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  1. NTU Theses and Dissertations Repository
  2. 生命科學院
  3. 生化科學研究所
請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76870
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dc.contributor.advisor徐尚德(Shang-Te Danny Hsu)
dc.contributor.authorYi-Hsiang Chiuen
dc.contributor.author邱怡翔zh_TW
dc.date.accessioned2021-07-10T21:39:04Z-
dc.date.available2021-07-10T21:39:04Z-
dc.date.copyright2020-08-28
dc.date.issued2020
dc.date.submitted2020-08-17
dc.identifier.citation1. Rankin, N. J.; Preiss, D.; Welsh, P.; Burgess, K. E.; Nelson, S. M.; Lawlor, D. A.; Sattar, N., The emergence of proton nuclear magnetic resonance metabolomics in the cardiovascular arena as viewed from a clinical perspective. Atherosclerosis 2014, 237 (1), 287-300.
2. Wang, H. W.; Wang, J. W., How cryo‐electron microscopy and X‐ray crystallography complement each other. Protein Science 2017, 26 (1), 32-39.
3. Gatan K2 Summit camera enables scientists to break the 3 Å barrier in structural biology using cryo-electron microscopy (cryo-EM). https://www.gatan.com/company/news/k2-summit-camera-enables-scientists-break-3-%C3%A5-barrier-structural-biology-using-cryo.
4. D'Imprima, E.; Floris, D.; Joppe, M.; Sánchez, R.; Grininger, M.; Kühlbrandt, W., Protein denaturation at the air-water interface and how to prevent it. Elife 2019, 8, e42747.
5. Carroni, M.; Saibil, H. R., Cryo electron microscopy to determine the structure of macromolecular complexes. Methods 2016, 95, 78-85.
6. Dubochet, J.; McDowall, A., Vitrification of pure water for electron microscopy. Journal of Microscopy 1981, 124 (3), 3-4.
7. Molecular weight statistics of single-particle released maps. https://www.ebi.ac.uk/pdbe/emdb/statistics_mol_wt.html/.
8. Herzik, M. A.; Wu, M.; Lander, G. C., High-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM. Nature communications 2019, 10 (1), 1-9.
9. Coscia, F.; Estrozi, L. F.; Hans, F.; Malet, H.; Noirclerc-Savoye, M.; Schoehn, G.; Petosa, C., Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein. Sci Rep 2016, 6, 30909.
10. Steiner, D.; Forrer, P.; Plückthun, A., Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display. Journal of molecular biology 2008, 382 (5), 1211-1227.
11. Liu, Y.; Gonen, S.; Gonen, T.; Yeates, T. O., Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system. Proc Natl Acad Sci U S A 2018, 115 (13), 3362-3367.
12. Yao, Q.; Weaver, S. J.; Mock, J. Y.; Jensen, G. J., Fusion of DARPin to Aldolase Enables Visualization of Small Protein by Cryo-EM. Structure 2019, 27 (7), 1148-1155 e3.
13. Sriramoju, M. K.; Chen, Y.; Lee, Y.-T. C.; Hsu, S.-T. D., Topologically knotted deubiquitinases exhibit unprecedented mechanostability to withstand the proteolysis by an AAA+ protease. Scientific reports 2018, 8 (1), 1-9.
14. Howard, B. R.; Endrizzi, J. A.; Remington, S. J., Crystal structure of Escherichia coli malate synthase G complexed with magnesium and glyoxylate at 2.0 Å resolution: mechanistic implications. Biochemistry 2000, 39 (11), 3156-3168.
15. Osaka, H.; Wang, Y.-L.; Takada, K.; Takizawa, S.; Setsuie, R.; Li, H.; Sato, Y.; Nishikawa, K.; Sun, Y.-J.; Sakurai, M., Ubiquitin carboxy-terminal hydrolase L1 binds to and stabilizes monoubiquitin in neuron. Human molecular genetics 2003, 12 (16), 1945-1958.
16. Saigoh, K.; Wang, Y.-L.; Suh, J.-G.; Yamanishi, T.; Sakai, Y.; Kiyosawa, H.; Harada, T.; Ichihara, N.; Wakana, S.; Kikuchi, T., Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice. Nature genetics 1999, 23 (1), 47-51.
17. Boudreaux, D. A.; Maiti, T. K.; Davies, C. W.; Das, C., Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation. Proceedings of the National Academy of Sciences 2010, 107 (20), 9117-9122.
18. Mallery, D. L.; Vandenberg, C. J.; Hiom, K., Activation of the E3 ligase function of the BRCA1/BARD1 complex by polyubiquitin chains. The EMBO journal 2002, 21 (24), 6755-6762.
19. Initial Analysis and Quality Assessment of Solution Scattering Data.
20. Scientific, T. F., Instructions for DSS and BS3 Crosslinkers.
21. Zheng, S. Q.; Palovcak, E.; Armache, J.-P.; Cheng, Y.; Agard, D. A., Anisotropic correction of beam-induced motion for improved single-particle electron cryo-microscopy. bioRxiv 2016, 061960.
22. Zivanov, J.; Nakane, T.; Forsberg, B. O.; Kimanius, D.; Hagen, W. J.; Lindahl, E.; Scheres, S. H., New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 2018, 7, e42166.
23. Zhang, K., Gctf: Real-time CTF determination and correction. Journal of structural biology 2016, 193 (1), 1-12.
24. Grigorieff, N., FREALIGN: high-resolution refinement of single particle structures. Journal of structural biology 2007, 157 (1), 117-125.
25. Punjani, A.; Rubinstein, J. L.; Fleet, D. J.; Brubaker, M. A., cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nature methods 2017, 14 (3), 290-296.
26. Franke, D.; Svergun, D. I., DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. Journal of applied crystallography 2009, 42 (2), 342-346.
27. Grant, T.; Rohou, A.; Grigorieff, N., cisTEM, user-friendly software for single-particle image processing. elife 2018, 7, e35383.
28. Scheres, S. H., Processing of structurally heterogeneous cryo-EM data in RELION. In Methods in enzymology, Elsevier: 2016; Vol. 579, pp 125-157.
29. Punjani, A.; Zhang, H.; Fleet, D. J., Non-uniform refinement: Adaptive regularization improves single particle cryo-EM reconstruction. BioRXiv 2019.
30. Merkley, E. D.; Rysavy, S.; Kahraman, A.; Hafen, R. P.; Daggett, V.; Adkins, J. N., Distance restraints from crosslinking mass spectrometry: mining a molecular dynamics simulation database to evaluate lysine–lysine distances. Protein science 2014, 23 (6), 747-759.
31. Andersson, F. I.; Werrell, E. F.; McMorran, L.; Crone, W. J.; Das, C.; Hsu, S.-T. D.; Jackson, S. E., The effect of Parkinson's-disease-associated mutations on the deubiquitinating enzyme UCH-L1. Journal of molecular biology 2011, 407 (2), 261-272.
dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76870-
dc.description.abstract隨著電子顯微鏡影像感測器和分析軟體的革新,冷凍低溫電子顯微鏡(cryo-EM)成為近期結構生物學中重要的工具。利用冷凍低溫電子顯微鏡收集樣品影線後,可使用分析軟體重建出蛋白質分子結構,達到近似原子級的解析度,這項技術稱為cryo-EM single particle reconstruction(SPR)。儘管能夠觀察到大分子結構的變化,但由於小於100 kD的蛋白的訊號和背景雜訊的訊號強度相似,小分子無法在顯微鏡下被清除觀察到,故對於分子量小於 100 kD 的樣品仍有困難性。為克服此苦難,結構生物學家設計支架蛋白(scaffold protein) 來幫助在顯微鏡下更易觀察到小蛋白。支架蛋白通常是對稱的同源多聚蛋白,其包括多個結合位置能以共價或非共價鍵與標的蛋白結合。在此研究中,我們設計了一種 scaffold protein,由鳥胺酸氨甲醯基轉移酶 (OTC) 組成。首先我們嘗試將OTC的C端與另一種蛋白,MSG的N端結合。使用冷凍低溫電子顯微鏡收集與分析數據後,OTC的機構可達近似原子級的解析度,但MSG的解析度仍非常低。儘管如此,OTC可望成為具有發展性的支架蛋白。接著,我們將OTC與泛素接在一起,形成OTC-Ub scaf-fold。UCHL1為一25 kD的蛋白,能與泛素結合。我們利用此結合性質,使用cryo-EM重建出OTC-Ub與UCHL1的復合物模型,其中泛素羧基末端水解酶-1 (UCHL1)部分的區塊可清楚被看見,並達到6.5 Å 解析度。因此,此研究提供了一種新支架蛋白來助於觀察小於30 kD蛋白的結構。zh_TW
dc.description.abstractThe recent resolution revolution in cryogenic-electron microscopy (cryo-EM) made it an increasingly powerful tool in structural biology. The revolution came with advances in the microscope optics, the direct electron detectors, and the analytical software. With the cur-rent pipeline, protein structures could be reconstructed to atomic resolutions through a se-ries of cryo-EM micrographs. This technique is known as cryo-EM single-particle recon-struction (SPR). Although capable of detecting macromolecular assemblies and their con-formational changes, there remained a resolution limit for samples with molecular weight below 100 kD. To overcome the size limit, scaffold proteins were developed to study sub-100 kD proteins by cryo-EM. Scaffold proteins are generally symmetrical homo-oligomeric proteins that include multiple fusion sites to attach covalently or non-covalently to the target proteins. We designed a scaffold system based on a homo-trimeric ornithine transcarbamylase (OTC). OTC was genetically fused at its C-terminus to an 80 kD target protein, malate synthase G (MSG). The scaffold protein, OTC, was visualized at near-atomic resolution, while MSG showed a diffused density. Nonetheless, the refined OTC density map showed its potential to serve as the scaffold for other proteins. We next fused OTC to ubiquitin to build an OTC-Ub fusion scaffold to visualize Ub-binding protein, such as ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1). UCHL1 is a 25 kD protein. The cryo-EM map showed clearly defined regions that could precisely house Ubiquitin and UCHL1. The currently attainable local resolution of UCHL1 was 6.5 Å, still larger than the required resolution for de novo model building. However, our results demonstrate the potential of OTC-Ub to aid visualization of the atomic structures of small (< 30 kD) ubiquitin-binding proteins.
a 26 kD protein indicated OTC-Ub may serve as a imaging scaffold for other ubiquitin-binding proteins.
en
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Previous issue date: 2020
en
dc.description.tableofcontents摘要: i
Abstract: ii
Abbreviations: iv
1. Introduction 1
1.1. Cryo-EM: an alternative approach to structure biology 1
1.1.1. Cryo-EM in comparison to NMR and X-ray crystallography 1
1.1.2. Sample preparation, imaging, and data analysis 2
1.1.3. Applications of cryo-EM on small proteins 3
1.2. Scaffolds: proteins assisting in imaging of small proteins 4
1.2.1. Literature review of Scaffold protein designs 4
1.2.2. Imaging scaffold of our choice: ornithine transcarbamylase 9
1.3. Cargo proteins: the small proteins selected to be imaged 9
1.3.1. Covalently linked cargo protein: malate synthase G 9
1.3.2. Noncovalently-bound cargo protein: ubiquitin-recognizing protein 10
1.4. Future directions: to determine atomic structure of another UCH of unknown structure 11
1.4.1. A scaffold for other deubiquitinating enzymes: BAP1 11
1.5. Biophysical tools: to aid cryo-EM analysis 12
1.5.1. Small-angle X-ray scattering (SAXS) 12
1.5.2. Size-exclusion chromatography-coupled multi-angle static light scattering (SEC-MALS) 13
1.5.3. A chemical crosslinker was used reduce conformational heterogeneity 14
1.6. Aims of this study 14
2. Material and Methods 16
2.1. Cloning 16
2.1.1. OTC-MSG 16
2.1.2. OTC-Ub 18
2.1.3. UCHL1 21
2.2. Protein purification 22
2.2.1. OTC scaffolds 22
2.2.2. UCHL1 23
2.3. SAXS analysis 23
2.4. BS3 crosslinking 24
2.5. EM sample preparation and data collection 25
2.6. Image processing 29
3. Results 31
3.1. Analysis of OTC-MSG 31
3.1.1. Protein purification and linker truncation to increase overall rigidity 31
3.1.2. SEC-SAXS analysis 33
3.1.3. Ab initio modeling through DAMMIF 34
3.1.4. CryoEM: data collection and analysis of OTC-MSG 34
3.1.5. Reduction of linker length reduces interdomain flexibility 38
3.2. Analysis of OTC-Ub: UCHL1 with cryo-EM and biophysical tools 40
3.2.1. Protein purification and linker truncation 40
3.2.2. Confirming binding of OTC Δ1 to UCHL1 41
3.2.3. SAXS data of OTCΔ1Ub:UCHL1 41
3.2.4. Building ab initio model with DAMMIF from SAXS data 43
3.2.5. Cryo-EM analysis of OTC-Ub:UCHL1 complex 44
3.2.6. Crosslink UCHL1 to OTCUbΔ1with BS3 to avoid dissociation 47
3.2.7. CryoEM analysis of OTC-Ub:UCHL1 crosslinked with BS3 48
3.2.8. Adding Tween 20 to overcome preferred orientation issue of BS3-treated OTCΔ1-Ub:UCHL1 50
3.2.9. Optimization of the conditions for BS3 crosslinking of OTCΔ1-Ub:UCHL1 52
3.2.10. Cryo-EM data analysis of OTC-Ub:UCHL1 crosslinked with excess BS3 54
4. Discussion 63
5. References 71
dc.language.isoen
dc.subject冷凍低溫電子顯微鏡zh_TW
dc.subject支架蛋白zh_TW
dc.subject鳥胺酸氨甲醯基轉移酶zh_TW
dc.subject泛素zh_TW
dc.subject泛素羧基末端水解酶-1zh_TW
dc.subjectornithine transcarbamylaseen
dc.subjectscaffold proteinen
dc.subjectubiquitinen
dc.subjectubiquitin carboxyl-terminal hydrolase isozyme L1en
dc.subjectcryogenic electron microscopyen
dc.title開發新型支架蛋白協助冷凍電子顯微鏡分析低分子量蛋白結構zh_TW
dc.titleImaging small proteins with a novel scaffold protein for cryo-EM structural analysisen
dc.typeThesis
dc.date.schoolyear108-2
dc.description.degree碩士
dc.contributor.oralexamcommittee吳昆峯(Kuen-Phon Wu),蘇士哲(Shih-Che Sue)
dc.subject.keyword冷凍低溫電子顯微鏡,支架蛋白,鳥胺酸氨甲醯基轉移酶,泛素,泛素羧基末端水解酶-1,zh_TW
dc.subject.keywordcryogenic electron microscopy,scaffold protein,ornithine transcarbamylase,ubiquitin,ubiquitin carboxyl-terminal hydrolase isozyme L1,en
dc.relation.page73
dc.identifier.doi10.6342/NTU202003123
dc.rights.note未授權
dc.date.accepted2020-08-18
dc.contributor.author-college生命科學院zh_TW
dc.contributor.author-dept生化科學研究所zh_TW
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