In situ cryo-ET dataset of S. cerevisiae prepared using cryo-plasmaFIB milling
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ABSTRACT:
SUBMITTER: Sagar Khavnekar
PROVIDER: EMPIAR-11658 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:We present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5?µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.
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Project description:Studying bacterial cell envelope architecture with electron microscopy is challenging due to the poor preservation of microbial ultrastructure with traditional methods. Here, we established and validated a super-resolution cryo-correlative light and electron microscopy (cryo-CLEM) method, and combined it with cryo-focused ion beam (cryo-FIB) milling and scanning electron microscopy (SEM) volume imaging to structurally characterize the bacterium Deinococcus radiodurans. Subsequent cryo-electron tomography (cryo-ET) revealed an unusual diderm cell envelope architecture with a thick layer of peptidoglycan (PG) between the inner and outer membranes, an additional periplasmic layer, and a proteinaceous surface S-layer. Cells grew in tetrads, and division septa were formed by invagination of the inner membrane (IM), followed by a thick layer of PG. Cytoskeletal filaments, FtsA and FtsZ, were observed at the leading edges of constricting septa. Numerous macromolecular complexes were found associated with the cytoplasmic side of the IM. Altogether, our study revealed several unique ultrastructural features of D. radiodurans cells, opening new lines of investigation into the physiology and evolution of the bacterium.
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Project description:Phycobilisome (PBS) is the main light-harvesting antenna in cyanobacteria and red algae. How PBS transfers the light energy to photosystem II (PSII) remains to be elucidated. Here we report the in situ structure of the PBS-PSII supercomplex from Porphyridium purpureum UTEX 2757 using cryo-electron tomography and subtomogram averaging. Our work reveals the organized network of hemiellipsoidal PBS with PSII on the thylakoid membrane in the native cellular environment. In the PBS-PSII supercomplex, each PBS interacts with six PSII monomers, of which four directly bind to the PBS, and two bind indirectly. Additional three 'connector' proteins also contribute to the connections between PBS and PSIIs. Two PsbO subunits from adjacent PSII dimers bind with each other, which may promote stabilization of the PBS-PSII supercomplex. By analyzing the interaction interface between PBS and PSII, we reveal that αLCM and ApcD connect with CP43 of PSII monomer and that αLCM also interacts with CP47' of the neighboring PSII monomer, suggesting the multiple light energy delivery pathways. The in situ structures illustrate the coupling pattern of PBS and PSII and the arrangement of the PBS-PSII supercomplex on the thylakoid, providing the near-native 3D structural information of the various energy transfer from PBS to PSII.
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Project description:Recent advances have made cryogenic (cryo) electron microscopy a key technique to achieve near-atomic-resolution structures of biochemically isolated macromolecular complexes. Cryo-electron tomography (cryo-ET) can give unprecedented insight into these complexes in the context of their natural environment. However, the application of cryo-ET is limited to samples that are thinner than most cells, thereby considerably reducing its applicability. Cryo-focused-ion-beam (cryo-FIB) milling has been used to carve (micromachining) out 100-250-nm-thin regions (called lamella) in the intact frozen cells. This procedure opens a window into the cells for high-resolution cryo-ET and structure determination of biomolecules in their native environment. Further combination with fluorescence microscopy allows users to determine cells or regions of interest for the targeted fabrication of lamellae and cryo-ET imaging. Here, we describe how to prepare lamellae using a microscope equipped with both FIB and scanning electron microscopy modalities. Such microscopes (Aquilos Cryo-FIB/Scios/Helios or CrossBeam) are routinely referred to as dual-beam microscopes, and they are equipped with a cryo-stage for all operations in cryogenic conditions. The basic principle of the described methodologies is also applicable for other types of dual-beam microscopes equipped with a cryo-stage. We also briefly describe how to integrate fluorescence microscopy data for targeted milling and critical considerations for cryo-ET data acquisition of the lamellae. Users familiar with cryo-electron microscopy who get basic training in dual-beam microscopy can complete the protocol within 2-3 d, allowing for several pause points during the procedure.
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Project description:The spike protein of the porcine epidemic diarrhea virus from various strains was generated from different mammalian expression systems. The integration of cryo-EM and MS delineated the glycosylation patterns on Spike proteins and also helped understand how individual glycans contribute to biological functions.
2022-10-15 | PXD035778 | Pride
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| EMPIAR-11718 | biostudies-other
Project description:The interior of eukaryotic cells is mysterious. How do the large communities of macromolecular machines interact with each other? How do the structures and positions of these nanoscopic entities respond to new stimuli? Questions like these can now be answered with the help of a method called electron cryotomography (cryo-ET). Cryo-ET will ultimately reveal the inner workings of a cell at the protein, secondary structure, and perhaps even side-chain levels. Combined with genetic or pharmacological perturbation, cryo-ET will allow us to answer previously unimaginable questions, such as how structure, biochemistry, and forces are related in situ. Because it bridges structural biology and cell biology, cryo-ET is indispensable for structural cell biology-the study of the 3-D macromolecular structure of cells. Here we discuss some of the key ideas, strategies, auxiliary techniques, and innovations that an aspiring structural cell biologist will consider when planning to ask bold questions.
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