气候变化领域集成服务门户(CCPortal): Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter

CCPortal

DOI	10.1073/pnas.2001849117
	Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter
	Dahlberg P.D.; Saurabh S.; Sartor A.M.; Wang J.; Wang J.; Mitchell P.G.; Chiu W.; Chiu W.; Shapiro L.; Moerner W.E.
发表日期	2020
ISSN	0027-8424
起始页码	13937
结束页码	13944
卷号	117 期号:25
英文摘要	Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves singlemolecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of ~30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus: McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET.While not directly discernable, PopZ fills a region at the cell poles that is devoid of electrondense ribosomes. We annotate the position of PopZ with singlemolecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region. © 2020 National Academy of Sciences. All rights reserved.
英文关键词	CIASM; CLEM; Correlative microscopy; Cryogenic electron tomography; Superresolution
语种	英语
scopus关键词	bacterial protein; protein McpA; protein PopZ; protein SpmX; unclassified drug; bacterial protein; Article; asymmetric cell division; Caulobacter vibrioides; controlled study; electron tomography; fluorescence microscopy; image reconstruction; measurement precision; nonhuman; priority journal; protein localization; ribosome; signal transduction; single molecule imaging; Caulobacter vibrioides; electron tomography; fluorescence microscopy; metabolism; procedures; protein transport; single molecule imaging; ultrastructure; Bacterial Proteins; Caulobacter crescentus; Electron Microscope Tomography; Microscopy, Fluorescence; Protein Transport; Single Molecule Imaging
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/160280
作者单位	Dahlberg, P.D., Department of Chemistry, Stanford University, Stanford, CA 94305, United States; Saurabh, S., Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, United States; Sartor, A.M., Department of Chemistry, Stanford University, Stanford, CA 94305, United States; Wang, J., Department of Chemistry, Stanford University, Stanford, CA 94305, United States; Wang, J., Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, United States; Mitchell, P.G., Division of Cryo-EM and Bioimaging, Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, United States; Chiu, W., Division of Cryo-EM and Bioimaging, Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, United States; Chiu, W., Department of Bioengineering, Stanford University, Stanford, CA 94305, United States; Shapiro, L., Department of Developmental Biology, ...
推荐引用方式 GB/T 7714	Dahlberg P.D.,Saurabh S.,Sartor A.M.,et al. Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter[J],2020,117(25).
APA	Dahlberg P.D..,Saurabh S..,Sartor A.M..,Wang J..,Wang J..,...&Moerner W.E..(2020).Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter.Proceedings of the National Academy of Sciences of the United States of America,117(25).
MLA	Dahlberg P.D.,et al."Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter".Proceedings of the National Academy of Sciences of the United States of America 117.25(2020).