42649-025-00111-9 |
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| author | Yoon Ho Park Myeong Seon Jeong Gang San Song Tak gwonbaek Young Kwan Kim Hyun Suk Jung |
| author_facet | Yoon Ho Park Myeong Seon Jeong Gang San Song Tak gwonbaek Young Kwan Kim Hyun Suk Jung |
| author_sort | Yoon Ho Park |
| collection | DOAJ |
| description | Abstract Negative staining electron microscopy remains a valuable tool for structural biology, particularly for initial characterization of large protein complexes. However, the limitations of single staining methods often result in incomplete structural information. Here, we present a novel multi-stain approach for negative staining electron microscopy, applied to the structural analysis of the pyruvate dehydrogenase E2 (PDH E2) complex. By integrating data from three distinct staining agents (uranyl acetate, ammonium phosphotungstate, and ammonium molybdate) we demonstrate significant improvements in structural resolution and detail. Our method improved the resolution from a range of approximately 27–31 Å (observed with individual stains) to about 21.7 Å in the combined dataset. This enhancement facilitated a clearer visualization of the complex’s icosahedral symmetry and allowed for a more precise determination of the overall shape and domain organization of the PDH E2 complex. The multi-stain approach revealed complementary structural information, with each stain highlighting different aspects of the protein complex. Uranyl acetate provided excellent overall contrast, while ammonium phosphotungstate and molybdate offered enhanced visibility of specific structural elements. The integration of these complementary data sets resulted in a more comprehensive structural model. Our findings suggest that this multi-stain negative staining approach can be a powerful tool for enhancing low-resolution structural information of large protein complexes, bridging the gap between initial characterization and high-resolution studies. This method holds promise for improving our understanding of challenging macromolecular assemblies and may serve as a valuable precursor to more advanced structural biology techniques. |
| format | Article |
| id | doaj-art-56241e8352eb4bea96a15afa4027c6f2 |
| institution | DOAJ |
| issn | 2287-4445 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | SpringerOpen |
| record_format | Article |
| series | Applied Microscopy |
| spelling | doaj-art-56241e8352eb4bea96a15afa4027c6f22025-08-20T03:22:27ZengSpringerOpenApplied Microscopy2287-44452025-05-015511910.1186/s42649-025-00111-9Technical approaches of 3D reconstruction from protein complex using the mixture of differently stained images: providing suggestive evidence for improving its resolutionYoon Ho Park0Myeong Seon Jeong1Gang San Song2Tak gwonbaek3Young Kwan Kim4Hyun Suk Jung5Department of Biochemistry, College of Natural Sciences, Kangwon National UniversityDepartment of Biochemistry, College of Natural Sciences, Kangwon National UniversityDepartment of Biochemistry, College of Natural Sciences, Kangwon National UniversityDepartment of Electrical and Electronic Engineering, College of Engineering, Kangwon National UniversityKangwon Center for Systems ImagingDepartment of Biochemistry, College of Natural Sciences, Kangwon National UniversityAbstract Negative staining electron microscopy remains a valuable tool for structural biology, particularly for initial characterization of large protein complexes. However, the limitations of single staining methods often result in incomplete structural information. Here, we present a novel multi-stain approach for negative staining electron microscopy, applied to the structural analysis of the pyruvate dehydrogenase E2 (PDH E2) complex. By integrating data from three distinct staining agents (uranyl acetate, ammonium phosphotungstate, and ammonium molybdate) we demonstrate significant improvements in structural resolution and detail. Our method improved the resolution from a range of approximately 27–31 Å (observed with individual stains) to about 21.7 Å in the combined dataset. This enhancement facilitated a clearer visualization of the complex’s icosahedral symmetry and allowed for a more precise determination of the overall shape and domain organization of the PDH E2 complex. The multi-stain approach revealed complementary structural information, with each stain highlighting different aspects of the protein complex. Uranyl acetate provided excellent overall contrast, while ammonium phosphotungstate and molybdate offered enhanced visibility of specific structural elements. The integration of these complementary data sets resulted in a more comprehensive structural model. Our findings suggest that this multi-stain negative staining approach can be a powerful tool for enhancing low-resolution structural information of large protein complexes, bridging the gap between initial characterization and high-resolution studies. This method holds promise for improving our understanding of challenging macromolecular assemblies and may serve as a valuable precursor to more advanced structural biology techniques.https://doi.org/10.1186/s42649-025-00111-93D reconstructionProtein complexNegative stainingTransmission electron microscopyResolution |
| spellingShingle | Yoon Ho Park Myeong Seon Jeong Gang San Song Tak gwonbaek Young Kwan Kim Hyun Suk Jung Technical approaches of 3D reconstruction from protein complex using the mixture of differently stained images: providing suggestive evidence for improving its resolution Applied Microscopy 3D reconstruction Protein complex Negative staining Transmission electron microscopy Resolution |
| title | Technical approaches of 3D reconstruction from protein complex using the mixture of differently stained images: providing suggestive evidence for improving its resolution |
| title_full | Technical approaches of 3D reconstruction from protein complex using the mixture of differently stained images: providing suggestive evidence for improving its resolution |
| title_fullStr | Technical approaches of 3D reconstruction from protein complex using the mixture of differently stained images: providing suggestive evidence for improving its resolution |
| title_full_unstemmed | Technical approaches of 3D reconstruction from protein complex using the mixture of differently stained images: providing suggestive evidence for improving its resolution |
| title_short | Technical approaches of 3D reconstruction from protein complex using the mixture of differently stained images: providing suggestive evidence for improving its resolution |
| title_sort | technical approaches of 3d reconstruction from protein complex using the mixture of differently stained images providing suggestive evidence for improving its resolution |
| topic | 3D reconstruction Protein complex Negative staining Transmission electron microscopy Resolution |
| url | https://doi.org/10.1186/s42649-025-00111-9 |
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