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Grant, T.

Publications and source records attributed to Grant, T..

At least 19 recordsLinked to original sources

A shark variable new antigen receptor recognizes an occluded epitope of fibroblast activation protein

Variable new antigen receptors (VNARs) are the smallest naturally occurring antibody binding domains. Their size allows VNARs to access sterically restricted epitopes that are inaccessible to conventional antibodies. We recently identified a suite of VNARs that target fibroblast activation protein (FAP), a stromal serine protease indicative of extracellular matrix remodeling. The presence of FAP on the surface of cancer-associated fibroblasts (CAFs) that promote immunosuppression has made FAP a compelling therapeutic target for cancer therapy. Although antibodies targeting FAP have been developed, there is a paucity of information on how biologics engage FAP. Here, we used single-particle cryogenic electron microscopy (cryo-EM) to compare FAP recognition of three antibody architectures: a shark-derived VNAR, variable heavy (VH) and light domains (VL) of a humanized Immunoglobulin G (IgG), and a camelid-derived VHH. The humanized VH-VL domains and camelid VHH both target a solvent-exposed {beta}-propeller domain, whereas the VNAR binds a highly conserved, topologically recessed epitope at the FAP dimer interface. Radical-footprinting mass spectrometry (MS) further mapped two additional immune-derived VNARs to distinct FAP surfaces outside the shared {beta}-propeller epitope. These findings demonstrate how unique VNAR architecture can expand access to underexplored FAP surfaces and establish a structural framework for rational multiepitope targeting strategies.

biochemistry↗

Diversifying particle-orientation distributions in cryo-EM with LEA protein additives

Preferential orientation at the air-water interface (AWI) remains a persistent challenge in cryo-electron microscopy, often requiring extensive optimization or specialized grid preparation strategies. Here, we show that late embryogenesis abundant (LEA) proteins, previously shown to protect fragile samples from AWI-induced damage, can be leveraged to resolve sample orientation bias. The addition of LEA proteins can yield either new orientation distributions or more isotropic ones in samples suffering from orientation biases. These findings establish LEA proteins as a practical class of cryo-EM additives that can reduce AWI-induced damage and improve particle-orientation distributions in single-particle analysis.

biophysics↗

Extending structural surfaceomics to identify aberrant conformations of tumor surface proteins as potential immunotherapy targets

The complement of tumor cell surface proteins, or "surfaceome", is a rich source of potential immunotherapy targets. To move beyond expression-based target discovery, we previously described "structural surfaceomics," combining crosslinking mass spectrometry (XL-MS) with surface protein biotinylation to identify conformation-selective targets. In our prior work, we applied this method to a single model of acute myeloid leukemia (AML), identifying active integrin beta-2 as a promising target. Here, we expand structural surfaceomics to identify additional immunotherapy targets and surface protein biology across additional models of AML, multiple myeloma, and prostate cancer, as well as donor peripheral blood mononuclear cells. Utilizing these models and different chemical crosslinkers, we compile an extensive database of 5,209 crosslinks. We characterize both shared and unique crosslink-based features, identifying 1,612 disease model-specific crosslinks, including 212 potentially defining tumor-specific conformations based on distance constraint violations relative to AlphaFold predictions. We further implement a suite of emerging modeling tools to predict tumor-specific protein structures. We probe crosslinking patterns suggesting multiple myeloma-specific CD48 and AML-specific integrin 1/{beta}4 heterodimer conformations. This work establishes a resource for cancer structural biology by implementation of structural surfaceomics. Our findings also point toward more realistic protein design models, potentially enabling systematic detection of targetable cancer-specific epitopes for next-generation immunotherapies.

cancer biology↗

Short communication: Oral microbiome as a potential proxy for grazing livestock methane emissions

Enteric methane emissions from ruminant livestock contribute to global warming, creating an urgent need for effective mitigation strategies that do not compromise animal productivity and welfare. Methanogenic archaea within the rumen microbiome drive enteric methane emissions. However, large-scale rumen-fluid sampling in commercial production systems is impractical, due to its invasive nature and the associated logistical challenges. This study hypothesised that rumination enables the capture of rumen microbial signals within the oral cavity and using oral microbiome profiles to provide a practical, non-invasive alternative method for proxy methane phenotyping in commercial production systems. To test the hypothesis, we estimated the oral microbiability, defined as the proportion of phenotypic variance in methane emissions explained by oral microbiome variation. Samples were collected from 209 animals across two trials in Queensland, Australia. Oral microbiome samples were obtained from all animals, with paired rumen samples in one trial, and methane emissions were measured using either the sulphur hexafluoride (SF6) tracer technique or the GreenFeed system. Microbial features were characterised using taxonomic and functional annotations, and microbiability was estimated using mixed linear models incorporating microbiome-based relationship matrices. Although the small sample size limited strong conclusions, the oral microbiability estimates reported in this study were comparable to those derived from rumen samples. Functional microbial profiles generally explained a greater proportion of methane variation than taxonomic profiles, suggesting that microbial function is more closely linked to methane production than community composition alone. However, these differences were not statistically significant due to large standard errors. These findings suggest that oral microbiome sampling potentially provides a practical, minimally invasive, scalable proxy method for methane emissions of individual cattle in grazing systems, where direct methane gas measurements are labour-intensive and difficult to implement. Integrating oral microbiome profiles in the existing breeding model with the host genetics, weight and environmental factors could provide a promising pathway for enabling selection for low emissions and advancing reduced emissions livestock farming under real-world production conditions. Lay summaryCattle produce methane as part of their normal digestion and this contributes to climate change. Reducing methane emission in grazing livestock systems is therefore important. However, measuring methane from individual grazing animals is difficult, costly, and often impractical under commercial conditions. The rumen microbiome has been used as a proxy for estimating methane emissions, but collecting rumen samples is invasive and impractical for large-scale use. Because rumination transfers material from the rumen to the mouth, we investigated whether microbes found in cattle mouths could also be used to estimate how much methane an individual animal produced. We suggest that mouth-swab sampling method can be an alternative to rumen fluid sampling because it was less invasive, relatively quick and practically applicable in commercial conditions. Importantly, the microbiome explained a meaningful proportion of the between-animal variation for methane emission. This suggests that collection of mouth swabs is a potentially scalable alternative proxy method to identify cattle that naturally produce less methane. Overall, our findings support the potential use of oral ruminant microbial information to improve breeding and management strategies aimed at reducing methane emissions while maintaining productive livestock systems. Teaser TextThis study demonstrates that collecting oral swabs from the mouths of grazing beef cattle could provide a scalable method to estimate individual methane emissions in commercial production systems, offering a practical alternative to invasive rumen sampling and complex gas measurement systems. These findings support the development of scalable breeding and management strategies for methane mitigation in large-scale livestock production systems.

microbiology↗

ImmTACs overcome cytotoxic T cell suppression

Immune mobilizing monoclonal TCR against cancer (ImmTAC) are cancer therapeutics that activate T cells through recognition of a tumor-associated antigenic MHC/peptide complex. A first-in-class ImmTAC, Tebentafusp, is approved for the treatment of metastatic uveal melanoma. While clinical efficacy is thus established, the cellular mechanisms underpinning ImmTAC action are not fully resolved. Using a recently established experimental strategy to generate suppressed human primary cytotoxic T lymphocytes (CTL), we have investigated an ImmTAC that recognizes a peptide derived from the tumor associated antigen NY-ESO-1 in comparison to direct engagement of a TCR recognizing the same MHC/peptide complex. In response to endogenous antigen presentation, ImmTACs could elicit tumor cell cytolysis by suppressed CTL, but not IFN{gamma} secretion, in a manner dependent on the engager affinity for CD3{varepsilon}. ImmTACs enhanced the efficient execution of subcellular CTL polarization steps required for effective cytolysis and could trigger calcium signaling. These data establish that ImmTACs activate CTL similarly to direct engagement of a TCR by MHC/peptide and are likely to retain this capability under suppressive conditions such as in the tumor microenvironment.

immunology↗

Visualization of the complete primosome reveals the structural mechanisms governing DNA replication restart

Replication restart pathways reinitiate DNA replication processes following their premature termination. In Escherichia coli, this essential process begins with the regulated assembly of the primosome complex, comprising the PriA, PriB, and DnaT proteins, onto an abandoned replication fork. Here, we present two distinct primosome structures. One represents an intermediate stage in primosome assembly with a single DnaT C-terminal domain (DnaTCTD) bound to PriA/PriB/DNA. The second captures the mature primosome, in which filamentation of multiple DnaTCTD molecules catalyzes the handoff of the single-stranded DNA lagging strand from PriB to DnaT. The DnaT N-terminal domain forms a separate, independent oligomer in the mature structure. Taken together, our results detail the molecular mechanisms underlying replication restart initiation and regulation and suggest an unexpected mechanistic similarity between DnaT and the canonical initiator protein DnaA.

biochemistry↗

Atlas of lysine acetylation in the mouse

Lysine acetylation has widespread ramifications from genetic regulatory effects to modulation of enzymatic function. With improved acetyl-lysine enrichment technologies and advances in mass spectrometer speed and sensitivity, we present a comprehensive atlas of the mouse acetylome comprising 17,952 unique lysine acetylation sites across 4,340 proteins and 15 tissues. This resource, which nearly doubles the known mouse acetyl-lysine catalog, shows at least 14% of the acetylome is shared across tissues. We focus our investigation on several acetylated proteins, including ribosomal acetylation and its potential to extend ribosomal half-life in the liver and pancreas. Additionally, we identify a novel acetylation event in the active site of carnitine O-acetyltransferase (Crat) that also mirrors tissue-specific Crat activity. By integrating these data with human pathogenic variants, we identify acetyl-lysine residues on cardiac troponin and homogentisate dioxygenase that likely mimic disease-causing mutations. This resource provides a foundational framework for investigating protein acetylation in metabolic health and disease.

biochemistry↗

Improved cryo-EM reconstruction of sub-50 kDa complexes using 2D template matching

Visualizing the structures of small proteins and complexes has been a longstanding challenge in single-particle cryo-EM. Some of these targets have been successfully resolved by binding to antibody fragments (Fabs) or fusing with external scaffolds to increase their size. Recent advances in conventional single-particle techniques have enabled the determination of an increasing number of structures smaller than 100 kDa, achieving resolutions relevant to drug research. Compared to X-ray crystallography, cryo-EM preserves the near-native states of biomolecules, can resolve structural heterogeneity, and has the potential to apply to a wide range of targets. In this work, we demonstrate that the alignment and reconstruction of small macromolecular complexes can be improved using high-resolution structures as priors combined with 2D template matching. Using this method, we reconstructed a previously intractable [~] 43 kDa protein kinase and improved the density of its ligand-binding site. Our theoretical analysis predicts that this method can further extend single-particle cryo-EM to important drug-binding complexes well below 50 kDa. Cryo-EM | 3D reconstruction | signal detection | template matching 61

biophysics↗

Top-Down Scoring of Spectral Fitness by Image Analysis for Protein Structure Validation

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for protein structure determination, but traditional approaches require extensive manual assignment of hundreds to thousands of resonances. Here we present NMRFAM-BPHON, a novel "top-down" approach that treats experimental NMR spectra as continuous grayscale images and quantitatively scores the agreement with simulated spectra generated from candidate protein structures. This method does not require complete resonance assignments, though it can incorporate experimental chemical shifts when available to improve performance. The simulated spectra are generated from postulated resonance assignments, which can be derived either from empirical database predictions, direct interpretation, or a hybrid combination. BPHON employs a physics-based approximate polarization transfer model to predict cross-peak intensities from the internuclear distances in the decoy structure, and models the peak lineshapes using empirical, bulk T2 relaxation rates and literature values for scalar couplings. The resulting simulated spectra are scored relative to the experimental data by normalized cross correlation, yielding a fitness score between 0 and 1. We demonstrate BPHONs ability to discriminate structural models, particularly in the case of 13C-detected magic angle spinning solid-state NMR spectra. The software is packaged with a user-friendly graphical user interface for ChimeraX, enabling advanced NMR analysis accessible without requiring extensive manual analysis.

biophysics↗

NMR Spectral Alignment Utilizing a CryoEM Motion Correction Algorithm

With recent advances in magic-angle spinning (MAS) solid-state NMR (SSNMR) resolution, precise spectral alignment has become a critical bottleneck in data processing workflows. While solution NMR employs deuterium lock systems, most SSNMR probes still lack this capability; though a lock corrects for magnet drift and instabilities, it is not alone sufficient to account for field gradients, sample temperature differences, and pulse sequence effects that can contribute to referencing errors among several data sets. These offsets become particularly problematic in the lengthy multidimensional experiments that provide the foundation for resonance assignment and structure determination procedures. Currently, researchers rely on manual alignment through visual peak inspection--a qualitative approach that often overemphasizes prominent, outlying peaks while overlooking subtle, global patterns. This subjective process becomes increasingly impractical for use cases with lower sensitivity, such as large proteins with thousands of peaks. To address these challenges, here we present Automated NMR Spectral Alignment (ANSA), a program that adapts cryo-electron microscopy motion correction principles to NMR spectroscopy. ANSA treats NMR spectra as images and applies cross-correlation functions to determine optimal alignment, improving cross-correlation scores from 0.33 to 1.00 in controlled tests and achieving 0.96 correlation in real-world applications with previously misaligned spectra. The algorithm successfully aligns spectra across varying experimental conditions, corrects shifts in long-duration experiments, and works with 2D and 3D datasets, with approaches that can be readily extended to additional dimensions. By eliminating human bias and providing objective, consistent spectral alignment, ANSA enhances scientific rigor, improves reproducibility between experiments, and enables automation of critical data processing steps. The software is freely available as an open-source tool, ready for integration into existing NMR workflows.

biophysics↗

Interaction with tumor cell spheroids induces suppression in primary human cytotoxic T cells

Cytotoxic T lymphocytes (CTL) are key effectors in the anti-tumor immune response. However, their function is commonly suppressed in tumors in the form of exhausted CTL. Understanding molecular mechanisms of suppression and of therapeutics to overcome them is of substantial basic and translational importance yet hindered by limited access to large numbers of exhausted CTL in vitro. Here we use three-dimensional tissue culture to generate primary human CTL with suppressed function. Using a 21-antibody flow cytometry panel and determination of calcium signaling and cell couple maintenance, we show that these cells closely resemble exhausted CTL from tumors. For better understanding of in vitro human primary CTL as key tools in therapeutic development, before and after induction of suppression, we have determined the dependence of CTL function on technicalities of in vitro CTL generation, antigen dose and affinity across two T cell receptors and multiple tumor cell lines. We have investigated morphology and subcellular F-actin distributions of CTL as a key regulators of effector function. Primary human CTL formed cell couples with tumor target cells even in the absence of antigen. Yet, gradual stabilization of such cell couples was associated with increasing CTL effector function. Induction of suppression substantially destabilized CTL tumor cell couples. This comprehensive characterization of the phenotype of in vitro primary human CTL, including a suppressed state, should facilitate their wider use in basic and translational research.

immunology↗

SPOT-RASTR - a cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface

In cryogenic electron microscopy (cryo-EM), specimen preparation remains a bottleneck despite recent advancements. Classical plunge freezing methods often result in issues like aggregation and preferred orientations at the air/water interface. Many alternative methods have been proposed, but there remains a lack a universal solution, and multiple techniques are often required for challenging samples. Here, we demonstrate the use of lipid nanotubes with nickel NTA headgroups as a platform for cryo-EM sample preparation. His-tagged specimens of interest are added to the tubules, and they can be frozen by conventional plunge freezing. We show that the nanotubes protect samples from the air/water interface and promote a wider range of orientations. The reconstruction of average subtracted tubular regions (RASTR) method allows for the removal of the nanotubule signal from the cryo-EM images resulting in isolated images of specimens of interest. Testing with {beta}-galactosidase validates the methods ability to capture particles at lower concentrations, overcome preferred orientations, and achieve near-atomic resolution reconstructions. Since the nanotubules can be identified and targeted automatically at low magnification, the method enables fully automated data collection. Furthermore, the particles on the tubes can be automatically identified and centered using 2D classification enabling particle picking without requiring prior information. Altogether, our approach that we call specimen preparation on a tube RASTR (SPOT-RASTR) holds promise for overcoming air-water interface and preferred orientation challenges and offers the potential for fully automated cryo-EM data collection and structure determination.

molecular biology↗

TIM3 is a context-dependent co-regulator of cytotoxic T cell function

TIM3 is a co-regulatory receptor that is highly expressed on multiple immune cell types, including on T cells after prolonged exposure to antigen. It marks functionally suppressed cytotoxic T lymphocytes (CTL) in the tumor microenvironment. However, it is unresolved whether TIM3 acts directly on suppressed CTL. Moreover, the nature of TIM3 ligands remains controversial. Paradoxically, TIM3 combines inhibitory function in vivo with costimulatory signaling capability in vitro. Here we have investigated TIM3 in the direct interaction of suppressed murine and human CTL with tumor target cell using spheroids. TIM3 directly inhibited the function of such CTL. TIM3 regulated the ability of suppressed CTL to polarize their cytoskeleton as a required step in cytolysis. Expression of CEACAM1 in cis, on the CTL, blocked TIM3 function, expression of CEACAM1 and galectin9 in trans, on the tumor target cells, enhanced TIM3 function. TIM3 only functioned as an inhibitory receptor on the spheroid-suppressed CTL, not on active CTL in a two-dimensional tissue culture model. These data suggest that TIM3 amplifies T cell function, serving as a co-inhibitory or co-stimulatory receptor depending on the functional context of the T cell it is expressed on.

immunology↗

Cryogenic Soft Landing Improves Structural Preservation of Protein Complexes

We describe an apparatus for the cryogenic landing of particles from the ion beam of a mass spectrometer onto transmission electron microscope grids for cryo-electron microscopy. This system also allows for the controlled formation of thin films of amorphous ice on the grid surface. We demonstrate that as compared to room temperature landings, use of this cryogenic landing device greatly improves the structural preservation of deposited protein-protein complexes. Further, landing under cryogenic conditions can increase the diversity of particle orientations, allowing for improved 3D structural interpretation. Finally, we conclude that this approach allows for the direct coupling of mass spectrometry with cryo-electron microscopy.

biochemistry↗

Detection of a geminate photoproduct of bovine cytochrome c oxidase by time-resolved serial femtosecond crystallography

Cytochrome c oxidase (CcO) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, CcO has a unique binuclear center (BNC) comprised of a copper atom (CuB) and a heme a3 iron, where O2 binds and is reduced to water. CO is a versatile O2 surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine CcO (bCcO) revealed that photolyzing CO from the heme a3 iron leads to a metastable intermediate (CuB-CO), where CO is bound to CuB, before it escapes out of the BNC. Here, with a time-resolved serial femtosecond X-ray crystallography-based pump-probe method, we detected a geminate photoproduct of the bCcO-CO complex, where CO is dissociated from the heme a3 iron and moved to a temporary binding site midway between the CuB and the heme a3 iron, while the locations of the two metal centers and the conformation of the Helix-X, housing the proximal histidine ligand of the heme a3 iron, remain in the CO complex state. This new structure, combined with other reported structures of bCcO, allows the full definition of the ligand dissociation trajectory, as well as the associated protein dynamics.

biophysics↗

Nodavirus RNA Replication Crown Architecture Reveals Proto-Crown Precursor and Viral Protein A Conformational Switching

Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesicles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first revealed viral RNA replication proteins forming a 12-fold symmetric "crown" at the vesicle opening to the cytosol, an arrangement recently confirmed to extend to distantly related alphaviruses. Using cryo-electron microscopy (cryo-EM), we show that mature nodavirus crowns comprise two stacked 12-mer rings of multi-domain viral RNA replication protein A. Each ring contains an ~19 nm circle of C-proximal polymerase domains, differentiated by strikingly diverged positions of N-proximal RNA capping/membrane binding domains. The lower ring is a "proto-crown" precursor that assembles prior to RNA template recruitment, RNA synthesis and replication vesicle formation. In this proto-crown, the N-proximal segments interact to form a toroidal central floor, whose 3.1 [A] resolution structure reveals many mechanistic details of the RNA capping/membrane binding domains. In the upper ring, cryo-EM fitting indicates that the N-proximal domains extend radially outside the polymerases, forming separated, membrane-binding "legs." The polymerase and N-proximal domains are connected by a long linker accommodating the conformational switch between the two rings and possibly also polymerase movements associated with RNA synthesis and non-symmetric electron density in the lower center of mature crowns. The results reveal remarkable viral protein multifunctionality, conformational flexibility and evolutionary plasticity and new insights into (+)RNA virus replication and control. SignificancePositive-strand RNA viruses - including coronaviruses, alphaviruses, flaviviruses and many other medically and economically important pathogens - replicate their RNA genomes by virus-encoded machinery that has been poorly characterized. Using an advanced nodavirus model, we identify a major precursor in RNA replication complex assembly and show it to be a 12-mer ring of viral RNA replication protein A, whose single particle cryo-EM structure reveals functional features of its membrane interaction, assembly, polymerase and RNA capping domains. We further show that fully functional RNA replication complexes acquire a second 12-mer ring of protein A in alternate conformation atop the first, and a central density likely to represent another polymerase conformation. These findings provide strong foundations for understanding, controlling and beneficially using such viruses.

microbiology↗

From primordial clocks to circadian oscillators

Circadian rhythms play an essential role in many biological processes and surprisingly only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and central component of the clock, with subsequent additions of KaiB and KaiA to regulate its phosphorylation state for time synchronization. The canonical KaiABC system in cyanobacteria is well understood, but little is known about more ancient systems that possess just KaiBC, except for reports that they might exhibit a basic, hourglass-like timekeeping mechanism. Here, we investigate the primordial circadian clock in Rhodobacter sphaeroides (RS) that contains only KaiBC to elucidate its inner workings despite the missing KaiA. Using a combination X-ray crystallography and cryo-EM we find a novel dodecameric fold for KaiCRS where two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the C-terminal extension of KaiCRS and serves as an ancient regulatory moiety later superseded by KaiA. A coiled-coil register shift between daytime- and nighttime-conformations is connected to the phosphorylation sites through a long-range allosteric network that spans over 160 [A]. Our kinetic data identify the difference in ATP-to-ADP ratio between day and night as the environmental cue that drives the clock and further unravels mechanistic details that shed light on the evolution of self-sustained oscillators.

biochemistry↗

3D Structure Determination of Protein Complexes using Matrix-Landing Mass Spectrometry

Native mass spectrometry (MS) is an emerging technology that can provide complementary data to electron microscopy (EM) for protein structure characterization. Beyond the ability to provide mass measurements of gas-phase biomolecular ions, MS instruments offer the ability to purify, select, and precisely control the spatial location of these ions. Here we present a modified Orbitrap MS system capable of depositing a native MS ion beam onto EM grids. We further describe use of a chemical landing matrix that both preserves and protects the structural integrity of the deposited particles. With this system we obtained the first 3D reconstructed structure of gas-phase, deposited biomolecular ions - the 800 KDa protein complex GroEL. These data provide direct evidence that non-covalent protein complexes can indeed retain their condensed-phase structures following ionization and vaporization. Finally, we describe how further developments of this technology could pave the way to an integrated MS-EM technology with promise to provide improved cryo-EM sample preparation over conventional plunge-freezing techniques.

molecular biology↗