scholarly journals A new-to-nature carboxylation module to improve natural and synthetic CO2 fixation

2021 ◽  
Author(s):  
Marieke Scheffen ◽  
Daniel G. Marchal ◽  
Thomas Beneyton ◽  
Sandra K. Schuller ◽  
Melanie Klose ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Active Site ◽  
Energy Demand ◽  
Rational Design ◽  
Co2 Fixation ◽  
Solution Space ◽  
Catalytic Efficiency ◽  
Atomic Resolution ◽  
Carbon Efficiency ◽  
Cryo Electron Microscopy

AbstractThe capture of CO2 by carboxylases is key to sustainable biocatalysis and a carbon-neutral bio-economy, yet currently limited to few naturally existing enzymes. Here, we developed glycolyl-CoA carboxylase (GCC), a new-to-nature enzyme, by combining rational design, high-throughput microfluidics and microplate screens. During this process, GCC’s catalytic efficiency improved by three orders of magnitude to match the properties of natural CO2-fixing enzymes. We verified our active-site redesign with an atomic-resolution, 1.96-Å cryo-electron microscopy structure and engineered two more enzymes that, together with GCC, form a carboxylation module for the conversion of glycolate (C2) to glycerate (C3). We demonstrate how this module can be interfaced with natural photorespiration, ethylene glycol conversion and synthetic CO2 fixation. Based on stoichiometrical calculations, GCC is predicted to increase the carbon efficiency of all of these processes by up to 150% while reducing their theoretical energy demand, showcasing how expanding the solution space of natural metabolism provides new opportunities for biotechnology and agriculture.

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 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 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 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 Cryo-EM Reveals the Structural Basis of Microtubule Depolymerization by Kinesin-13s

2017 ◽  
Author(s):  
Matthieu P. M. H. Benoit ◽  
Ana B. Asenjo ◽  
Hernando Sosa
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Distinct Group ◽  
Atomic Resolution ◽  
Structural Basis ◽  
Structural Elements ◽  
Microtubule Depolymerization ◽  
Cryo Electron Microscopy ◽  
Kinesin Superfamily ◽  
Motile Activity

SummaryKinesin-13s constitute a distinct group within the kinesin superfamily of motor proteins that promotes microtubule depolymerization and lacks motile activity. The molecular mechanism by which the kinesins depolymerize microtubules and are adapted to perform a seemingly very different activity from other kinesins is still unclear. To address this issue we obtained near atomic resolution cryo-electron microscopy (cryo-EM) structures of Drosophila melanogaster kinesin-13 KLP10A constructs bound to curved or straight tubulin in different nucleotide states. The structures show how nucleotide induced conformational changes near the catalytic site are coupled with kinesin-13-specific structural elements to induce tubulin curvature leading to microtubule depolymerization. The data highlight a modular structure that allows similar kinesin core motor-domains to be used for different functions, such as motility or microtubule depolymerization.

scholarly journals Cryo-EM structure of alpha-synuclein fibrils

2018 ◽  
Author(s):  
Ricardo Guerrero-Ferreira ◽  
Nicholas M. I. Taylor ◽  
Daniel Mona ◽  
Philippe Ringler ◽  
Matthias E. Lauer ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rational Design ◽  
Alpha Synuclein ◽  
Diagnosis And Treatment ◽  
Cryo Electron Microscopy ◽  
Cell Propagation ◽  
Hydrophobic Cleft

AbstractIntracellular inclusions of alpha-synuclein are the neuropathological hallmark of progressive disorders called synucleinopathies. Alpha-synuclein fibrils are associated with transmissive cell-to-cell propagation of pathology. We report the structure of an alpha-synuclein fibril (residues 1-121) determined by cryo-electron microscopy at 3.4Å resolution. Two protofilaments form a polar fibril composed of staggered β-strands. The backbone of residues 38 to 95, including the fibril core and the non-amyloid component region, are well resolved in the EM map. Residues 50-57, containing three mutation sites associated with familial synucleinopathies, form the interface between the two protofilaments and contribute to fibril stability. A hydrophobic cleft may have implications for fibril elongation, and inform the rational design of molecules for diagnosis and treatment of synucleinopathies.

scholarly journals An ensemble of cryo-EM structures of TRiC reveal its conformational landscape and subunit specificity

2019 ◽  
Vol 116 (39) ◽  
pp. 19513-19522 ◽  
Author(s):  
Mingliang Jin ◽  
Wenyu Han ◽  
Caixuan Liu ◽  
Yunxiang Zang ◽  
Jiawei Li ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ring Closure ◽  
Atomic Resolution ◽  
Protein Homeostasis ◽  
Structural Detail ◽  
Experimental Conditions ◽  
Closed State ◽  
Cryo Electron Microscopy ◽  
Conformational Landscape ◽  
Substrate Processing

TRiC/CCT assists the folding of ∼10% of cytosolic proteins through an ATP-driven conformational cycle and is essential in maintaining protein homeostasis. Here, we determined an ensemble of cryo-electron microscopy (cryo-EM) structures of yeast TRiC at various nucleotide concentrations, with 4 open-state maps resolved at near-atomic resolutions, and a closed-state map at atomic resolution, revealing an extra layer of an unforeseen N-terminal allosteric network. We found that, during TRiC ring closure, the CCT7 subunit moves first, responding to nucleotide binding; CCT4 is the last to bind ATP, serving as an ATP sensor; and CCT8 remains ADP-bound and is hardly involved in the ATPase-cycle in our experimental conditions; overall, yeast TRiC consumes nucleotide in a 2-ring positively coordinated manner. Our results depict a thorough picture of the TRiC conformational landscape and its allosteric transitions from the open to closed states in more structural detail and offer insights into TRiC subunit specificity in ATP consumption and ring closure, and potentially in substrate processing.

scholarly journals Dimeric structures of quinol-dependent nitric oxide reductases (qNORs) revealed by cryo–electron microscopy

2019 ◽  
Vol 5 (8) ◽  
pp. eaax1803 ◽  
Author(s):  
Chai C. Gopalasingam ◽  
Rachel M. Johnson ◽  
George N. Chiduza ◽  
Takehiko Tosha ◽  
Masaki Yamamoto ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Electron Microscopy ◽  
Cytochrome C ◽  
Active Site ◽  
Proton Transport ◽  
Water Channel ◽  
Water Molecules ◽  
Cryo Electron Microscopy ◽  
Proton Source ◽  
Key Residues

Quinol-dependent nitric oxide reductases (qNORs) are membrane-integrated, iron-containing enzymes of the denitrification pathway, which catalyze the reduction of nitric oxide (NO) to the major ozone destroying gas nitrous oxide (N2O). Cryo–electron microscopy structures of active qNOR from Alcaligenes xylosoxidans and an activity-enhancing mutant have been determined to be at local resolutions of 3.7 and 3.2 Å, respectively. They unexpectedly reveal a dimeric conformation (also confirmed for qNOR from Neisseria meningitidis) and define the active-site configuration, with a clear water channel from the cytoplasm. Structure-based mutagenesis has identified key residues involved in proton transport and substrate delivery to the active site of qNORs. The proton supply direction differs from cytochrome c–dependent NOR (cNOR), where water molecules from the cytoplasm serve as a proton source similar to those from cytochrome c oxidase.

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