RESUMEN
Visual proteomics attempts to build atlases of the molecular content of cells but the automated annotation of cryo electron tomograms remains challenging. Template matching (TM) and methods based on machine learning detect structural signatures of macromolecules. However, their applicability remains limited in terms of both the abundance and size of the molecular targets. Here we show that the performance of TM is greatly improved by using template-specific search parameter optimization and by including higher-resolution information. We establish a TM pipeline with systematically tuned parameters for the automated, objective and comprehensive identification of structures with confidence 10 to 100-fold above the noise level. We demonstrate high-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, fatty acid synthases, lipid membranes and microtubules, and individual subunits inside crowded eukaryotic cells. We provide software tools for the generic implementation of our method that is broadly applicable towards realizing visual proteomics.
Asunto(s)
Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Complejo de la Endopetidasa Proteasomal , Proteómica , Ribosomas , Programas Informáticos , Tomografía con Microscopio Electrónico/métodos , Microscopía por Crioelectrón/métodos , Ribosomas/ultraestructura , Ribosomas/metabolismo , Complejo de la Endopetidasa Proteasomal/ultraestructura , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Humanos , Proteómica/métodos , Poro Nuclear/ultraestructura , Poro Nuclear/metabolismo , Microtúbulos/ultraestructura , Microtúbulos/metabolismo , Ácido Graso Sintasas/metabolismo , Aprendizaje Automático , Imagenología Tridimensional/métodos , Algoritmos , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyze biomolecules in situ by subtomogram averaging, yet data quality critically depends on specimen thickness. Cells that are too thick for transmission imaging can be thinned into lamellae by cryo-focused ion beam (cryo-FIB) milling. Despite being a crucial parameter directly affecting attainable resolution, optimal lamella thickness has not been systematically investigated nor the extent of structural damage caused by gallium ions used for FIB milling. We thus systematically determined how resolution is affected by these parameters. We find that ion-induced damage does not affect regions more than 30 nanometers from either lamella surface and that up to ~180-nanometer lamella thickness does not negatively affect resolution. This shows that there is no need to generate very thin lamellae and lamella thickness can be chosen such that it captures cellular features of interest, thereby opening cryo-ET also for studies of large complexes.
Asunto(s)
Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Galio/químicaRESUMEN
Ribosomes catalyze protein synthesis by cycling through various functional states. These states have been extensively characterized in vitro, but their distribution in actively translating human cells remains elusive. We used a cryo-electron tomography-based approach and resolved ribosome structures inside human cells with high resolution. These structures revealed the distribution of functional states of the elongation cycle, a Z transfer RNA binding site, and the dynamics of ribosome expansion segments. Ribosome structures from cells treated with Homoharringtonine, a drug used against chronic myeloid leukemia, revealed how translation dynamics were altered in situ and resolve the small molecules within the active site of the ribosome. Thus, structural dynamics and drug effects can be assessed at high resolution within human cells.
Asunto(s)
Antineoplásicos , Neoplasias , Biosíntesis de Proteínas , Humanos , Antineoplásicos/farmacología , Sitios de Unión , Microscopía por Crioelectrón , Neoplasias/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Ribosomas/química , Ribosomas/metabolismo , ARN de Transferencia/metabolismoRESUMEN
Ribosomes translate genetic information into primary structure. During translation, various cofactors transiently bind to the ribosome that undergoes prominent conformational and structural changes. Different translational states of ribosomes have been well characterized in vitro. However, to which extent the known translational states are representative of the native situation inside cells has thus far only been addressed in prokaryotes. Here, we apply cryo-electron tomography to cryo-FIB milled Dictyostelium discoideum cells combined with subtomogram averaging and classification. We obtain an in situ structure that is locally resolved up to 3 Angstrom, the distribution of eukaryotic ribosome translational states, and unique arrangement of rRNA expansion segments. Our work demonstrates the use of in situ structural biology techniques for identifying distinct ribosome states within the cellular environment.