scholarly journals Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy

2015 ◽  
Vol 112 (43) ◽  
pp. 13237-13242 ◽  
Author(s):  
Lorenzo Sborgi ◽  
Francesco Ravotti ◽  
Venkata P. Dandey ◽  
Mathias S. Dick ◽  
Adam Mazur ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nmr Spectroscopy ◽  
Specific Binding ◽  
3D Structure ◽  
High Mobility ◽  
Atomic Resolution ◽  
Receptor Protein ◽  
Filament Formation ◽  
Cryo Electron Microscopy

Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.

scholarly journals Introduction to high-resolution cryo-electron microscopy

2016 ◽  
Vol 62 (3) ◽  
pp. 383-394
Author(s):  
Mariusz Czarnocki-Cieciura ◽  
Marcin Nowotny
Keyword(s):  
Electron Microscopy ◽  
Nmr Spectroscopy ◽  
Structural Biology ◽  
Atomic Resolution ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Electron Microscopes

For many years two techniques have dominated structural biology – X-ray crystallography and NMR spectroscopy. Traditional cryo-electron microscopy of biological macromolecules produced macromolecular reconstructions at resolution limited to 6–10 Å. Recent development of transmission electron microscopes, in particular the development of direct electron detectors, and continuous improvements in the available software, have led to the “resolution revolution” in cryo-EM. It is now possible to routinely obtain near-atomic-resolution 3D maps of intact biological macromolecules as small as ~100 kDa. Thus, cryo-EM is now becoming the method of choice for structural analysis of many complex assemblies that are unsuitable for structure determination by other methods.

scholarly journals A resolution record for cryoEM

2021 ◽  
Vol 10 ◽  
Author(s):  
Jonathan Ashmore ◽  
Bridget Carragher ◽  
Peter B Rosenthal ◽  
William Weis
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Structure Determination ◽  
Test Specimen ◽  
Structural Information ◽  
Atomic Resolution ◽  
Fast Growing ◽  
Cryo Electron Microscopy ◽  
New Instrument ◽  
Resolution Structure

Cryo electron microscopy (cryoEM) is a fast-growing technique for structure determination. Two recent papers report the first atomic resolution structure of a protein obtained by averaging images of frozen-hydrated biomolecules. They both describe maps of symmetric apoferritin assemblies, a common test specimen, in unprecedented detail. New instrument improvements, different in the two studies, have contributed better images, and image analysis can extract structural information sufficient to resolve individual atomic positions. While true atomic resolution maps will not be routine for most proteins, the studies suggest structures determined by cryoEM will continue to improve, increasing their impact on biology and medicine.

Forge Wednesday Roundup - June 10, 2020

2021 ◽  
Author(s):  
H.L. Cu Si
Keyword(s):  
Electron Microscopy ◽  
Facial Recognition ◽  
Academic Research ◽  
Atomic Resolution ◽  
Rapa Nui ◽  
Social Distancing ◽  
Cryo Electron Microscopy

In today’s Roundup: COVID is still with us; pair of papers quantifies effects of social distancing; IBM bails on facial recognition, warns of harms; questioning “collapse” of Rapa Nui; attention turns to antibodies for COVID-19 therapies; WHO walks back confusing statement on asymptomatic COVID transmission; cryo-electron microscopy reveals proteins at atomic resolution; unstoppable newsbot runs amok, propagates bias; exploring the lure of misinformation; academic research goes on strike against racism; much more...

scholarly journals Cryo-EM structure and polymorphism of Aβ amyloid fibrils purified from Alzheimer’s brain tissue

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Marius Kollmer ◽  
William Close ◽  
Leonie Funk ◽  
Jay Rasmussen ◽  
Aref Bsoul ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Brain Tissue ◽  
Amyloid Fibrils ◽  
Structural Basis ◽  
Amyloid Angiopathy ◽  
Cryo Electron Microscopy ◽  
Aβ Amyloid ◽  
Cerebral Amyloid ◽  
Aβ Fibrils

Abstract The formation of Aβ amyloid fibrils is a neuropathological hallmark of Alzheimer’s disease and cerebral amyloid angiopathy. However, the structure of Aβ amyloid fibrils from brain tissue is poorly understood. Here we report the purification of Aβ amyloid fibrils from meningeal Alzheimer’s brain tissue and their structural analysis with cryo-electron microscopy. We show that these fibrils are polymorphic but consist of similarly structured protofilaments. Brain derived Aβ amyloid fibrils are right-hand twisted and their peptide fold differs sharply from previously analyzed Aβ fibrils that were formed in vitro. These data underscore the importance to use patient-derived amyloid fibrils when investigating the structural basis of the disease.

scholarly journals Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display

2019 ◽  
Vol 5 (9) ◽  
pp. eaaw2853 ◽  
Author(s):  
Charles Vragniau ◽  
Joshua C. Bufton ◽  
Frédéric Garzoni ◽  
Emilie Stermann ◽  
Fruzsina Rabi ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Electron Microscopy ◽  
High Performance ◽  
Vaccine Candidate ◽  
Cost Effective ◽  
Atomic Resolution ◽  
Self Assembling ◽  
Cryo Electron Microscopy ◽  
Architectural Features ◽  
Multimeric Protein

Self-assembling virus-like particles represent highly attractive tools for developing next-generation vaccines and protein therapeutics. We created ADDomer, an adenovirus-derived multimeric protein-based self-assembling nanoparticle scaffold engineered to facilitate plug-and-play display of multiple immunogenic epitopes from pathogens. We used cryo–electron microscopy at near-atomic resolution and implemented novel, cost-effective, high-performance cloud computing to reveal architectural features in unprecedented detail. We analyzed ADDomer interaction with components of the immune system and developed a promising first-in-kind ADDomer-based vaccine candidate to combat emerging Chikungunya infectious disease, exemplifying the potential of our approach.

scholarly journals Cryo-electron Microscopy Structures of Novel Viruses from Mud Crab Scylla paramamosain with Multiple Infections

2019 ◽  
Vol 93 (7) ◽  
Author(s):  
Yuanzhu Gao ◽  
Shanshan Liu ◽  
Jiamiao Huang ◽  
Qianqian Wang ◽  
Kunpeng Li ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
3D Structure ◽  
Structural Features ◽  
Economic Losses ◽  
Scylla Paramamosain ◽  
Multiple Infections ◽  
Mud Crab ◽  
Cryo Electron Microscopy ◽  
Novel Virus ◽  
Icosahedral Capsid

ABSTRACT Viruses associated with sleeping disease (SD) in crabs cause great economic losses to aquaculture, and no effective measures are available for their prevention. In this study, to help develop novel antiviral strategies, single-particle cryo-electron microscopy was applied to investigate viruses associated with SD. The results not only revealed the structure of mud crab dicistrovirus (MCDV) but also identified a novel mud crab tombus-like virus (MCTV) not previously detected using molecular biology methods. The structure of MCDV at a 3.5-Å resolution reveals three major capsid proteins (VP1 to VP3) organized into a pseudo-T=3 icosahedral capsid, and affirms the existence of VP4. Unusually, MCDV VP3 contains a long C-terminal region and forms a novel protrusion that has not been observed in other dicistrovirus. Our results also reveal that MCDV can release its genome via conformation changes of the protrusions when viral mixtures are heated. The structure of MCTV at a 3.3-Å resolution reveals a T= 3 icosahedral capsid with common features of both tombusviruses and nodaviruses. Furthermore, MCTV has a novel hydrophobic tunnel beneath the 5-fold vertex and 30 dimeric protrusions composed of the P-domains of the capsid protein at the 2-fold axes that are exposed on the virion surface. The structural features of MCTV are consistent with a novel type of virus. IMPORTANCE Pathogen identification is vital for unknown infectious outbreaks, especially for dual or multiple infections. Sleeping disease (SD) in crabs causes great economic losses to aquaculture worldwide. Here we report the discovery and identification of a novel virus in mud crabs with multiple infections that was not previously detected by molecular, immune, or traditional electron microscopy (EM) methods. High-resolution structures of pathogenic viruses are essential for a molecular understanding and developing new disease prevention methods. The three-dimensional (3D) structure of the mud crab tombus-like virus (MCTV) and mud crab dicistrovirus (MCDV) determined in this study could assist the development of antiviral inhibitors. The identification of a novel virus in multiple infections previously missed using other methods demonstrates the usefulness of this strategy for investigating multiple infectious outbreaks, even in humans and other animals.

scholarly journals Atomic Resolution Structures of Human Bufaviruses Determined by Cryo-Electron Microscopy

Viruses ◽  
2018 ◽  
Vol 10 (1) ◽  
pp. 22 ◽  
Author(s):  
Maria Ilyas ◽  
Mario Mietzsch ◽  
Shweta Kailasan ◽  
Elina Väisänen ◽  
Mengxiao Luo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atomic Resolution ◽  
Cryo Electron Microscopy

scholarly journals Maturation of the Human Papillomavirus 16 Capsid

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Giovanni Cardone ◽  
Adam L. Moyer ◽  
Naiqian Cheng ◽  
Cynthia D. Thompson ◽  
Israel Dvoretzky ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Disulfide Bonds ◽  
Mammalian Cells ◽  
Structural Integrity ◽  
Regular Structure ◽  
Human Papillomaviruses ◽  
Cryo Electron Microscopy ◽  
Cross Links ◽  
Risk Of Cancer

ABSTRACTPapillomaviruses are a family of nonenveloped DNA viruses that infect the skin or mucosa of their vertebrate hosts. The viral life cycle is closely tied to the differentiation of infected keratinocytes. Papillomavirus virions are released into the environment through a process known as desquamation, in which keratinocytes lose structural integrity prior to being shed from the surface of the skin. During this process, virions are exposed to an increasingly oxidative environment, leading to their stabilization through the formation of disulfide cross-links between neighboring molecules of the major capsid protein, L1. We used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV16 capsids assembled in mammalian cells and exposed to an oxidizing environment after cell lysis. Initially, the virion is a loosely connected procapsid that, underin vitroconditions, condenses over several hours into the more familiar 60-nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by ~5% in diameter, its pentameric capsomers change in structure (most markedly in the axial region), and the interaction surfaces between adjacent capsomers are consolidated. A C175S mutant that cannot achieve normal inter-L1 disulfide cross-links shows maturation-related shrinkage but does not achieve the fully condensed 60-nm form. Pseudoatomic modeling based on a 9-Å resolution reconstruction of fully mature capsids revealed C-terminal disulfide-stabilized “suspended bridges” that form intercapsomeric cross-links. The data suggest a model in which procapsids exist in a range of dynamic intermediates that can be locked into increasingly mature configurations by disulfide cross-linking, possibly through a Brownian ratchet mechanism.IMPORTANCEHuman papillomaviruses (HPVs) cause nearly all cases of cervical cancer, a major fraction of cancers of the penis, vagina/vulva, anus, and tonsils, and genital and nongenital warts. HPV types associated with a high risk of cancer, such as HPV16, are generally transmitted via sexual contact. The nonenveloped virion of HPVs shows a high degree of stability, allowing the virus to persist in an infectious form in environmental fomites. In this study, we used cryo-electron microscopy to elucidate the structure of the HPV16 capsid at different stages of maturation. The fully mature capsid adopts a rigid, highly regular structure stabilized by intermolecular disulfide bonds. The availability of a pseudoatomic model of the fully mature HPV16 virion should help guide understanding of antibody responses elicited by HPV capsid-based vaccines.

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