Journal Of Structural Biology[JOURNAL]
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200 papers
Selective signal enhancement in Fourier space as a tool for discovering ultrastructural organization of macromolecules from in situ TEM.
Matsko NB, Schorb M, Schwab Y
J Struct Biol · 2024 Dec · PMID 39284397 · Publisher ↗
We present a Fourier transform (FT) based analytical method that allows to obtain of ultrastructural details from TEM images at sub-nanometer scale applying a selective filtering for singular macromolecule electron micro...
We present a Fourier transform (FT) based analytical method that allows to obtain of ultrastructural details from TEM images at sub-nanometer scale applying a selective filtering for singular macromolecule electron microscopy density information. It can be applied to high-pressure frozen, frozen hydrated and epoxy freeze substituted and embedded biological species. Both 2D projections and orthoslices from reconstructed tomograms can be used as a source of structural information. The key to the method is to select the macromolecule or organelle of interest with an accuracy of ≥ 7 - 3 nm (depending on pixel size of initial tilt series or singular image acquisition) and explore both the central low frequency FT intensity and diffraction regions to obtain the spatial structural organization and its dimensional characteristics, respectively. We also introduce a structure-specific selective mask FT filtering approach that can significantly improve image information even in poorly contrasted TEM of resin sections without heavy metal been used. The described method elucidates chromatin architecture without the need of averaging. A zigzag symmetry of 30 nm diameter chromatin fibers which in general is a controversial topic of research has been identified for C. elegans cells in vivo with sub-nanometer details being preserved in the images.
Three-dimensional cellular architecture of the sigmoid filament in Trichomonas vaginalis.
Ortiz S, Verdan R, Benchimol M
J Struct Biol · 2024 Dec · PMID 39244171 · Publisher ↗
Trichomonas vaginalis is a parasite protozoan that causes human trichomoniasis, a sexually transmitted infection (STI) that affects more than 156 million people worldwide. T. vaginalis contains an uncommon and complex cy...
Trichomonas vaginalis is a parasite protozoan that causes human trichomoniasis, a sexually transmitted infection (STI) that affects more than 156 million people worldwide. T. vaginalis contains an uncommon and complex cytoskeleton constituting the mastigont system, formed by several fibers and proteinaceous structures associated with basal bodies. Among these structures is the pelta-axostylar complex made of microtubules and striated filaments such as the costa and the parabasal filaments. In addition, some structures are poorly known and studied, such as the sigmoid filament and the X-filament. Here, we have isolated the Trichomonas vaginalis cytoskeleton and used UHR-SEM (ultra-high resolution scanning electron microscopy), tomography, immunofluorescence, immunolabeling, and backscattered electrons on SEM, negative staining to model the three-dimensional architecture and possible function of the sigmoid.
Comparison of the global crystallographic texture of minerals in the shells of Bathymodiolus thermophilus Kenk et B.R. Wilson, 1985 and species of the genus Mytilus Linnaeus, 1758.
Pakhnevich A, Nikolayev D, Lychagina T
J Struct Biol · 2024 Dec · PMID 39244170 · Publisher ↗
The global crystallographic texture of calcite and aragonite in the shells of the bivalves Bathymodiolus thermophilus, Mytilus galloprovincialis, M. edulis and M. trossulus was studied by means of neutron diffraction. It...
The global crystallographic texture of calcite and aragonite in the shells of the bivalves Bathymodiolus thermophilus, Mytilus galloprovincialis, M. edulis and M. trossulus was studied by means of neutron diffraction. It was revealed that the general appearance of pole figures isolines of both minerals coincides for the studied species. The crystallographic texture sharpness evaluated by means of pole density on the calcite pole figures ((0006), (101¯4)) and aragonite pole figures ((012)/(121), (040)/(221)) coincides or has close values for deep-sea hydrothermal species B. thermophilus and the studied shallow-water species of the genus Mytilus. The calcite pole figures (0006) and (101¯4) of B. thermophilus show a shift in the position of texture maximum values compared to corresponding pole figures of other mussels. The shell microstructure of all studied mollusks is similar, only the shape of the fibers of B. thermophilus differs. Global crystallographic texture is a stable feature of the family Mytilidae. The extreme habitat conditions of the hydrothermal biotope do not significantly affect the crystallographic texture of B. thermophilus.
Studying protein-protein interactions: Latest and most popular approaches.
Akbarzadeh S, Coşkun Ö, Günçer B
J Struct Biol · 2024 Dec · PMID 39214321 · Publisher ↗
PPIs, or protein-protein interactions, are essential for many biological processes. According to the findings, abnormal PPIs have been linked to several diseases, such as cancer and infectious and neurological disorders....
PPIs, or protein-protein interactions, are essential for many biological processes. According to the findings, abnormal PPIs have been linked to several diseases, such as cancer and infectious and neurological disorders. Consequently, focusing on PPIs is a path toward disease treatment and a crucial tool for producing novel medications. Many methods exist to investigate PPIs, including low- and high-throughput studies. Since many PPIs have been discovered using in vitro and in vivo experimental approaches, the use of computational methods to predict PPIs has grown due to the expanding scale of PPI data and the intrinsic complexity of interacting mechanisms. Recognizing PPI networks offers a systematic means of predicting protein functions, and pathways that are included. These investigations can help uncover the underlying molecular mechanisms of complex phenotypes and clarify the biological processes related to health and diseases. Therefore, our goal in this study is to provide an overview of the latest and most popular approaches for investigating PPIs. We also overview some important clinical approaches based on the PPIs and how these interactions can be targeted.
Structural analysis of the human C5a-C5aR1 complex using cryo-electron microscopy.
Yang T, Li J, Cheng X … +13 more , Lu Q, Farooq Z, Fu Y, Lv S, Nan W, Yu B, Duan J, Zhang Y, Fu Y, Jiang H, McCormick PJ, Li Y, Zhang J
J Struct Biol · 2024 Sep · PMID 39153560 · Publisher ↗
The complement system is a complex network of proteins that plays a crucial role in the innate immune response. One important component of this system is the C5a-C5aR1 complex, which is critical in the recruitment and ac...
The complement system is a complex network of proteins that plays a crucial role in the innate immune response. One important component of this system is the C5a-C5aR1 complex, which is critical in the recruitment and activation of immune cells. In-depth investigation of the activation mechanism as well as biased signaling of the C5a-C5aR1 system will facilitate the elucidation of C5a-mediated pathophysiology. In this study, we determined the structure of C5a-C5aR1-Gi complex at a high resolution of 3 Å using cryo-electron microscopy (Cryo-EM). Our results revealed the binding site of C5a, which consists of a polar recognition region on the extracellular side and an amphipathic pocket within the transmembrane domain. Furthermore, we found that C5a binding induces conformational changes of C5aR1, which subsequently leads to the activation of G protein signaling pathways. Notably, a key residue (M265) located on transmembrane helix 6 (TM6) was identified to play a crucial role in regulating the recruitment of β-arrestin driven by C5a. This study provides more information about the structure and function of the human C5a-C5aR1 complex, which is essential for the proper functioning of the complement system. The findings of this study can also provide a foundation for the design of new pharmaceuticals targeting this receptor with bias or specificity.
Cryo-EM reconstruction of oleate hydratase bound to a phospholipid membrane bilayer.
Oldham ML, Zuhaib Qayyum M, Kalathur RC … +2 more , Rock CO, Radka CD
J Struct Biol · 2024 Sep · PMID 39151742 · Full text
Oleate hydratase (OhyA) is a bacterial peripheral membrane protein that catalyzes FAD-dependent water addition to membrane bilayer-embedded unsaturated fatty acids. The opportunistic pathogen Staphylococcus aureus uses O...
Oleate hydratase (OhyA) is a bacterial peripheral membrane protein that catalyzes FAD-dependent water addition to membrane bilayer-embedded unsaturated fatty acids. The opportunistic pathogen Staphylococcus aureus uses OhyA to counteract the innate immune system and support colonization. Many Gram-positive and Gram-negative bacteria in the microbiome also encode OhyA. OhyA is a dimeric flavoenzyme whose carboxy terminus is identified as the membrane binding domain; however, understanding how OhyA binds to cellular membranes is not complete until the membrane-bound structure has been elucidated. All available OhyA structures depict the solution state of the protein outside its functional environment. Here, we employ liposomes to solve the cryo-electron microscopy structure of the functional unit: the OhyA•membrane complex. The protein maintains its structure upon membrane binding and slightly alters the curvature of the liposome surface. OhyA preferentially associates with 20-30 nm liposomes with multiple copies of OhyA dimers assembling on the liposome surface resulting in the formation of higher-order oligomers. Dimer assembly is cooperative and extends along a formed ridge of the liposome. We also solved an OhyA dimer of dimers structure that recapitulates the intermolecular interactions that stabilize the dimer assembly on the membrane bilayer as well as the crystal contacts in the lattice of the OhyA crystal structure. Our work enables visualization of the molecular trajectory of membrane binding for this important interfacial enzyme.
A glimpse into the hidden world of the flexible C-terminal protein binding domains of human RAD52.
Struble LR, Lovelace JJ, Borgstahl GEO
J Struct Biol · 2024 Sep · PMID 39117045 · Full text
Human RAD52 protein binds DNA and is involved in genomic stability maintenance and several forms of DNA repair, including homologous recombination and single-strand annealing. Despite its importance, there are very few s...
Human RAD52 protein binds DNA and is involved in genomic stability maintenance and several forms of DNA repair, including homologous recombination and single-strand annealing. Despite its importance, there are very few structural details about the variability of the RAD52 ring size and the RAD52 C-terminal protein-protein interaction domains. Even recent attempts to employ cryogenic electron microscopy (cryoEM) methods on full-length yeast and human RAD52 do not reveal interpretable structures for the C-terminal half that contains the replication protein A (RPA) and RAD51 binding domains. In this study, we employed the monodisperse purification of two RAD52 deletion constructs and small angle X-ray scattering (SAXS) to construct a structural model that includes RAD52's RPA binding domain. This model is of interest to DNA repair specialists as well as for drug development against HR-deficient cancers.
Relationship between thermal stability of collagens and the fraction of hydrophobic residues in their molecules.
Meshcheryakova OV, Bogdanov MA, Efimov AV
J Struct Biol · 2024 Sep · PMID 39094716 · Publisher ↗
In this study, a database of the thermal stability of collagens and their synthetic analogues has been compiled taking into account literature sources. In total, our database includes 1200 records. As a result of a compa...
In this study, a database of the thermal stability of collagens and their synthetic analogues has been compiled taking into account literature sources. In total, our database includes 1200 records. As a result of a comparative theoretical analysis of the collected experimental data, the relationship between the melting temperature (T) or denaturation temperature (T) of collagens and the fraction of hydrophobic residues (f) in their molecules has been established. It is shown that this relationship is linear: the larger the f value, the higher the denaturation or melting temperature of a given collagen.
Crystal structure of the GluK1 ligand-binding domain with kainate and the full-spanning positive allosteric modulator BPAM538.
Bay Y, Cabello FJM, Koens CC … +8 more , Frantsen SM, Pickering DS, Frydenvang K, Francotte P, Pirotte B, Kristensen AS, Bowie D, Kastrup JS
J Struct Biol · 2024 Sep · PMID 39079583 · Publisher ↗
Kainate receptors play an important role in the central nervous system by mediating postsynaptic excitatory neurotransmission and modulating the release of the inhibitory neurotransmitter GABA through a presynaptic mecha...
Kainate receptors play an important role in the central nervous system by mediating postsynaptic excitatory neurotransmission and modulating the release of the inhibitory neurotransmitter GABA through a presynaptic mechanism. To date, only three structures of the ligand-binding domain (LBD) of the kainate receptor subunit GluK1 in complex with positive allosteric modulators have been determined by X-ray crystallography, all belonging to class II modulators. Here, we report a high-resolution structure of GluK1-LBD in complex with kainate and BPAM538, which belongs to the full-spanning class III. One BPAM538 molecule binds at the GluK1 dimer interface, thereby occupying two allosteric binding sites simultaneously. BPAM538 stabilizes the active receptor conformation with only minor conformational changes being introduced to the receptor. Using a calcium-sensitive fluorescence-based assay, a 5-fold potentiation of the kainate response (100 μM) was observed in presence of 100 μM BPAM538 at GluK1(Q), whereas no potentiation was observed at GluK2(VCQ). Using electrophysiology recordings of outside-out patches excised from HEK293 cells, BPAM538 increased the peak response of GluK1(Q) co-expressed with NETO2 to rapid application of 10 mM L-glutamate with 130 ± 20 %, and decreased desensitization determined as the steady-state/peak response ratio from 23 ± 2 % to 90 ± 4 %. Based on dose-response relationship experiments on GluK1(Q) the EC of BPAM538 was estimated to be 58 ± 29 μM.
From viruses to humans - Exploring the structure-function relationship of the Kesv protein for the future of biomedicine.
Asrani P, Seebohm G, Stoll R
J Struct Biol · 2024 Sep · PMID 39069032 · Publisher ↗
Viruses often use ion channel proteins to initialise host infections. Defects in ion channel proteins are also linked to several metabolic disorders in humans. In that instance, modulation of ion channel activities becom...
Viruses often use ion channel proteins to initialise host infections. Defects in ion channel proteins are also linked to several metabolic disorders in humans. In that instance, modulation of ion channel activities becomes central to development of antiviral therapies and drug design. Kesv, a potassium-selective ion channel protein expressed by Ectocarpus siliculosus virus (EsV), possesses remarkable properties which can help to characterise the molecular basis of the functional processes relevant to virus biology and human physiology. The small structural features of this ion channel could serve as a fundamental primer to study more complex ion channels from humans. Therefore, in spite of their evolutionary distance, the potential link between viral and human ion channel proteins could provide opportunities for therapeutic and biotechnological applications.
3D quantification of the lacunocanalicular network on human femoral diaphysis through synchrotron radiation-based nanoCT.
Yu B, Gauthier R, Olivier C … +4 more , Villanova J, Follet H, Mitton D, Peyrin F
J Struct Biol · 2024 Sep · PMID 39059753 · Publisher ↗
Osteocytes are the major actors in bone mechanobiology. Within bone matrix, they are trapped close together in a submicrometric interconnected network: the lacunocanalicular network (LCN). The interstitial fluid circulat...
Osteocytes are the major actors in bone mechanobiology. Within bone matrix, they are trapped close together in a submicrometric interconnected network: the lacunocanalicular network (LCN). The interstitial fluid circulating within the LCN transmits the mechanical information to the osteocytes that convert it into a biochemical signal. Understanding the interstitial fluid dynamics is necessary to better understand the bone mechanobiology. Due to the submicrometric dimensions of the LCN, making it difficult to experimentally investigate fluid dynamics, numerical models appear as a relevant tool for such investigation. To develop such models, there is a need for geometrical and morphological data on the human LCN. This study aims at providing morphological data on the human LCN from measurement of 27 human femoral diaphysis bone samples using synchrotron radiation nano-computed tomography with an isotropic voxel size of 100 nm. Except from the canalicular diameter, the canalicular morphological parameters presented a high variability within one sample. Some differences in terms of both lacunar and canalicular morphology were observed between the male and female populations. But it has to be highlighted that all the canaliculi cannot be detected with a voxel size of 100 nm. Hence, in the current study, only a specific population of large canaliculi that could be characterize. Still, to the authors knowledge, this is the first time such a data set was introduced to the community. Further processing will be achieved in order to provide new insight on the LCN permeability.
Quantitative 3D electron microscopy characterization of mitochondrial structure, mitophagy, and organelle interactions in murine atrial fibrillation.
Guttipatti P, Saadallah N, Ji R … +3 more , Avula UMR, Goulbourne CN, Wan EY
J Struct Biol · 2024 Sep · PMID 39009246 · Full text
Atrial fibrillation (AF) is the most common clinical arrhythmia, however there is limited understanding of its pathophysiology including the cellular and ultrastructural changes rendered by the irregular rhythm, which li...
Atrial fibrillation (AF) is the most common clinical arrhythmia, however there is limited understanding of its pathophysiology including the cellular and ultrastructural changes rendered by the irregular rhythm, which limits pharmacological therapy development. Prior work has demonstrated the importance of reactive oxygen species (ROS) and mitochondrial dysfunction in the development of AF. Mitochondrial structure, interactions with other organelles such as sarcoplasmic reticulum (SR) and T-tubules (TT), and degradation of dysfunctional mitochondria via mitophagy are important processes to understand ultrastructural changes due to AF. However, most analysis of mitochondrial structure and interactome in AF has been limited to two-dimensional (2D) modalities such as transmission electron microscopy (EM), which does not fully visualize the morphological evolution of the mitochondria during mitophagy. Herein, we utilize focused ion beam-scanning electron microscopy (FIB-SEM) and perform reconstruction of three-dimensional (3D) EM from murine left atrial samples and measure the interactions of mitochondria with SR and TT. We developed a novel 3D quantitative analysis of FIB-SEM in a murine model of AF to quantify mitophagy stage, mitophagosome size in cardiomyocytes, and mitochondrial structural remodeling when compared with control mice. We show that in our murine model of spontaneous and continuous AF due to persistent late sodium current, left atrial cardiomyocytes have heterogenous mitochondria, with a significant number which are enlarged with increased elongation and structural complexity. Mitophagosomes in AF cardiomyocytes are located at Z-lines where they neighbor large, elongated mitochondria. Mitochondria in AF cardiomyocytes show increased organelle interaction, with 5X greater contact area with SR and are 4X as likely to interact with TT when compared to control. We show that mitophagy in AF cardiomyocytes involves 2.5X larger mitophagosomes that carry increased organelle contents. In conclusion, when oxidative stress overcomes compensatory mechanisms, mitophagy in AF faces a challenge of degrading bulky complex mitochondria, which may result in increased SR and TT contacts, perhaps allowing for mitochondrial Ca maintenance and antioxidant production.
Exploring α-synuclein stability under the external electrostatic field: Effect of repeat unit.
Khursandov J, Mashalov R, Makhkamov M … +3 more , Turgunboev F, Sharipov A, Razzokov J
J Struct Biol · 2024 Sep · PMID 38964522 · Publisher ↗
Parkinson's disease (PD) is a category of neurodegenerative disorders (ND) that currently lack comprehensive and definitive treatment strategies. The etiology of PD can be attributed to the presence and aggregation of a...
Parkinson's disease (PD) is a category of neurodegenerative disorders (ND) that currently lack comprehensive and definitive treatment strategies. The etiology of PD can be attributed to the presence and aggregation of a protein known as α-synuclein. Researchers have observed that the application of an external electrostatic field holds the potential to induce the separation of the fibrous structures into peptides. To comprehend this phenomenon, our investigation involved simulations conducted on the α-synuclein peptides through the application of Molecular Dynamics (MD) simulation techniques under the influence of a 0.1 V/nm electric field. The results obtained from the MD simulations revealed that in the presence of external electric field, the monomer and oligomeric forms of α-synuclein are experienced significant conformational changes which could prevent them from further aggregation. However, as the number of peptide units in the model system increases, forming trimers and tetramers, the stability against the electric field also increases. This enhanced stability in larger aggregates indicates a critical threshold in α-synuclein assembly where the electric field's effectiveness in disrupting the aggregation diminishes. Therefore, our findings suggest that early diagnosis and intervention could be crucial in preventing PD progression. When α-synuclein predominantly exists in its monomeric or dimeric form, applying even a lower electric field could effectively disrupt the initial aggregation process. Inhibition of α-synuclein fibril formation at early stages might serve as a viable solution to combat PD by halting the formation of more stable and pathogenic α-synuclein fibrils.
High-resolution single-particle imaging at 100-200 keV with the Gatan Alpine direct electron detector.
Chan LM, Courteau BJ, Maker A … +11 more , Wu M, Basanta B, Mehmood H, Bulkley D, Joyce D, Lee BC, Mick S, Czarnik C, Gulati S, Lander GC, Verba KA
J Struct Biol · 2024 Sep · PMID 38944401 · Full text
Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments indicate advantages to imagi...
Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments indicate advantages to imaging at 100 keV, energies for which the current detectors have not been optimized. In this study, we evaluated the Gatan Alpine detector, designed for operation at 100 and 200 keV. Compared to the Gatan K3, Alpine demonstrated a significant DQE improvement at these energies, specifically a ∼ 4-fold improvement at Nyquist at 100 keV. In single-particle cryo-EM experiments, Alpine datasets yielded better than 2 Å resolution reconstructions of apoferritin at 120 and 200 keV on a ThermoFisher Scientific (TFS) Glacios microscope fitted with a non-standard SP-Twin lens. We also achieved a ∼ 3.2 Å resolution reconstruction of a 115 kDa asymmetric protein complex, proving Alpine's effectiveness with complex biological samples. In-depth analysis revealed that Alpine reconstructions are comparable to K3 reconstructions at 200 keV, and remarkably, reconstruction from Alpine at 120 keV on a TFS Glacios surpassed all but the 300 keV data from a TFS Titan Krios with GIF/K3. Additionally, we show Alpine's capability for high-resolution data acquisition and screening on lower-end systems by obtaining ∼ 3 Å resolution reconstructions of apoferritin and aldolase at 100 keV and detailed 2D averages of a 55 kDa sample using a side-entry cryo holder. Overall, we show that Gatan Alpine performs well with the standard 200 keV imaging systems and may potentially capture the benefits of lower accelerating voltages, bringing smaller sized particles within the scope of cryo-EM.
High-quality AFM image acquisition of living cells by modified residual encoder-decoder network.
Wang J, Yang F, Wang B … +5 more , Liu M, Wang X, Wang R, Song G, Wang Z
J Struct Biol · 2024 Sep · PMID 38906499 · Publisher ↗
Atomic force microscope enables ultra-precision imaging of living cells. However, atomic force microscope imaging is a complex and time-consuming process. The obtained images of living cells usually have low resolution a...
Atomic force microscope enables ultra-precision imaging of living cells. However, atomic force microscope imaging is a complex and time-consuming process. The obtained images of living cells usually have low resolution and are easily influenced by noise leading to unsatisfactory imaging quality, obstructing the research and analysis based on cell images. Herein, an adaptive attention image reconstruction network based on residual encoder-decoder was proposed, through the combination of deep learning technology and atomic force microscope imaging supporting high-quality cell image acquisition. Compared with other learning-based methods, the proposed network showed higher peak signal-to-noise ratio, higher structural similarity and better image reconstruction performances. In addition, the cell images reconstructed by each method were used for cell recognition, and the cell images reconstructed by the proposed network had the highest cell recognition rate. The proposed network has brought insights into the atomic force microscope-based imaging of living cells and cell image reconstruction, which is of great significance in biological and medical research.
Multimodal analysis and comparison of stoichiometric and structural characteristics of parosteal and conventional osteosarcoma with massive sclerosis in human bone.
Zanghellini B, Zechmann N, Baurecht D … +6 more , Grünewald TA, Burghammer M, Liegl-Atzwanger B, Leithner A, Davydok A, Lichtenegger H
J Struct Biol · 2024 Sep · PMID 38871094 · Publisher ↗
Osteosarcoma (OS) is the most common malignant primary bone tumor in humans and occurs in various subtypes. Tumor formation happens through malignant osteoblasts producing immature bone. In the present paper we studied t...
Osteosarcoma (OS) is the most common malignant primary bone tumor in humans and occurs in various subtypes. Tumor formation happens through malignant osteoblasts producing immature bone. In the present paper we studied two different subtypes of osteosarcoma, from one individual with conventional OS with massive sclerosis and one individual with parosteal OS, based on a multimodal approach including small angle x-ray scattering (SAXS), wide angle x-ray diffraction (WAXS), backscattered electron imaging (BEI) and Raman spectroscopy. It was found that both tumors showed reduced mineral particle sizes and degree of orientation of the collagen-mineral composite in the affected areas, alongside with a decreased crystallinity. Distinct differences between the tumor material from the two individuals were found in the degree of mineralization. Further differences were observed in the carbonate to phosphate ratio, which is related to the degree of carbonate substitution in bone mineral and indicative of the turnover rate. The contraction of the c-axis of the bone mineral crystals proved to be a further, very sensitive parameter, potentially indicative of malignancy.
The CryoEM structure of human serum albumin in complex with ligands.
Catalano C, Lucier KW, To D … +7 more , Senko S, Tran NL, Farwell AC, Silva SM, Dip PV, Poweleit N, Scapin G
J Struct Biol · 2024 Sep · PMID 38852682 · Publisher ↗
Human serum albumin (HSA) is the most prevalent plasma protein in the human body, accounting for 60 % of the total plasma protein. HSA plays a major pharmacokinetic function, serving as a facilitator in the distribution...
Human serum albumin (HSA) is the most prevalent plasma protein in the human body, accounting for 60 % of the total plasma protein. HSA plays a major pharmacokinetic function, serving as a facilitator in the distribution of endobiotics and xenobiotics within the organism. In this paper we report the cryoEM structures of HSA in the apo form and in complex with two ligands (salicylic acid and teniposide) at a resolution of 3.5, 3.7 and 3.4 Å, respectively. We expand upon previously published work and further demonstrate that sub-4 Å maps of ∼60 kDa proteins can be routinely obtained using a 200 kV microscope, employing standard workflows. Most importantly, these maps allowed for the identification of small molecule ligands, emphasizing the practical applicability of this methodology and providing a starting point for subsequent computational modeling and in silico optimization.
Honeycomb gold specimen supports enabling orthogonal focussed ion beam-milling of elongated cells for cryo-ET.
Hale VL, Hooker J, Russo CJ … +1 more , Löwe J
J Struct Biol · 2024 Jun · PMID 38772448 · Full text
Cryo-focussed ion beam (FIB)-milling is a powerful technique that opens up thick, cellular specimens to high-resolution structural analysis by electron cryotomography (cryo-ET). FIB-milled lamellae can be produced from c...
Cryo-focussed ion beam (FIB)-milling is a powerful technique that opens up thick, cellular specimens to high-resolution structural analysis by electron cryotomography (cryo-ET). FIB-milled lamellae can be produced from cells on grids, or cut from thicker, high-pressure frozen specimens. However, these approaches can put geometrical constraints on the specimen that may be unhelpful, particularly when imaging structures within the cell that have a very defined orientation. For example, plunge frozen rod-shaped bacteria orient parallel to the plane of the grid, yet the Z-ring, a filamentous structure of the tubulin-like protein FtsZ and the key organiser of bacterial division, runs around the circumference of the cell such that it is perpendicular to the imaging plane. It is therefore difficult or impractical to image many complete rings with current technologies. To circumvent this problem, we have fabricated monolithic gold specimen supports with a regular array of cylindrical wells in a honeycomb geometry, which trap bacteria in a vertical orientation. These supports, which we call "honeycomb gold discs", replace standard EM grids and when combined with FIB-milling enable the production of lamellae containing cross-sections through cells. The resulting lamellae are more stable and resistant to breakage and charging than conventional lamellae. The design of the honeycomb discs can be modified according to need and so will also enable cryo-ET and cryo-EM imaging of other specimens in otherwise difficult to obtain orientations.
Structure and conformational variability of the HER2-trastuzumab-pertuzumab complex.
Ruedas R, Vuillemot R, Tubiana T … +7 more , Winter JM, Pieri L, Arteni AA, Samson C, Jonic S, Mathieu M, Bressanelli S
J Struct Biol · 2024 Jun · PMID 38723875 · Publisher ↗
Single particle analysis from cryogenic transmission electron microscopy (cryo-EM) is particularly attractive for complexes for which structure prediction remains intractable, such as antibody-antigen complexes. Here we...
Single particle analysis from cryogenic transmission electron microscopy (cryo-EM) is particularly attractive for complexes for which structure prediction remains intractable, such as antibody-antigen complexes. Here we obtain the detailed structure of a particularly difficult complex between human epidermal growth factor receptor 2 (HER2) and the antigen-binding fragments from two distinct therapeutic antibodies binding to distant parts of the flexible HER2, pertuzumab and trastuzumab (HTP). We highlight the strengths and limitations of current data processing software in dealing with various kinds of heterogeneities, particularly continuous conformational heterogeneity, and in describing the motions that can be extracted from our dataset. Our HTP structure provides a more detailed view than the one previously available for this ternary complex. This allowed us to pinpoint a previously overlooked loop in domain IV that may be involved both in binding of trastuzumab and in HER2 dimerization. This finding may contribute to explain the synergistic anticancer effect of the two antibodies. We further propose that the flexibility of the HTP complex, beyond the difficulties it causes for cryo-EM analysis, actually reflects regulation of HER2 signaling and its inhibition by therapeutic antibodies. Notably we obtain our best data with ultra-thin continuous carbon grids, showing that with current cameras their use to alleviate particle misdistribution is compatible with a protein complex of only 162 kDa. Perhaps most importantly, we provide here a dataset for such a smallish protein complex for further development of software accounting for continuous conformational heterogeneity in cryo-EM images.
Cell-induced collagen alignment in a 3D in vitro culture during extracellular matrix production.
Schaart JM, Kea-Te Lindert M, Roverts R … +3 more , Nijhuis WH, Sommerdijk N, Akiva A
J Struct Biol · 2024 Jun · PMID 38697586 · Publisher ↗
The bone extracellular matrix consists of a highly organized collagen matrix that is mineralized with carbonated hydroxyapatite. Even though the structure and composition of bone have been studied extensively, the mechan...
The bone extracellular matrix consists of a highly organized collagen matrix that is mineralized with carbonated hydroxyapatite. Even though the structure and composition of bone have been studied extensively, the mechanisms underlying collagen matrix organization remain elusive. In this study, we used a 3D cell culture system in which osteogenic cells deposit and orient the collagen matrix that is subsequently mineralized. Using live fluorescence imaging combined with volume electron microscopy, we visualize the organization of the cells and collagen in the cell culture. We show that the osteogenically induced cells are organizing the collagen matrix during development. Based on the observation of tunnel-like structures surrounded by aligned collagen in the center of the culture, we propose that osteoblasts organize the deposited collagen during migration through the culture. Overall, we show that cell-matrix interactions are involved in collagen alignment during early-stage osteogenic differentiation and that the matrix is organized by the osteoblasts in the absence of osteoclast activity.
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