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Instruments / Technical Area	Publication Details	Published
Krios I-Titan Krios I at Diamond M01-Polara at OPIC (Oxford)	The aggregation of alpha-synuclein and its disaggregation by the Hsp70 disaggregation machinery Jim Emilien Jacques Monistrol Show Abstract ▼ Abstract: Neurodegenerative disorders are characterised by the deposition of proteinaceous aggregates in the brain. In the case of synucleinopathies, such as Parkinson’s disease, the aggregates are mainly composed of α-synuclein (αSyn). The aggregation process of αSyn is toxic for neurons but the cause of the toxicity remains elusive. In vitro, the constitutively expressed cytosolic heat shock 70 protein, Hsc70, together with two cochaperones, the J-domain protein DNAJB1 and the nucleotide exchange factor Apg2, can disassemble αSyn amyloid fibres and small oligomers. However, the model is incomplete, lacking structural as well as mechanistic details. To address these two issues (αSyn aggregation in cells and the incomplete model of αSyn disaggregation in vitro), I have used electron and fluorescence microscopy and biochemical assays. Correlating fluorescence images and cryo tomograms, I have investigated the structure of αSyn aggregates in a yeast model. αSyn colocalises at the plasma membrane and forms cytosolic inclusions rich in vesicles and lipid droplets. Using cryo electron microscopy (cryo-EM) and tomography, I have shown that recombinant αSyn does not form large oligomers on the surface of tubulated liposomes. Using single particle cryo-EM, I have studied the complex formed of αSyn amyloid fibres and a mutant of DNAJB1 lacking the J-domain and G/F linker (ΔJ-DNAJB1). A low-resolution reconstruction of the complex was generated in which horseshoe-like densities, identified as ΔJ-DNAJB1, surrounded the fibre. I also produced a high-resolution map of the αSyn fibre showing a new conformation. Lastly, using cryo-EM, fluorescence microscopy and biochemical assays, I have investigated the function of Apg2 in disaggregation. Unexpectedly, Apg2 enhances Hsc70 recruitment to the fibres, preferentially at one end. This Apg2-induced recruitment regulates the disaggregation activity. The work in this thesis provides new information about αSyn aggregation, its toxicity and its disaggregation by the Hsc70 system.	Jul 2022
Krios I-Titan Krios I at Diamond	Cooperative amyloid fibre binding and disassembly by the Hsp70 disaggregase Joseph G. Beton , Jim Monistrol , Anne Wentink , Erin C. Johnston , Anthony J. Roberts , Bernd G. Bukau , Bart W. Hoogenboom , Helen R. Saibil The Embo Journal 9 DOI: 10.15252/embj.2021110410 Diamond Proposal Number(s): [20287] Open Access Show Abstract ▼ Abstract: Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of α-synuclein, huntingtin, or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70 family member, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Clustering of Hsc70 on the fibrils appears to be necessary for disassembly. Here we use atomic force microscopy to show that segments of in vitro assembled α-synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo-electron tomography and total internal reflection fluorescence microscopy reveal fibrils with regions of densely bound chaperones, preferentially at one end of the fibre. Sub-stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of ~ 4 subunits per second. The observed unidirectional bursts of Hsc70 loading and unravelling may be explained by differences between the two ends of the polar fibre structure.	Jun 2022
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Drosophila OTK is a glycosaminoglycan-binding protein with high conformational flexibility Daniel Rozbesky , Jim Monistrol , Vitul Jain , James Hillier , Sergi Padilla-Parra , E. Yvonne Jones Structure DOI: 10.1016/j.str.2020.02.008 Diamond Proposal Number(s): [14744] Open Access Show Abstract ▼ Abstract: The transmembrane protein OTK plays an essential role in plexin and Wnt signaling during Drosophila development. We have determined a crystal structure of the last three domains of the OTK ectodomain and found that OTK shows high conformational flexibility resulting from mobility at the interdomain interfaces. We failed to detect direct binding between Drosophila Plexin A (PlexA) and OTK, which was suggested previously. We found that, instead of PlexA, OTK directly binds semaphorin 1a. Our binding analyses further revealed that glycosaminoglycans, heparin and heparan sulfate, are ligands for OTK and thus may play a role in the Sema1a-PlexA axon guidance system.	Mar 2020

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