Biology subjects

Noble, A. J.

Publications and source records attributed to Noble, A. J..

18 recordsLinked to original sources

bioRxiv2024

VirtualIce: Half-synthetic CryoEM Micrograph Generator

Single particle cryo-electron microscopy (cryoEM) is going through a phase of rapid optimization focused on increasing the efficiency, accuracy, and automation of every step in the data pipeline. Machine learning models in particular have been making substantial advances in cryoEM, however their impact has been limited. This limitation is due in part to the lack of availability of realistic ground-truth datasets for training and evaluation of cryoEM machine learning models. To address this limitation and accelerate this phase, we introduce VirtualIce which generates half-synthetic micrographs by projecting proteins onto real, curated micrographs of vitrified buffer. VirtualIce provides configurable features including noise simulation, realistic particle distributions, particle overlapping, particle aggregation, filtering obscured regions, and multiple structures per micrograph. VirtualIce may be a valuable resource to help visualize unknown proteins, accelerate the development of automated data collection and processing pipelines, and develop cryoEM algorithms.

molecular biology↗

bioRxiv2024

Myosin forces elicit an F-actin structural landscape that mediates mechanosensitive protein recognition

Cells mechanically interface with their surroundings through cytoskeleton-linked adhesions, allowing them to sense physical cues that instruct development and drive diseases such as cancer. Contractile forces generated by myosin motor proteins mediate these mechanical signal transduction processes through unclear protein structural mechanisms. Here, we show that myosin forces elicit structural changes in actin filaments (F-actin) that modulate binding by the mechanosensitive adhesion protein -catenin. Using correlative cryo-fluorescence microscopy and cryo-electron tomography, we identify F-actin featuring domains of nanoscale oscillating curvature at cytoskeleton-adhesion interfaces enriched in zyxin, a marker of actin-myosin generated traction forces. We next introduce a reconstitution system for visualizing F-actin in the presence of myosin forces with cryo-electron microscopy, which reveals morphologically similar superhelical F-actin spirals. In simulations, transient forces mimicking tugging and release of filaments by motors produce spirals, supporting a mechanistic link to myosins ATPase mechanochemical cycle. Three-dimensional reconstruction of spirals uncovers extensive asymmetric remodeling of F-actins helical lattice. This is recognized by -catenin, which cooperatively binds along individual strands, preferentially engaging interfaces featuring extended inter-subunit distances while simultaneously suppressing rotational deviations to regularize the lattice. Collectively, we find that myosin forces can deform F-actin, generating a conformational landscape that is detected and reciprocally modulated by a mechanosensitive protein, providing a direct structural glimpse at active force transduction through the cytoskeleton.

biophysics↗

bioRxiv2024

CryoCycle your grids: Plunge vitrifying and reusing clipped grids to advance cryoEM democratization

CryoEM democratization is hampered by access to costly plunge-freezing supplies. We introduce methods, called CryoCycle, for reliably blotting, vitrifying, and reusing clipped cryoEM grids. We demonstrate that vitreous ice may be produced by plunging clipped grids with purified proteins into liquid ethane and that clipped grids may be reused several times for different protein samples. Furthermore, we demonstrate the vitrification of thin areas of cells prepared on gold-coated, pre-clipped grids.

molecular biology↗

bioRxiv2023

Smart Leginon enables cryoEM screening automation across multiple grids

SummaryCryoEM multi-grid screening is often a tedious process that demands hours of attention. Here, this protocol shows how to set up standard Leginon collection and Smart Leginon Autoscreen to automate this process. This protocol can be applied to the majority of cryoEM holey foil grids. Advancements in cryo-electron microscopy (cryoEM) techniques over the past decade have allowed structural biologists to routinely resolve macromolecular protein complexes to near-atomic resolution. The general workflow of the entire cryoEM pipeline involves iterating between sample preparation, cryoEM grid preparation, and sample/grid screening before moving on to high-resolution data collection. Iterating between sample/grid preparation and screening is typically a major bottleneck for researchers, as every iterative experiment must optimize for sample concentration, buffer conditions, grid material, grid hole size, ice thickness, and protein particle behavior in the ice, amongst other variables. Furthermore, once these variables are satisfactorily determined, grids prepared under identical conditions vary widely in whether they are ready for data collection, so additional screening sessions prior to selecting optimal grids for high-resolution data collection are recommended. This sample/grid preparation and screening process often consumes several dozen grids and days of operator time at the microscope. Furthermore, the screening process is limited to operator/microscope availability and microscope accessibility. Here, we demonstrate how to use Leginon and Smart Leginon Autoscreen to automate the majority of cryoEM grid screening. Autoscreen combines machine learning, computer vision algorithms, and microscope-handling algorithms to remove the need for constant manual operator input. Autoscreen can autonomously load and image grids with multi-scale imaging using an automated specimen-exchange cassette system, resulting in unattended grid screening for an entire cassette. As a result, operator time for screening 12 grids may be reduced to [~]10 minutes with Autoscreen compared to [~]6 hours using previous methods which are hampered by their inability to account for high variability between grids. This protocol and video tutorial first introduces basic Leginon setup and functionality, then demonstrates Autoscreen functionality step-by-step from the creation of a template session to the end of a 12 grid automated screening session.

molecular biology↗

bioRxiv2022

Fully Automated Multi-Grid Cryo-EM Screening using Smart Leginon

Single particle cryo-electron microscopy (cryoEM) is a swiftly growing method for understanding protein structure. With increasing demand for high-throughput, high-resolution cryoEM services comes greater demand for rapid and automated cryoEM grid and sample screening. During screening, optimal grids and sample conditions are identified for subsequent high-resolution data collection. Screening is a major bottleneck for new cryoEM projects because grids must be optimized over several factors, including grid type, grid hole size, sample concentration, buffer conditions, ice thickness, and particle behaviors. Even for mature projects, multiple grids are commonly screened to select a subset for high-resolution data collection. Here, machine learning and novel, purpose-built image processing and microscope-handling algorithms are incorporated into the automated data collection software, Leginon, to provide an open-source solution for fully automated, high-throughput grid screening. This new version, broadly called Smart Leginon, emulates the actions of an operator in identifying areas on the grid to explore as potentially useful for data collection. Smart Leginon Autoscreen sequentially loads and examines grids from an automated specimen exchange system to provide completely unattended grid screening across a set of grids. Comparisons between a multi-grid Autoscreen session and conventional manual screening by five expert microscope operators are presented. On average, Autoscreen reduces operator time from [~]6 hours to <10 minutes and provides a comparable percentage of suitable images for evaluation as the best operator. Smart Leginons ability to target holes that are particularly difficult to identify is analyzed. Finally, Smart Leginons utility is illustrated with three real-world multi-grid user screening/collection sessions, demonstrating the efficiency and flexibility of the software package. Smart Leginons fully automated functionality significantly reduces the burden on operator screening time, improves the throughput of screening, and recovers idle microscope time, thereby improving availability of cryoEM services.

biophysics↗

bioRxiv2022

Architecture of the human erythrocyte ankyrin-1 complex

The stability and shape of the erythrocyte membrane is provided by the ankyrin-1 complex, but how it tethers the spectrin-actin cytoskeleton to the lipid bilayer and the nature of its association with the band 3 anion exchanger and the Rhesus glycoproteins remains unknown. Here we present structures of ankyrin-1 complexes purified from human erythrocytes. We reveal the architecture of a core complex of ankyrin-1, the Rhesus proteins RhAG and RhCE, the band 3 anion exchanger, protein 4.2 and glycophorin A. The distinct T-shaped conformation of membrane-bound ankyrin-1 facilitates recognition of RhCE and unexpectedly, the water channel aquaporin-1. Together, our results uncover the molecular details of ankyrin-1 association with the erythrocyte membrane, and illustrate the mechanism of ankyrin-mediated membrane protein clustering.

biochemistry↗

bioRxiv2021

Hypomethylation in FASTKD1 detected in the association between in utero tobacco exposure and conduct problem in a New Zealand longitudinal study

Despite the known adverse effects of in utero tobacco exposure on offspring health, maternal tobacco use during pregnancy remains prevalent and is a major driver of health inequalities. One such health inequality is the development of conduct problem (CP) in exposed offspring which may be mediated by methylation changes that persist into adulthood. Here we apply a genome-wide approach to probe the association between maternal tobacco use during pregnancy and CP outcomes in exposed offspring. We examined maternal tobacco use during pregnancy (in utero exposure) in the Christchurch Health and Development Study, a longitudinal birth cohort studied for over 40 years. We then evaluated the interaction between methylation effects of in utero exposure and CP score. When modelling this interaction between in utero exposure and CP score we detected nominal DNA methylation differences, at FASTKD1 which has roles in early development. Our observations are consistent with DNA methylation mediating the development of CP following in utero tobacco exposure. In addition, we detected nominal significance in FRMDA4 and MYO1G between individuals exposed to tobacco in utero and those that were unexposed, however these did not reach significance after adjustment for multiple testing. However due to limited power in our analysis, further studies are needed to investigate the interaction between in utero tobacco exposure and high CP health outcomes.

genomics↗

bioRxiv2021

Heterogeneous non-canonical nucleosomes predominate in yeast cells in situ

Nuclear processes depend on the organization of chromatin, whose basic units are cylinder-shaped complexes called nucleosomes. A subset of mammalian nucleosomes in situ (inside cells) resembles the canonical structure determined in vitro 25 years ago. Nucleosome structure in situ is otherwise poorly understood. Using cryo-ET and 3-D classification analysis of budding yeast cells, here we find that canonical nucleosomes account for less than 10% of total nucleosomes expected in situ. In a strain in which H2A-GFP is the sole source of histone H2A, class averages that resemble canonical nucleosomes both with and without GFP densities are found ex vivo (in nuclear lysates), but not in situ. These data suggest that the budding yeast intranuclear environment favors multiple non-canonical nucleosome conformations. Using the structural observations here and the results of previous genomics and biochemical studies, we propose a model in which the average budding yeast nucleosomes DNA is partially detached in situ.

cell biology↗

bioRxiv2020

Waffle method: A general and flexible approach for FIB-milling small and anisotropically oriented samples

Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens that do not vitrify well, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and cellular specimens that distribute poorly across grid squares. Here we present a general approach we call the Waffle Method which leverages high-pressure freezing to address these challenges. We illustrate the mitigation of these challenges by applying the Waffle Method and cryo-ET to reveal the macrostructure of the polar tube in microsporidian spores in multiple complementary orientations, which was previously not possible due to preferred orientation of the spores on the grid. We demonstrate the broadness of the Waffle Method by applying it to three additional cellular samples and a single particle sample using a variety of cryo-FIB-milling hardware, with both manual and automated approaches. We also present a unique and critical stress-relief gap designed specifically for waffled lamellae. Additionally, we describe applications of the Waffle Method which are currently being explored. We propose the Waffle Method as a way to achieve many of the advantages of cryo-liftout on the specimen grid while avoiding the long, challenging, and technically-demanding process required for cryo-liftout.

microbiology↗

bioRxiv2020

Epigenetic signatures associated with the observed link between maternal tobacco use during pregnancy, and offspring conduct problems in childhood and adolescence

Maternal tobacco smoking during pregnancy is a large driver of health inequalities and a higher prevalence of conduct problem has been observed in exposed offspring. Further, maternal tobacco use during pregnancy can also alter offspring DNA methylation. However, currently, limited molecular evidence have been found to support this observation. Thus we aim to examine the association between maternal tobacco use in pregnancy and whether offspring Conduct problems is mediated by tobacco exposure-induced via DNA methylation differences. Understanding the etiology of the causal link will be crucial in the early identification and treatment of CP in children and adolescents. DNA was sourced from the Christchurch Health and Development Study, a longitudinal birth cohort studied for over 40 years in New Zealand. Bisulfite-based amplicon sequencing of 10 loci known to play a role in neurodevelopment, or with associations with CP phenotypes, was undertaken. We identified nominally significant differential DNA methylation at specific CpG sites in CYP1A1, ASH2L and MEF2C in individuals with Conduct problems who were exposed to tobacco in utero. We conclude that environmentally-induced DNA methylation differences could play a role in the observed link between maternal tobacco use during pregnancy and childhood/adolescent Conduct problems However, larger sample sizes are needed to produce an adequate amount of power to investigate this interaction further.

genomics↗

bioRxiv2020

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs, and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.

biochemistry↗

bioRxiv2020

A validation of Illumina EPIC array system with bisulfite-based amplicon sequencing

The Illumina Infinium(R) MethylationEPIC BeadChip system (hereafter EPIC array) is considered to be the current gold standard detection method for assessing DNA methylation at the genome-wide level. EPIC arrays are used for hypothesis generation or pilot studies, the natural conclusion is to validate methylation candidates and expand these in a larger cohort, in a targeted manner. As such, an accurate smaller-scale, targeted technique, that generates data at the individual CpG level that is equivalent to the EPIC array, is needed. Here, we tested an alternative DNA methylation detection technique, known as bisulfite-based amplicon sequencing (BSAS), to determine its ability to validate CpG sites detected in EPIC array studies. BSAS was able to detect differential DNA methylation at CpG sites to a degree which correlates highly with the EPIC array system. However, BSAS correlated less well with EPIC array data when the magnitude of change via EPIC array was greater than 5%, suggesting that this lower specificity at larger differential methylation values is a consequence of PCR amplification that BSAS requires. However, our data suggests that BSAS does offer advantages that the EPIC array: BSAS amplifies a region of the genome (~500bp) around a CpG of interest, allowing analyses of other CpGs in the region that may not be present on the EPIC array, aiding discovery of novel CpG sites and differentially methylated regions of interest. We conclude that BSAS offers a valid investigative tool for specific regions of the genome that are currently not contained on the array system.

genomics↗

bioRxiv2019

Topaz-Denoise: general deep denoising models for cryoEM

Cryo-electron microscopy (cryoEM) is becoming the preferred method for resolving protein structures. Low signal-to-noise (SNR) in cryoEM images reduces the confidence and throughput of structure determination during several steps of data processing, resulting in impediments such as missing particle orientations. Denoising cryoEM images can not only improve downstream analysis but also accelerate the time-consuming data collection process by allowing lower electron dose micrographs to be used for analysis. Here, we present Topaz-Denoise, a deep learning method for reliably and rapidly increasing the SNR of cryoEM images and cryoET tomograms. By training on a dataset composed of thousands of micrographs collected across a wide range of imaging conditions, we are able to learn models capturing the complexity of the cryoEM image formation process. The general model we present is able to denoise new datasets without additional training. Denoising with this model improves micrograph interpretability, enabling us to solve the first 3D single particle closed and partially open structures of clustered protocadherin, an elongated particle with previously-elusive views. We then show for the first time that low dose collection, enabled by Topaz-Denoise, improves downstream analysis in addition to reducing data collection time. We also present the first general 3D denoising model for cryoET. Topaz-Denoise and pre-trained general models are now included in Topaz. We expect that Topaz-Denoise will be of broad utility to the cryoEM community for improving micrograph and tomogram interpretability and accelerating analysis.

bioinformatics↗

bioRxiv2019

Genome-wide DNA methylation analysis of heavy cannabis use in a New Zealand longitudinal cohort

Cannabis use is of increasing public health interest globally. Here we examined the effect of cannabis use, with and without tobacco, on genome-wide DNA methylation in a longitudinal birth cohort (Christchurch Health and Development Study). We found the most differentially methylated sites in cannabis with tobacco users were in the AHRR and F2RL3 genes, replicating previous studies on the effects of tobacco. Cannabis-only users had no evidence of differential methylation in these genes, or at any other loci at the epigenomewide significance level (P<10-7). However, there were 521 sites differentially methylated at P<0.001 which were enriched for genes involved in cardiomyopathy and neuronal signalling. Further, the most differentially methylated loci were associated with genes with reported roles in brain function (e.g. TMEM190, MUC3L, CDC20 and SP9). We conclude that the effects of cannabis use on the mature human blood methylome differ from, and are less pronounced than, the effects of tobacco use, and that larger sample sizes are required to investigate this further.

genomics↗

bioRxiv2018

Eliminating effects of particle adsorption to the air/water interface in single-particle cryo-electron microscopy: Bacterial RNA polymerase and CHAPSO

Preferred particle orientation presents a major challenge for many single particle cryoelectron microscopy (cryo-EM) samples. Orientation bias limits the angular information used to generate three-dimensional maps and thus affects the reliability and interpretability of the structural models. The primary cause of preferred orientation is presumed to be due to adsorption of the particles at the air/water interface during cryo-EM grid preparation. To ameliorate this problem, detergents are often added to cryo-EM samples to alter the properties of the air/water interface. We have found that many bacterial transcription complexes suffer severe orientation bias when examined by cryo-EM. The addition of non-ionic detergents, such as NP-40, does not remove the orientation bias but the Zwitter-ionic detergent CHAPSO significantly broadens the particle orientation distributions, yielding isotropically uniform maps. We used cryoelectron tomography to examine the particle distribution within the ice layer of cryo-EM grid preparations of Escherichia coli 6S RNA/RNA polymerase holoenzyme particles. In the absence of CHAPSO, essentially all of the particles are located at the ice surfaces. CHAPSO at the critical micelle concentration eliminates particle absorption at the air/water interface and allows particles to randomly orient in the vitreous ice layer. We find that CHAPSO eliminates orientation bias for a wide range of bacterial transcription complexes containing E. coli or Mycobacterium tuberculosis RNA polymerases. Findings of this study confirm the presumed basis for how detergents can help remove orientation bias in cryo-EM samples and establishes CHAPSO as a useful tool to facilitate cryo-EM studies of baterial transcription complexes.

biophysics↗

bioRxiv2018

Routine Determination of Ice Thickness for Cryo-EM Grids

Recent advances in instrumentation and automation have made cryo-EM a popular method for producing near-atomic resolution structures of a variety of proteins and complexes. Sample preparation is still a limiting factor in collecting high quality data. Thickness of the vitreous ice in which the particles are embedded is one of the many variables that need to be optimized for collection of the highest quality data. Here we present two methods, using either an energy filter or scattering outside the objective aperture, to measure ice thickness for potentially every image collected. Unlike geometrical or tomographic methods, these can be implemented directly in the single particle collection workflow without interrupting or significantly slowing down data collection. We describe the methods as implemented into the Leginon/Appion data collection workflow, along with some examples from test cases. Routine monitoring of ice thickness should prove helpful for optimizing sample preparation, data collection, and data processing.

biophysics↗

bioRxiv2018

Reducing effects of particle adsorption to the air-water interface in cryoEM

Most protein particles prepared in vitreous ice for single particle cryo-electron microscopy are adsorbed to air-water or substrate-water interfaces, potentially causing particles to adopt preferred orientations. Using the Spotiton robot and nanowire grids, we can significantly reduce air-water interface issues by decreasing the dwell time of particles in thin liquid films. We demonstrate this by using single particle cryoEM and cryoET on three biological samples.

biophysics↗

bioRxiv2017

Routine Single Particle CryoEM Sample and Grid Characterization by Tomography

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are freely floating away from the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1,000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

biophysics↗