The chaperonin GroEL assists the folding of nascent or stress-denatured polypeptides by actions of binding and encapsulation. ATP binding initiates a series of conformational changes triggering the ...association of the cochaperonin GroES, followed by further large movements that eject the substrate polypeptide from hydrophobic binding sites into a GroES-capped, hydrophilic folding chamber. We used cryo-electron microscopy, statistical analysis, and flexible fitting to resolve a set of distinct GroEL-ATP conformations that can be ordered into a trajectory of domain rotation and elevation. The initial conformations are likely to be the ones that capture polypeptide substrate. Then the binding domains extend radially to separate from each other but maintain their binding surfaces facing the cavity, potentially exerting mechanical force upon kinetically trapped, misfolded substrates. The extended conformation also provides a potential docking site for GroES, to trigger the final, 100° domain rotation constituting the “power stroke” that ejects substrate into the folding chamber.
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► Single-particle cryo-EM identifies seven ATP-bound structures of GroEL chaperonin ► Analysis of structures suggests trajectory of domain movements triggered by ATP binding ► The structures delineate a mechanism for mechanical unfolding of polypeptide substrate ► Model suggests an intermediate conformation binding substrate and GroES simultaneously
Cryo-EM analysis reveals a series of conformational steps through which GroEL binds polypeptide substrates, stretches and unfolds them, and then moves them into a folding chamber that promotes adoption of the native state.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In this paper, we analyze two mainstream children's films, Storks (2016), and The Boss Baby (2017), both of which answer the proverbial question, "Where do babies come from?" with "They come from ...corporate factories above the clouds." We argue that these films bypass the gendered labor of reproduction, forwarding instead a politics of neoliberal multiculturalism in which the entrepreneurial spirit of masculinized reproductive industries appears to facilitate a post-racial utopia. These films tacitly reinstate traditional familial and heteronormative whiteness as the path toward the intertwined goals of capital accumulation and happiness. As such, they support the socialization of children into a policy landscape that increasingly devalues the physical, emotional, and economic labor of gestation, birth, and parenting, which in turn contributes to the conditions for disinvestment in the structures and policies that would ensure reproductive rights, health, and justice for marginalized groups.
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BFBNIB, NUK, PILJ, SAZU, UL, UM, UPUK
Chaperonins are intricate allosteric machines formed of two back-to-back, stacked rings of subunits presenting end cavities lined with hydrophobic binding sites for nonnative polypeptides. Once ...bound, substrates are subjected to forceful, concerted movements that result in their ejection from the binding surface and simultaneous encapsulation inside a hydrophilic chamber that favors their folding. Here, we review the allosteric machine movements that are choreographed by ATP binding, which triggers concerted tilting and twisting of subunit domains. These movements distort the ring of hydrophobic binding sites and split it apart, potentially unfolding the multiply bound substrate. Then, GroES binding is accompanied by a 100° twist of the binding domains that removes the hydrophobic sites from the cavity lining and forms the folding chamber. ATP hydrolysis is not needed for a single round of binding and encapsulation but is necessary to allow the next round of ATP binding in the opposite ring. It is this remote ATP binding that triggers dismantling of the folding chamber and release of the encapsulated substrate, whether folded or not.
The basis for these ordered actions is an elegant system of nested cooperativity of the ATPase machinery. ATP binds to a ring with positive cooperativity, and movements of the interlinked subunit domains are concerted. In contrast, there is negative cooperativity between the rings, so that they act in alternation. It is remarkable that a process as specific as protein folding can be guided by the chaperonin machine in a way largely independent of substrate protein structure or sequence.
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► The chaperonin GroEL provides essential assistance to cellular protein folding. ► Here, we review its allosteric mechanism of action. ► Coordinated subunit movements are driven by ATPase cycles. ► We describe the structural basis of positive intra-ring and negative inter-ring cooperativity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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•Most sub-100 kDa membrane proteins solved by cryo-EM are encapsulated in detergent.•DDM is the most popular detergent for small membrane proteins.•Carbon on gold grids are the most ...popular for sub- 100 kDa membrane proteins.•Many different approaches are taken for data processing.
Membrane proteins (MPs) are essential components of all biological membranes, contributing to key cellular functions that include signalling, molecular transport and energy metabolism. Consequently, MPs are important biomedical targets for therapeutics discovery. Despite hardware and software developments in cryo-electron microscopy, as well as MP sample preparation, MPs smaller than 100 kDa remain difficult to study structurally. Significant investment is required to overcome low levels of naturally abundant protein, MP hydrophobicity as well as conformational and compositional instability. Here we have reviewed the sample preparation approaches that have been taken to successfully express, purify and prepare small MPs for analysis by cryo-EM (those with a total solved molecular weight of under 100 kDa), as well as examining the differing approaches towards data processing and ultimately obtaining a structural solution. We highlight common challenges at each stage in the process as well as strategies that have been developed to overcome these issues. Finally, we discuss future directions and opportunities for the study of sub-100 kDa membrane proteins by cryo-EM.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable ...chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale—including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy—we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
As the resolutions of Three Dimensional Electron Microscopic reconstructions of biological macromolecules are being improved, there is a need for better fitting and refinement methods at high ...resolutions and robust approaches for model assessment. Flex-EM/MODELLER has been used for flexible fitting of atomic models in intermediate-to-low resolution density maps of different biological systems. Here, we demonstrate the suitability of the method to successfully refine structures at higher resolutions (2.5–4.5Å) using both simulated and experimental data, including a newly processed map of Apo-GroEL. A hierarchical refinement protocol was adopted where the rigid body definitions are relaxed and atom displacement steps are reduced progressively at successive stages of refinement. For the assessment of local fit, we used the SMOC (segment-based Manders’ overlap coefficient) score, while the model quality was checked using the Qmean score. Comparison of SMOC profiles at different stages of refinement helped in detecting regions that are poorly fitted. We also show how initial model errors can have significant impact on the goodness-of-fit. Finally, we discuss the implementation of Flex-EM in the CCP-EM software suite.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the ...human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (K
of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
In the aftermath of Dobbs v. Jackson Women's Health Organization, which overturned the federal constitutional right to abortion, states have begun to recriminalize the procedure. These abortion bans ...raise important questions about the political and social status of women and pregnant people in the United States. Moreover, restrictions in social welfare programs such as the Special Supplemental Nutrition Assistance Program for Women, Infants, and Children and Temporary Assistance for Needy Families, which serve low-income pregnant people and parents, raise similar questions. The regulation and administration of all three are framed by race, class, and gender. To understand how these restrictions (a) claim to protect women but ultimately function to control, police, and surveil and (b) rely on imagined, stereotype-laden psychological states such as vulnerability, irresponsibility, or irrationality, we turn to the British Common Law doctrine of coverture, which subsumed a married woman's legal, financial, and political identities under her husband's. The American colonies, and later, states of the United States, drew from British Common Law to craft laws that regulated relationships between men and women. Taken together, this analysis can provide a more comprehensive accounting of the cumulative harms experienced by women, poor people, people of color, and pregnant people in today's health and social welfare landscape. We conclude with recommendations for psychologists and other mental health providers to address, in practice and advocacy, the ethical dilemmas and obligations raised by the reach of coverture's logics in people's lives.
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NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. ...Here, we directly visualise an unsuspected 'single shelled' intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is 'collapsed' compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses.
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