Cryo-electron Tomography of Microtubules Assembled In Vitro from Purified Components

Rapid increase of intracellular synthesis of specific histone-like Dps protein that binds DNA to protect the genome against deleterious factors leads to in cellulo crystallization—one of the most curious processes in the area of life science at the moment. However, the actual structure of the Dps–DNA co-crystals remained uncertain in the details for more than two decades. Cryo-electron tomography and small-angle X-ray scattering revealed polymorphous modifications of the co-crystals depending on the buffer parameters. Two different types of the Dps–DNA co-crystals are formed in vitro: triclinic and cubic. Three-dimensional reconstruction revealed DNA and Dps molecules in cubic co-crystals, and the unit cell parameters of cubic lattice were determined consistently by both methods.

Download Full-text

Protective Dps–DNA co‐crystallization in stressed cells: an in vitro structural study by small‐angle X‐ray scattering and cryo‐electron tomography

FEBS Letters ◽

10.1002/1873-3468.13439 ◽

2019 ◽

Vol 593 (12) ◽

pp. 1360-1371 ◽

Cited By ~ 7

Author(s):

Liubov A. Dadinova ◽

Yurii M. Chesnokov ◽

Roman A. Kamyshinsky ◽

Ivan A. Orlov ◽

Maxim V. Petoukhov ◽

...

Keyword(s):

Structural Study ◽

Small Angle ◽

Electron Tomography ◽

X Ray ◽

X Ray Scattering ◽

Cryo Electron Tomography ◽

Stressed Cells ◽

Ray Scattering

Download Full-text

PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility

Molecular Biology of the Cell ◽

10.1091/mbc.e19-01-0063 ◽

2019 ◽

Vol 30 (15) ◽

pp. 1805-1816 ◽

Cited By ~ 14

Author(s):

Erin E. Dymek ◽

Jianfeng Lin ◽

Gang Fu ◽

Mary E. Porter ◽

Daniela Nicastro ◽

...

Keyword(s):

Cell Motility ◽

Electron Tomography ◽

Structural Studies ◽

Ciliary Beating ◽

Ciliary Motility ◽

Functional Studies ◽

Cryo Electron Tomography ◽

Motile Cilia

We previously demonstrated that PACRG plays a role in regulating dynein-driven microtubule sliding in motile cilia. To expand our understanding of the role of PACRG in ciliary assembly and motility, we used a combination of functional and structural studies, including newly identified Chlamydomonas pacrg mutants. Using cryo-electron tomography we show that PACRG and FAP20 form the inner junction between the A- and B-tubule along the length of all nine ciliary doublet microtubules. The lack of PACRG and FAP20 also results in reduced assembly of inner-arm dynein IDA b and the beak-MIP structures. In addition, our functional studies reveal that loss of PACRG and/or FAP20 causes severe cell motility defects and reduced in vitro microtubule sliding velocities. Interestingly, the addition of exogenous PACRG and/or FAP20 protein to isolated mutant axonemes restores microtubule sliding velocities, but not ciliary beating. Taken together, these studies show that PACRG and FAP20 comprise the inner junction bridge that serves as a hub for both directly modulating dynein-driven microtubule sliding, as well as for the assembly of additional ciliary components that play essential roles in generating coordinated ciliary beating.

Download Full-text

The structure of the COPI coat determined within the cell

eLife ◽

10.7554/elife.32493 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 60

Author(s):

Yury S Bykov ◽

Miroslava Schaffer ◽

Svetlana O Dodonova ◽

Sahradha Albert ◽

Jürgen M Plitzko ◽

...

Keyword(s):

Focused Ion Beam ◽

Structural Model ◽

Electron Tomography ◽

Ion Beam ◽

Membrane Thickness ◽

In Vitro Reconstitution ◽

Cryo Electron Tomography ◽

Subtomogram Averaging

COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β’–COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.

Download Full-text

Current data processing strategies for cryo-electron tomography and subtomogram averaging

Biochemical Journal ◽

10.1042/bcj20200715 ◽

2021 ◽

Vol 478 (10) ◽

pp. 1827-1845

Author(s):

Euan Pyle ◽

Giulia Zanetti

Keyword(s):

Data Processing ◽

Electron Tomography ◽

Signal To Noise Ratio ◽

Three Dimensional ◽

Current Data ◽

Processing Strategies ◽

Cryo Electron Tomography ◽

Subtomogram Averaging ◽

The Individual

Cryo-electron tomography (cryo-ET) can be used to reconstruct three-dimensional (3D) volumes, or tomograms, from a series of tilted two-dimensional images of biological objects in their near-native states in situ or in vitro. 3D subvolumes, or subtomograms, containing particles of interest can be extracted from tomograms, aligned, and averaged in a process called subtomogram averaging (STA). STA overcomes the low signal to noise ratio within the individual subtomograms to generate structures of the particle(s) of interest. In recent years, cryo-ET with STA has increasingly been capable of reaching subnanometer resolution due to improvements in microscope hardware and data processing strategies. There has also been an increase in the number and quality of software packages available to process cryo-ET data with STA. In this review, we describe and assess the data processing strategies available for cryo-ET data and highlight the recent software developments which have enabled the extraction of high-resolution information from cryo-ET datasets.

Download Full-text

In situ architecture of neuronal α-Synuclein inclusions

10.1101/2020.08.07.234138 ◽

2020 ◽

Author(s):

Victoria A. Trinkaus ◽

Irene Riera-Tur ◽

Antonio Martínez-Sánchez ◽

Felix J.B. Bäuerlein ◽

Qiang Guo ◽

...

Keyword(s):

Amyloid Fibrils ◽

Electron Tomography ◽

Structural Flexibility ◽

Multiple Systems ◽

Cryo Electron Tomography ◽

Multiple Systems Atrophy ◽

The Brain ◽

Cellular Organelles

Summaryα-Synuclein (α-Syn) aggregation is a hallmark of devastating neurodegenerative disorders including Parkinson’s disease (PD) and multiple systems atrophy (MSA)1,2. α-Syn aggregates spread throughout the brain during disease progression2, suggesting mechanisms of intercellular seeding. Formation of α-Syn amyloid fibrils is observed in vitro3,4 and fibrillar α-Syn has been purified from patient brains5,6, but recent reports questioned whether disease-relevant α-Syn aggregates are fibrillar in structure7-9. Here we use cryo-electron tomography (cryo-ET) to image neuronal Lewy body-like α-Syn inclusions in situ at molecular resolution. We show that the inclusions consist of α-Syn fibrils crisscrossing a variety of cellular organelles such as the endoplasmic reticulum (ER), mitochondria and autophagic structures, without interacting with membranes directly. Neuronal inclusions seeded by recombinant or MSA patient-derived α-Syn aggregates have overall similar architecture, although MSA-seeded fibrils show higher structural flexibility. Using gold-labeled seeds we find that aggregate nucleation is predominantly mediated by α-Syn oligomers, with fibrils growing unidirectionally from the seed. Our results conclusively demonstrate that neuronal α-Syn inclusions contain α-Syn fibrils intermixed with cellular membranes, and illuminate the mechanism of aggregate nucleation.

Download Full-text

Topological Insights into Mutant Huntingtin Exon 1 and PolyQ Aggregates by Cryo-Electron Tomography

10.1101/2020.12.08.417048 ◽

2020 ◽

Author(s):

Jesús G. Galaz-Montoya ◽

Sarah H. Shahmoradian ◽

Koning Shen ◽

Judith Frydman ◽

Wah Chiu

Keyword(s):

Trinucleotide Repeat ◽

Electron Tomography ◽

Mutant Huntingtin ◽

And Topology ◽

Cryo Electron Tomography ◽

Exon 1 ◽

Subtomogram Averaging ◽

The Brain ◽

Polyq Tract

ABSTRACTHuntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Yet, we observed some filaments in both types of aggregates under ∼2 nm in width, thinner than previously reported, while other regions form large sheets. In addition, our data show a prevalent subpopulation of filaments exhibiting a lumpy, slab-shaped morphology in both aggregates, supportive of the “polyQ core” model. This provides a basis for future cryoET studies of various aggregated mHTT and polyQ constructs to improve their structure-based modeling and their identification in cells without fusion tags.

Download Full-text

Cryo-electron Tomography of in vitro Translation Systems

10.1240/sav_gbm_2005_m_001003 ◽

2005 ◽

Vol 2005 (Spring) ◽

Author(s):

Karoline Bopp ◽

Markus Stemp ◽

Friedrich F�rster ◽

Julio Ortiz ◽

Vishwas Agashe ◽

...

Keyword(s):

Electron Tomography ◽

In Vitro Translation ◽

Cryo Electron Tomography ◽

Translation Systems

Download Full-text

CryoET structures of immature HIV Gag reveal six-helix bundle

Communications Biology ◽

10.1038/s42003-021-01999-1 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Luiza Mendonça ◽

Dapeng Sun ◽

Jiying Ning ◽

Jiwei Liu ◽

Abhay Kotecha ◽

...

Keyword(s):

Electron Tomography ◽

Single Amino Acid ◽

Helical Conformation ◽

Particle Assembly ◽

Virus Maturation ◽

Helix Bundle ◽

Cryo Electron Tomography ◽

Subtomogram Averaging ◽

Single Amino Acid Mutation

AbstractGag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. Gag is a polyprotein composed of MA (matrix), CA (capsid), SP1, NC (nucleocapsid), SP2 and p6 domains. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. However, the structure of the complete six-helix bundle has been elusive. Here, we determined the structures of both Gag in vitro assemblies and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions using cryo-electron tomography and subtomogram averaging by emClarity. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain. These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and thus block HIV-1 infection.

Download Full-text