Summary L eucine R ich R epeat K inase 2 ( LRRK2 ) is the most commonly mutated gene in familial Parkinson’s disease (PD) 1 and is also linked to its idiopathic form 2 . LRRK2 is proposed to function in membrane trafficking 3 and co-localizes with microtubules 4 . Despite LRRK2’s fundamental importance for understanding and treating PD, there is limited structural information on it. Here we report the 3.5Å structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported 14Å cryo-electron tomography in situ structure 5 . We propose that the conformation of LRRK2’s kinase domain regulates its microtubule interaction, with a closed conformation favoring oligomerization on microtubules. We show that the catalytic half of LRRK2 is sufficient for filament formation and blocks the motility of the microtubule-based motors kinesin-1 and cytoplasmic dynein-1 in vitro . Kinase inhibitors that stabilize an open conformation relieve this interference and reduce LRRK2 filament formation in cells, while those that stabilize a closed conformation do not. Our findings suggest that LRRK2 can act as a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.

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Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using in situ cryo-electron tomography and subtomogram averaging, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double-helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase points towards the microtubule, while the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to obtain structures of previously unsolved proteins in their cellular environment and provides insights into LRRK2 function and pathogenicity.

Leucine Rich Repeat Kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinson's disease. LRRK2 is proposed to function in membrane trafficking and co-localizes with microtubules. We report the 3.5Å structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported 14Å cryo-electron tomography in situ structure. We propose that the conformation of LRRK2's kinase domain regulates its microtubule interaction, with a closed conformation favoring binding. We show that the catalytic half of LRRK2 is sufficient for microtubule binding and blocks the motility of the microtubule-based motors kinesin and dynein in vitro. Kinase inhibitors that stabilize an open conformation relieve this interference and reduce LRRK2 filament formation in cells, while those that stabilize a closed conformation do not. Our findings suggest that LRRK2 is a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.

Despite tremendous advances in sample preparation and classification algorithms for electron cryomicroscopy (cryo-EM) and single-particle analysis (SPA), sample heterogeneity remains a major challenge and can prevent access to high-resolution structures. In addition, optimization of preparation conditions for a given sample can be time consuming. In the current work, it is demonstrated that native electrospray ion-beam deposition (native ES-IBD) is an alternative, reliable approach for preparation of extremely high-purity samples, based on mass selection in vacuum. Folded protein ions are generated by native electrospray ionization, separated from other proteins, contaminants, aggregates, and fragments, gently deposited on cryo-EM grids, frozen in liquid nitrogen, and subsequently imaged by cryo-EM. We demonstrate homogeneous coverage of ice-free cryo-EM grids with mass-selected protein complexes. SPA reveals that the complexes remain folded and assembled, but variations in secondary and tertiary structure are currently limiting information in 2D classes and 3D EM density maps. We identify and discuss challenges that need to be addressed to obtain a resolution comparable to that of the established cryo-EM workflow. Our results show the potential of native ES-IBD to increase the scope and throughput of cryo-EM for protein structure determination and provide an essential link between gas phase and solution phase protein structures.

In situ structural biology aims to resolve macromolecular structures and collective behaviors of macromolecular machineries within the cell. In bacteria, transcription mediated by RNA polymerase is known to be functionally coupled to the leading ribosome, a process crucial for efficient gene expression. Direct ribosome-RNA polymerase interaction has been demonstrated in vitro, but how these two gigantic molecular machines coordinate is yet unclear, especially within the context of crowded and complex cellular environments. Using Mycoplasma pneumonia as model system, we integrated cellular cryo-electron tomography followed by sub-tomogram analysis (STA) and in-cell crosslinking mass spectrometry (CLMS) to investigate the coupling in native cells, without resorting to labelling or cell disruption. By averaging sub-tomograms extracted in silico from cellular volumes, we solved the 70S ribosome structure at 6.4 A resolution, so far the highest resolution reported for in-cell cryo-ET. After large-scale classification, we determined the structure of a super-complex consisting of ribosome, RNA polymerase and other auxiliary proteins. Proteinprotein interaction networks revealed by in-cell CLMS indicated an essential transcription elongation factor bridging RNA polymerase and the translating ribosome. Integrative modelling suggests a novel, indirect transcriptiontranslation coupling mechanism, which advances our understanding of key machineries of the central dogma of molecular biology in bacteria. Methodology wise, our work demonstrates the feasibility and superiority of integrative structural biology performed directly inside cells. Integration of two approaches, cryo-ET/STA and in-cell CLMS, holds great potential to delineate cellular processes in a quantitative and structural view.

Many bacteria in nature exist in multicellular communities termed biofilms, where cells are embedded in an extracellular matrix that provides rigidity to the biofilm and protects cells from chemical and mechanical stresses. In the Gram-positive model bacterium Bacillus subtilis, TasA is the major protein component of the biofilm matrix, where it has been reported to form functional amyloid fibres contributing to biofilm structure and stability. Here, we present electron cryomicroscopy structures of TasA fibres, which show that, rather than forming amyloid fibrils, TasA monomers assemble into fibres through donor-strand exchange, with each subunit donating a β-strand to complete the fold of the next subunit along the fibre. Combining electron cryotomography, atomic force microscopy, and mutational studies, we show how TasA fibres congregate in three dimensions to form abundant fibre bundles that are essential for B. subtilis biofilm formation. Our study explains the previously observed biochemical properties of TasA and shows how a bacterial extracellular globular protein can assemble from monomers into β-sheet-rich fibres, and how such fibres assemble into bundles in biofilms.

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