Biology subjects

Hällberg, B. M.

Publications and source records attributed to Hällberg, B. M..

8 recordsLinked to original sources

bioRxiv2026

In-Chamber Sublimation: A Practical Approach for Mitigating Ice and Curtaining in Cryo-Electron Tomography Lamellae Preparation

Surface ice contamination is a persistent challenge in cryo-electron tomography (cryo-ET) workflows, where it can obscure regions of interest and contribute to curtaining artefacts during focused ion beam (FIB) milling. We demonstrate using high-pressure frozen yeast cells that a sublimation step within the scanning electron microscope (SEM) chamber before lamella milling visually removes surface ice and reduces sample roughness without detectable devitrification. While sublimation has been widely applied in cryo-SEM and volume imaging, it is not common on cryo-ET samples due to concerns about devitrification. Using tomographic reconstructions, we show that controlled sublimation improves lamella quality by reducing surface roughness and minimizing curtaining without compromising sample vitrification. Furthermore, subtomogram averaging of the 80S ribosome confirmed lamellae quality are preserved after sublimation. This approach offers a practical refinement to existing cryo-ET preparation protocols, requiring no additional instrumentation or workflow modifications.

cell biology↗

bioRxiv2024

Structural basis of 3'-tRNA maturation by the human mitochondrial RNAse Z complex

Human mitochondrial tRNA maturation is essential for cellular energy production, yet the underlying mechanisms remain only partially understood. Here, we present several cryo-EM structures of the mitochondrial RNase Z complex (ELAC2/SDR5C1/TRMT10C) bound to different maturation states of mitochondrial tRNAHis, showing the molecular basis for tRNA-substrate selection and catalysis. Our structural insights provide a molecular rationale for the 5'-to-3' tRNA processing order in mitochondria, the 3'-CCA antideterminant effect, and the basis for sequence-independent recognition of mitochondrial tRNA substrates. Furthermore, the study links mutations in ELAC2 to clinically relevant mitochondrial diseases, offering a deeper understanding of the molecular defects contributing to these conditions.

biochemistry↗

bioRxiv2024

Engineering multivalent Fc display for FcγR blockade

Autoimmune diseases, driven by Fc{gamma} receptor (Fc{gamma}R) activation through autoantibody immune complexes (IC), present a complex therapeutic challenge of achieving pharmacological blockade of Fc{gamma}R without triggering receptor activation. The assembly of ICs into polydisperse, higher-order structures is required for Fc{gamma}R activation. However, engineered multimeric, monodisperse Fc assemblies have been reported to prevent Fc{gamma}R activation, suggesting that Fc spatial organization determines Fc{gamma}R activation. In this study, we engineered a functional single-chain Fc domain protein (scFc) for unidirectional, multivalent presentation by virus-like particles (VLPs), used as a display platform. We found that the multivalent display of scFc on the VLPs elicited distinct cellular responses compared with monovalent scFc, highlighting the importance of the structural context of scFc on its function. scFc-VLPs had minimal impact on the nanoscale spatial organization of Fc{gamma}R at the cell membrane and caused limited receptor activation and internalization. In contrast, the monovalent scFc acted as an Fc{gamma}R agonist, inducing receptor clustering, activation, and internalization. Increasing scFc valency in scFc-VLPs was associated with increased binding to monocytes, reaching a plateau at high valencies. Notably, the ability of scFc-VLPs to block IC-mediated phagocytosis in vitro increased with scFc valency. In a murine model of passive immune thrombocytopenia (ITP), a high valency scFc-VLP variant with a desirable immunogenicity profile induced attenuation of thrombocytopenia. Here we show that multivalent presentation of an engineered scFc on a display platform can be tailored to promote suppression of IC-mediated phagocytosis while preventing Fc{gamma}R activation. This work introduces a new paradigm that can contribute to the development of therapies for autoimmune diseases.

bioengineering↗

bioRxiv2022

Mass spectrometry of RNA-binding proteins during liquid-liquid phase separation reveals distinct assembly mechanisms and droplet architectures

Phase separation of heterogeneous ribonucleoproteins (hRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility-mass spectrometry, and molecular dynamics simulations. We used a phase separation-compatible spider silk domain and pH changes to control the self-assembly of the hRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with phase separation. We find that NT*-FUS monomers undergo an unfolded-to-globular transition, whereas NT*-TDP-43 oligomerizes into partially disordered dimers and trimers. NT*-hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over phase separation. The divergent assembly mechanisms result in structurally distinct complexes, indicating differences in RNA processing and translation depending on biological context.

biophysics↗

bioRxiv2022

Structural basis of Omicron neutralization by affinity-matured public antibodies

The SARS-CoV-2 Omicron1 Variant of Concern (B.1.1.529) has spread rapidly in many countries. With a spike that is highly diverged from that of the pandemic founder, it escapes most available monoclonal antibody therapeutics2,3 and erodes vaccine protection4. A public class of IGHV3-53-using SARS-CoV-2 neutralizing antibodies5,6 typically fails to neutralize variants carrying mutations in the receptor-binding motif7-11, including Omicron. As antibodies from this class are likely elicited in most people following SARS-CoV-2 infection or vaccination, their subsequent affinity maturation is of particular interest. Here, we isolated IGHV3-53-using antibodies from an individual seven months after infection and identified several antibodies capable of broad and potent SARS-CoV-2 neutralization, extending to Omicron without loss of potency. By introducing select somatic hypermutations into a germline-reverted form of one such antibody, CAB-A17, we demonstrate the potential for commonly elicited antibodies to develop broad cross-neutralization through affinity maturation. Further, we resolved the structure of CAB-A17 Fab in complex with Omicron spike at an overall resolution of 2.6 [A] by cryo-electron microscopy and defined the structural basis for this breadth. Thus, public SARS-CoV-2 neutralizing antibodies can, without modified spike vaccines, mature to cross-neutralize exceptionally antigenically diverged SARS-CoV-2 variants.

immunology↗

bioRxiv2021

A dual allosteric pathway drives human mitochondrial Lon

The hexameric, barrel-forming, AAA+ protease Lon is critical for maintaining mitochondrial matrix protein homeostasis. Efficient substrate processing by Lon requires the coordinated action of six protomers. Despite Lons importance for human health, the molecular bases for Lons substrate recognition and processing remain unclear. Here, we use a combination of biochemistry and electron cryomicroscopy (cryo-EM) to unveil the structural and functional basis for full-length human mitochondrial Lons degradation of mitochondrial transcription factor A (TFAM). We show how opposing protomers in the Lon hexamer barrel interact through their N-terminal domains to give what resembles three feet above the barrel and help to form a triangular pore located just above the entry pore to the barrel. The interactions between opposing protomers constitute a primary allosteric regulation of Lon activity. A secondary allosteric regulation consists of an inter-subunit signaling element in the ATPase domains. By considering the ATP or ADP load in each protomer, we show how this dual allosteric mechanism in Lon achieves coordinated ATP hydrolysis and substrate processing. This mechanism enforces sequential anti-clockwise ATP hydrolysis resulting in a coordinated hand-over-hand translocation of the substrate towards the protease active sites.

biochemistry↗

bioRxiv2020

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 {micro}g/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed a novel conformation of the spike where two RBDs are in the up ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

biophysics↗

bioRxiv2020

An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction

SARS-CoV-2 is the etiologic agent of COVID-19, currently causing a devastating pandemic for which pharmacological interventions are urgently needed. The virus enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. The nanobody binds with high affinity in the low nM range to the RBD, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the ‘up’ and ‘down’ conformations and that Ty1 sterically hinders RBD-ACE2 binding. This 12.8 kDa nanobody does not need an Fc domain to neutralize SARS-CoV-2, and can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗