J Struct Biol
· 2025 Sep · PMID 40669763
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Emerging evidence highlights the importance of the interactions between amyloidogenic proteins and nucleic acids in both pathological and functional amyloid systems. Here, we review the current knowledge on the mechanism...Emerging evidence highlights the importance of the interactions between amyloidogenic proteins and nucleic acids in both pathological and functional amyloid systems. Here, we review the current knowledge on the mechanisms by which nucleic acids modulate amyloid assembly and structure, highlighting conserved paradigms that govern these interactions. Drawing from studies of prion protein, amyloid-β, α-synuclein, and functional bacterial amyloids, we describe how nucleic acids act as cofactors in amyloidogenesis and influence the biological roles of these systems. Despite these studies, key questions remain regarding the structural specificity, sequence dependence, and biophysical principles underlying these interactions. Biophysical and structural tools such as NMR spectroscopy and cryo-EM offer exciting opportunities to resolve these gaps and deepen our understanding of how nucleic acids shape amyloid formation, function, and pathology.
The interaction between the membrane (M) protein and the nucleocapsid (N) protein of coronaviruses plays a crucial role in virus assembly and morphogenesis. Previous studies indicate that one M-N interaction occurs betwe...The interaction between the membrane (M) protein and the nucleocapsid (N) protein of coronaviruses plays a crucial role in virus assembly and morphogenesis. Previous studies indicate that one M-N interaction occurs between M protein and the carboxy-terminus of N protein. However, the mechanistic details of M-N interactions remain unclear. Here, we present a complex structure of an N protein carboxy-terminal peptide bound to M protein from Pipistrellus bat coronavirus HKU5. The structure shows that the M-N peptide binding site includes a "horizontal" groove located between the carboxy-terminal domain and the transmembrane domain of M protein. Combined with molecular docking and binding analysis, our results provide structural insight into the binding mechanism between M and N proteins of a coronavirus.
Spatial organization of chromosomes is crucial for genome stability, transcription, and proper mitotic segregation. By employing a range of imaging technologies, including random illumination microscopy and single molecu...Spatial organization of chromosomes is crucial for genome stability, transcription, and proper mitotic segregation. By employing a range of imaging technologies, including random illumination microscopy and single molecule localization microscopy (SMLM), we conducted an in-depth exploration of the chromatin organization in budding yeast, with optical resolutions ranging from 250 nm to 50 nm. In silico models based on passively moving polymer chains and local tethering to nuclear landmarks explained much of the experimental data in yeast chromatin. We compared these models with our new imaging data of the nucleoplasmic and nucleolar chromatin. Chromatin fibers observed in the nucleoplasm showed some similarity with model prediction with a resolution of 150 nm. However, we visualized local clustering of chromatin in both the nucleoplasm and nucleolus, rather than the tube-like appearance predicted by polymer chain models. In the nucleolus, local clustering of ribosomal DNA (rDNA) chromatin is consistently observed from 150 nm resolution down to 50 nm. We also observed that actively transcribed rDNA spatially segregates from bulk nucleolar chromatin. Using correlative light and electron microscopy (CLEM), we found that local rDNA clustering is forming a specific nucleolar subdomain visible in transmission electron microscopy, the yeast equivalent of metazoan fibrillar center. We conclude that nucleolar chromatin forms a distinct sub-nucleolar compartment in yeast, supporting the model of a tripartite structural organization of the yeast nucleolus.
J Struct Biol
· 2025 Sep · PMID 40618940
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Full text
This study examines the validity of an assay that is used to report on the retainment of functional competence by ribosomes as they pass a microsprayer. We find a reproducible increase, rather than the expected decrease...This study examines the validity of an assay that is used to report on the retainment of functional competence by ribosomes as they pass a microsprayer. We find a reproducible increase, rather than the expected decrease in GFP production as monitored by fluorescence, which may suggest heterogeneity or partial aggregation of ribosomes in solution. An even larger increase in functional activity is observed when sonication is used, pointing to mechanical agitation as the decisive factor in both scenarios. The results have a bearing on the design and interpretation of validation experiments in time-resolved cryo-EM based on microfluidic chips.
We presents the discovery and molecular characterization of a novel lytic enzyme from the extremophilic Thermus thermophilus MAT72 phage vB_Tt72. The protein of 346-aa (MW = 39,705) functions as phage vB_Tt72 endolysin a...We presents the discovery and molecular characterization of a novel lytic enzyme from the extremophilic Thermus thermophilus MAT72 phage vB_Tt72. The protein of 346-aa (MW = 39,705) functions as phage vB_Tt72 endolysin and shows low sequence identity (<37 %) to members of M23 family of peptidoglycan hydrolases, except for two uncharacterized endopeptidases of T. thermophilus phages: φYS40 (87 %) and φTMA (88 %). The enzyme exhibits lytic activity mainly against bacteria of the genus Thermus and, to a lesser extent, against other Gram-negative and Gram-positive bacteria. The protein is monomeric in solution and is highly thermostable (T = 98.3 °C). It retains ∼ 50 % of its lytic activity after 90 min of incubation at 99 °C. Crystallographic analysis, at 2.2 Å resolution, revealed a fold characteristic of M23 metallopeptidases, accounting for 40 % of the structure. The remaining parts of the molecule are folded in a manner that was previously undescribed. The M23 fold contains a Zn ion coordinated by a conserved His-Asp-His triad, and two conserved His residues essential for catalysis. The active site is occupied by a phosphate or a sulfate anion, while the substrate-binding groove contains a ligand, which is a fragment of E. coli peptidoglycan. The common sequence-based criteria failed to identify the protein as (hyper)thermophilic. It is likely that the protein's thermal stability is owed to peculiar features of its three-dimensional structure. Instead of trimmed surface loops, observed in many thermostable proteins, the catalytic domain contains two long loops that interlace and form an α-helical bundle with its own hydrophobic core.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogenic agent responsible for the coronavirus disease 2019 (COVID-19) pandemic, uses the trimeric spike protein to gain entry into the host cell. Struc...Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogenic agent responsible for the coronavirus disease 2019 (COVID-19) pandemic, uses the trimeric spike protein to gain entry into the host cell. Structural studies have revealed that the spike protein is comprised of the S1 and S2 subunits. The S1 subunit of the spike protein contains the receptor-binding domain (RBD), which binds to the human angiotensin-converting enzyme 2 (ACE2) receptor. The interaction between the RBD and ACE2 facilitates membrane fusion and host cell infection. The SARS-CoV-2 spike protein also contains a unique insertion of four amino acids that results in the 682-RRAR↓S-686 polybasic furin cleavage motif at the boundary of the S1 and S2 subunits. The furin cleavage motif contributes to the high infectivity and transmissibility of SARS-CoV-2. This review provides a comprehensive analysis of the molecular interactions of the spike protein, with a specific focus on the RBD and furin cleavage site. In addition to examining the binding characteristics with ACE2, the interactions with alternative receptors, such as neuropilin-1 (NRP1) and the nicotinic acetylcholine receptors (nAChRs) are highlighted. The ability of the spike protein to bind alternative receptors and host factors has been linked to the pathophysiology of COVID-19 and the persistence of symptoms in the post COVID-19 condition. Furthermore, we examine the impact of spike protein mutations on receptor affinity and disease severity. SARS-CoV-2 continues to evolve, with variants remaining an ongoing threat to public health. Understanding these molecular interactions is critical for the development of novel therapeutic interventions.
Lipid membranes are uniquely complex biological structures with large and still undisclosed regulatory potential in many living processes caused by versatile changes in their structure while adsorption of various guest m...Lipid membranes are uniquely complex biological structures with large and still undisclosed regulatory potential in many living processes caused by versatile changes in their structure while adsorption of various guest molecules (dopants). This work is devoted to exploring spontaneous dopant-driven formation of lipid domains in a monolipid membrane observed experimentally for dopants with bimodal adsorption. The work offers the results obtained for a wide range of different cases exploiting our proposed original simulation method and numerical model. The central idea of the approach is dopant binding 'like the surroundings', i.e. preferential binding. The value range of the preferential binding extent was determined, where stable domains are formed and their size distribution becomes steady. The density of domain size distribution is power-law, i.e. the domain patterns possesses self-similarity. Outside this range, only one phase dominates if the extent is too large, whereas if it is too small, great dispersion of membrane was observed, so the membrane is physically homogeneous. Various neighboring as well as different methods of calculation of dopant binding probabilities are considered. The results obtained differed quantitatively but not qualitatively. The suggested model and the domain definition are similar to those used in percolation theory. Thus, the results can be applicated to percolation problems. Grounding on analysis of literature data on domain patterns formed in various lipid systems, we suggested that the preferential binding mechanism is in line with the mechanism of preferential neighboring which is implicitly assumed in such systems irrespective of their specific nature.
Cannabis sativa is a high-value plant renowned for its diverse chemical composition and abundant terpene content, contributing to its unique aroma, flavour, and therapeutic effects. Terpenes significantly influence consu...Cannabis sativa is a high-value plant renowned for its diverse chemical composition and abundant terpene content, contributing to its unique aroma, flavour, and therapeutic effects. Terpenes significantly influence consumer preference for C. sativa products, driving scientific interest in optimising terpene expression profiles and shaping the selective breeding of terpene profiles in C. sativa cultivars. In particular, the monoterpene, terpinolene, is influential in defining the sensory and therapeutic qualities of many C. sativa strains due to its woody, citrus-like aroma. Here we report the 2.5 Å resolution crystal structure of terpinolene synthase (CsTOS) from C. sativa in its apo form. The structure exhibits the class I monoterpene synthase fold with an open active site conformation. Using site-directed mutagenesis, we identified H618 as a key residues in determining product specificity. Substituting H618 with charged residues resulted in the preferential formation of limonene over terpinolene, highlighting its critical role in stabilising the substrate intermediate. Additionally, novel mutations uncovered an extended epistatic network of residues within 5 Å of the active site, spanning the α-helical bundle of the terpene synthase fold. These interactions contribute to monoterpene formation by modulating substrate positioning and catalytic activity. These insights advance our understanding of monoterpene biosynthesis and enable the targeted engineering of terpene synthases for customised terpene production, offering significant potential for the C. sativa industry.
Adenylyl cyclase 9 (AC9) regulates many physiologic functions through the production of cAMP, an important second messenger that regulates downstream effectors. The activation of AC9 is highly regulated by GPCR signaling...Adenylyl cyclase 9 (AC9) regulates many physiologic functions through the production of cAMP, an important second messenger that regulates downstream effectors. The activation of AC9 is highly regulated by GPCR signaling. For example, AC9 is activated by the binding of Gαs, which, in turn, is activated by Gs-driven GPCRs. The structure of bovine AC9 (bAC9) was reported in 2019 using single-particle cryo-electron microscopy (cryo-EM). The structure of human AC9 (hAC9), however, has not been reported to date despite its potential benefit for drug development. Here, we analyzed the structures of hAC9 and hAC9 in complex with Gαs (hAC9-Gαs) using single-particle cryo-EM. The soluble domain of AC9-Gαs, the transmembrane (TM) domain of AC9-Gαs, and AC9 alone were analyzed at resolutions of 2.7 Å, 3.4 Å, and 3.2 Å, respectively. The results revealed three key aspects of the activation mechanism of hAC9 and its cAMP-generating function. First, a conformational change of the soluble domain was observed upon Gαs binding, resulting in a widely open catalytic site. Second, we analyzed the exact position of the C-terminus occluding the catalytic site in the hAC9-Gαs complex. Finally, we unexpectedly identified an elongated density suggestive of a single acyl chain in the TM domain. Consistent with recent reports on the allosteric regulation of AC by lipids, this finding suggests that the TM domain could serve as a potential drug target.These structural findings enhance our understanding of the structure and function of AC9 and other ACs and will provide a foundation for future AC-target drug discovery.
We present the Volume Segmentation Tool (VST), a deep learning software tool that implements volumetric image segmentation in volume electron microscopy image stack data from a wide range of biological sample types. VST...We present the Volume Segmentation Tool (VST), a deep learning software tool that implements volumetric image segmentation in volume electron microscopy image stack data from a wide range of biological sample types. VST automates the handling of data preprocessing, data augmentation, and network building, as well as the configuration for model training, while adapting to the specific dataset. We have tried to make VST more accessible by designing it to operate entirely on local hardware and have provided a browser-based interface with additional features for visualizations of the networks and augmented datasets. VST can utilise contour map prediction to support instance segmentation on top of semantic segmentation. Through examples from various resin-embedded sample derived transmission electron microscopy and scanning electron microscopy datasets, we demonstrate that VST achieves state of the art performance compared to existing approaches.
Biomolecular image analysis and data interpretation is significantly improved through the application of advanced visualization techniques. Numerous visualization packages are currently available, spanning a broad spectr...Biomolecular image analysis and data interpretation is significantly improved through the application of advanced visualization techniques. Numerous visualization packages are currently available, spanning a broad spectrum of applications. Recently, we developed a plugin called ArtiaX which extended the capabilities of UCSF ChimeraX to address the specific demands of cryo-electron tomography. Here, we introduce the evolution of ArtiaX, that can now generate models to facilitate particle selection, define camera recording paths, and execute particle selection routines. Diverse models can be generated and populated with putative particle positions and orientations. In addition, models can be used to drive the camera position, thereby simplifying the process of movie creation. The plugin incorporates fundamental image filtering options for the on-the-fly analysis of tomographic data and provides compatibility of particle lists with RELION-5 .star files. Collectively, this update of ArtiaX comprehensively encompasses essential tools for the analysis and visualization of electron tomograms. It retains its hallmark attributes of speed, reliability, and user-friendliness, fostering seamless human-machine interaction.
Xeg5A from Aspergillus oryzae belongs to glycoside hydrolase family 5 subfamily 4. This enzyme has been characterized as a xyloglucan-specific endo-β-1,4-glucanase (xyloglucanase) that cleaves the main chain of xylogluca...Xeg5A from Aspergillus oryzae belongs to glycoside hydrolase family 5 subfamily 4. This enzyme has been characterized as a xyloglucan-specific endo-β-1,4-glucanase (xyloglucanase) that cleaves the main chain of xyloglucan at both unbranched and xylosylated glucosyl residues in an endo-processive mode of action. X-ray crystallography revealed that Xeg5A is a tri-modular enzyme composed of a catalytic, an Ig-like, and a C-terminal CBM46-like domains. Xeg5A structures complexed with branched xyloglucan oligosaccharides at subsites -4 to +4 showed that the recognition of xyloglucan side-chain moieties is important for Xeg5A activity. The crystal structure also provided structural insights into the role of the CBM46-like domain in contributing to regiospecificity and, possibly, processivity.
Fascins are crucial actin-binding proteins linked to carcinomas, such as cancer metastasis. Fascins crosslink unipolar actin filaments into linear and rigid parallel bundles, which play essential roles in the formation o...Fascins are crucial actin-binding proteins linked to carcinomas, such as cancer metastasis. Fascins crosslink unipolar actin filaments into linear and rigid parallel bundles, which play essential roles in the formation of filopodia, stereocilia and other membrane protrusions. However, the mechanism of how fascin bundles actin filaments has remained elusive. Here, we studied the organization of reconstituted fascin-actin bundles by cryo-electron tomography and determined the structure of the fascin-actin complex at 9 Å resolution by subtomogram averaging. Consistent with earlier findings, fascin molecules decorate adjacent actin filaments, positioned at regular intervals corresponding to the half-pitch of actin filaments. The fascin-actin complex structure allows us to verify the binding orientation of fascin between the two actin filaments. Fitting of the previously solved fascin crystal structure facilitates the analysis of the interaction surfaces. Our structural models serve as a blueprint to understand the detailed interactions between fascin and actins and provide new insights for the development of drugs targeting fascin proteins.
The members of the PadR family of transcriptional regulators are important for cell survival in toxic environments and play an important role in detoxification, pathogenicity, and multi-drug resistance. Rv0047c of Mycoba...The members of the PadR family of transcriptional regulators are important for cell survival in toxic environments and play an important role in detoxification, pathogenicity, and multi-drug resistance. Rv0047c of Mycobacterium tuberculosis H37Rv is annotated as a PadR family protein. We have characterized the stability and structure of Rv0047c. Rv0047c forms a stable dimer in solution. Its stability is characterized by a thermal melting transition temperature (Tm) of 55.3 °C. The crystal structure of Rv0047c was determined at a resolution of 3.15 Å. The structure indicates the biological unit to be a dimer with each monomer having a characteristic N-terminal winged-helix-turn-helix DNA binding domain and a C-terminal dimerization domain. The N-terminal domain is composed of four helices, α1, α2, α3, and α4 and two beta strands β1 and β2. The C-terminal dimerization domain (CTD) consists two long helices α6 and α7. The two domains are connected by helix α5. A short helical turn (helix αa, residue 89-92), leads to compaction of the α4-α5 loop. Rv0047c exhibits specificity in binding to an upstream region having an inverted repeat sequence. This binding is dependent upon Y18 and Y40 residue of Rv0047c, which are highly conserved among the PadR family. Overall, our results suggest a transcription regulatory role for Rv0047c, similar to other PadR family proteins.
Dental caries is the most prevalent oral disease that causes structural and compositional changes of the dental hard tissues due to a chronic demineralisation (combined with possible phases of remineralisation) process....Dental caries is the most prevalent oral disease that causes structural and compositional changes of the dental hard tissues due to a chronic demineralisation (combined with possible phases of remineralisation) process. Though considerable efforts have been directed at studying natural and artificial carious lesions, most characterisations remain either constrained to 2D analyses or have been unable to achieve fine resolution in 3D due to limited field of view. To overcome this challenge, the present study combined X-ray diffraction (XRD) and scanning transmission X-ray microscopy (STXM) tomography techniques to analyse the mineral density, scattering intensity, and crystallite size in normal, carious, 30 % artificially demineralised, and 50 % artificially demineralised dentine. Combined XRD and STXM tomography was performed on the I18 beamline at Diamond Light Source, using a 15 keV monochromatic beam with 2 × 2 µm spotsize and scanning with translation steps of 2 µm, providing a reconstructed voxel size of 2 × 2 × 2 µm. Natural carious dentine showed a reduction in hydroxyapatite (HAp) crystallite size due to chronic demineralisation. This was unlike artificially demineralised dentine samples that underwent short, continuous demineralisation, which created a zone of fully demineralised dentine, near the sample surface, and a zone of partially demineralised dentine that had a reduced mineral density but an increased average crystallite size.
We investigated the biomineralization process of calcium carbonate deposition in the spicules of the calcareous sponge Heteropia glomerosa (Porifera, Calcarea). The finely polished spicules, composed of Mg-calcite, prese...We investigated the biomineralization process of calcium carbonate deposition in the spicules of the calcareous sponge Heteropia glomerosa (Porifera, Calcarea). The finely polished spicules, composed of Mg-calcite, present a pattern of concentric lines spaced 400 nm apart when observed by scanning electron microscopy. We showed by electron backscattered diffraction that the whole spicule length has the same crystallographic orientation. Still, misorientation of up to 1.8° in adjacent regions (∼ 2 µm) and a continuous increase in the misalignment of up to 4.5° in regions separated by 300 µm were present. The sponge cells (mainly sclerocytes and pinacocytes) near the mineralization zone contain a high number of vesicles rich in calcium, which could be involved in the spicule biomineralization. We showed by electron and ion microscopies that the spicule growth occurs through the addition calcium carbonate granules, which form near the membrane of the sclerocyte, the cell responsible for biomineralization. The granules were deposited layer by layer on the surface of the spicule, increasing the biomineral thickness. Domains of 1-3 µm containing facets partially connected and surrounded by organic material were observed in an intermediate stage of the spicule growth. Misorientation between these domains was approximately 2°, similar to the misorientation obtained by electron backscattered diffraction, indicating that the spicule is formed by the addition of granules fusing in a predominant orientation.
J Struct Biol
· 2025 Jun · PMID 40378936
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Cryo-electron tomography (cryo-ET) is an essential tool in structural biology, uniquely capable of visualizing three-dimensional macromolecular complexes within their native cellular environments, thereby providing profo...Cryo-electron tomography (cryo-ET) is an essential tool in structural biology, uniquely capable of visualizing three-dimensional macromolecular complexes within their native cellular environments, thereby providing profound molecular-level insights. Despite its significant promise, cryo-ET faces persistent challenges in the systematic localization, identification, segmentation, and structural recovery of three-dimensional subcellular components, necessitating the development of efficient and accurate large-scale image analysis methods. In response to these complexities, this paper introduces AITom, an open-source artificial intelligence platform tailored for cryo-ET researchers. AITom integrates a comprehensive suite of public and proprietary algorithms, supporting both traditional template-based and template-free approaches, alongside state-of-the-art deep learning methodologies for cryo-ET data analysis. By incorporating diverse computational strategies, AITom enables researchers to more effectively tackle the complexities inherent in cryo-ET, facilitating precise analysis and interpretation of complex biological structures. Furthermore, AITom provides extensive tutorials for each analysis module, offering valuable guidance to users in utilizing its comprehensive functionalities.
Thermostable enzymes have significant advantages in industries, yet uncovering novel candidates with superior properties remains a scientific pursuit. This study identified rMxyl, a glycoside hydrolase 11 family thermoph...Thermostable enzymes have significant advantages in industries, yet uncovering novel candidates with superior properties remains a scientific pursuit. This study identified rMxyl, a glycoside hydrolase 11 family thermophilic xylanase from compost-soil metagenome, which exhibited a high specific activity of 5954 U·mg at pH 5.5 and 80°C. rMxyl was crystallized and diffracted to 1.5 Å resolution. Compared to the mesophilic xylanase Xyn II, rMxyl exhibits a more compact architecture. Notably, B-factor analysis reveals a uniquely flexible thumb region, hinting at its critical role in the enzyme's catalytic mechanism. rMxyl contains two disulfide bonds in the thumb and the N-terminal regions. Breaking these disulfide bonds by mutagenesis has dramatically decreased activities and thermostability. Conversely, introducing an extra disulfide bond at the N-terminal region of its α-helix extended its half-life for more than five folds at 80°C. Our studies firmly establish that the disulfide bonds are essential for its high thermal stability and the flexibility of the thumb region is crucial for its activity. Comparing the rMxyl crystal structure with the AlphaFold2-predicted model shows overall similarity, but the crystal structure offers higher local accuracy, especially in key functional regions. These findings not only deepen our understanding of the structure-function relationship of thermophilic xylanases but also inform a rational design of industrial enzymes.
Single-particle cryo-electron microscopy (cryo-EM) has significantly advanced macromolecular structure reconstruction. However, a key limitation is the conventional reliance on micrographs obtained by motion correction a...Single-particle cryo-electron microscopy (cryo-EM) has significantly advanced macromolecular structure reconstruction. However, a key limitation is the conventional reliance on micrographs obtained by motion correction and averaging, which inherently loses the richness of information contained within each frame of the original movie. The future of cryo-EM reconstruction ideally involves harnessing the raw signal from every frame to unlock potentially higher quality structures. In this paper, we present a first essential step toward this paradigm shift, that is, a novel, non-parametric method for detecting tomographic projections across all movie frames, using temporal consistency. Our method is inspired by Structure-from-Motion (SfM), and independent of motion correction, CTF estimation, and initial reconstruction. Our experimental results demonstrate reduced outlier rate and accurate particle localization comparable to existing approaches throughout the entire movie sequence.
Single particle cryo-electron microscopy (cryoEM) is a powerful tool for elucidating the structures of biological macromolecules without requiring crystallisation or fixation. However, certain barriers to obtaining high-...Single particle cryo-electron microscopy (cryoEM) is a powerful tool for elucidating the structures of biological macromolecules without requiring crystallisation or fixation. However, certain barriers to obtaining high-resolution structures persist, particularly during grid preparation when samples are in a thin liquid film. At this stage, extensive exposure to the air-water interface (AWI) can lead to subunit dissociation, denaturation, and preferred orientation of particles. Another obstacle to high-resolution cryoEM is molecular flexibility, which introduces heterogeneity in the dataset, weakening the signal during image processing. This study explores the effects of AWI interactions and molecular flexibility on the cryoEM density maps of KtrA, the soluble regulatory subunit of the potassium transporter KtrAB from Bacillus subtilis. From grids prepared using a standard blotting technique, we observed a lack of density in the C-lobe domains and preferred orientation. Modifications such as reducing AWI exposure through faster vitrification times (6 s vs ≤100 ms) notably improved C-lobe density. Moreover, the addition of cyclic di-AMP, which binds to the C-lobes, combined with a 100 ms plunge time, further enhanced C-lobe density and eliminated preferred orientation. These findings demonstrate that both AWI interactions and flexibility had to be addressed to obtain density for the C-lobe domains of KtrA. This study underscores the ongoing complexities in achieving high-resolution cryoEM for many samples.