Aquilos-CryoFIB at Diamond
Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[33808]
Open Access
Abstract: Inorganic minerals that form via regulated biological processes exhibit remarkable properties. This is due to the involvement of macromolecules that control biomineralization. Even though the interactions of these biopolymers with solid mineral phases are intensely studied, not much is known about their involvement in the preceding steps of intracellular transport of the mineral building blocks. In this work, the model system of coccolith calcite crystallization is utilized to address the role of mineral-associated polysaccharides in the transport of calcium ions. State-of-the-art cryo-electron tomography is used to image in situ ion-rich dense phases in the wild-type and in two mutant strains, defected in coccolith production. The results show that the abundance and solubility of the calcium-rich condensates need to be finely tuned for proper crystallization. When the native macromolecular assemblage is compromised, calcium is still present in the calcifying fluid as a solute, but this is not sufficient for coccolith development. These results suggest that biomineralizing systems achieve superior regulation of crystallization due to the use of dense macromolecule-rich phases.
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Nov 2024
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Krios II-Titan Krios II at Diamond
Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[23047]
Open Access
Abstract: The widespread adoption of cryoEM technologies for structural biology has pushed the discipline to new frontiers. A significant worldwide effort has refined the single-particle analysis (SPA) workflow into a reasonably standardized procedure. Significant investments of development time have been made, particularly in sample preparation, microscope data-collection efficiency, pipeline analyses and data archiving. The widespread adoption of specific commercial microscopes, software for controlling them and best practices developed at facilities worldwide has also begun to establish a degree of standardization to data structures coming from the SPA workflow. There is opportunity to capitalize on this moment in the maturation of the field, to capture metadata from SPA experiments and correlate the metadata with experimental outcomes, which is presented here in a set of programs called EMinsight. This tool aims to prototype the framework and types of analyses that could lead to new insights into optimal microscope configurations as well as to define methods for metadata capture to assist with the archiving of cryoEM SPA data. It is also envisaged that this tool will be useful to microscope operators and facilities looking to rapidly generate reports on SPA data-collection and screening sessions.
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Apr 2024
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Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
Krios V-Titan Krios V at Diamond
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Open Access
Abstract: Electron cryo-microscopy image-processing workflows are typically composed of elements that may, broadly speaking, be categorized as high-throughput workloads which transition to high-performance workloads as preprocessed data are aggregated. The high-throughput elements are of particular importance in the context of live processing, where an optimal response is highly coupled to the temporal profile of the data collection. In other words, each movie should be processed as quickly as possible at the earliest opportunity. The high level of disconnected parallelization in the high-throughput problem directly allows a completely scalable solution across a distributed computer system, with the only technical obstacle being an efficient and reliable implementation. The cloud computing frameworks primarily developed for the deployment of high-availability web applications provide an environment with a number of appealing features for such high-throughput processing tasks. Here, an implementation of an early-stage processing pipeline for electron cryotomography experiments using a service-based architecture deployed on a Kubernetes cluster is discussed in order to demonstrate the benefits of this approach and how it may be extended to scenarios of considerably increased complexity.
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Mar 2024
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Open Access
Abstract: Simulations of cryo-electron microscopy (cryo-EM) images of biological samples can be used to produce test datasets to support the development of instrumentation, methods, and software, as well as to assess data acquisition and analysis strategies. To be useful, these simulations need to be based on physically realistic models which include large volumes of amorphous ice. The gold standard model for EM image simulation is a physical atom-based ice model produced using molecular dynamics simulations. Although practical for small sample volumes; for simulation of cryo-EM data from large sample volumes, this can be too computationally expensive. We have evaluated a Gaussian Random Field (GRF) ice model which is shown to be more computationally efficient for large sample volumes. The simulated EM images are compared with the gold standard atom-based ice model approach and shown to be directly comparable. Comparison with experimentally acquired data shows the Gaussian random field ice model produces realistic simulations. The software required has been implemented in the Parakeet software package and the underlying atomic models are available online for use by the wider community.
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Nov 2023
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Krios I-Titan Krios I at Diamond
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Open Access
Abstract: Membrane proteins (MPs) are essential components of all biological membranes, contributing to key cellular functions that include signalling, molecular transport and energy metabolism. Consequently, MPs are important biomedical targets for therapeutics discovery. Despite hardware and software developments in cryo-electron microscopy, as well as MP sample preparation, MPs smaller than 100 kDa remain difficult to study structurally. Significant investment is required to overcome low levels of naturally abundant protein, MP hydrophobicity as well as conformational and compositional instability. Here we have reviewed the sample preparation approaches that have been taken to successfully express, purify and prepare small MPs for analysis by cryo-EM (those with a total solved molecular weight of under 100 kDa), as well as examining the differing approaches towards data processing and ultimately obtaining a structural solution. We highlight common challenges at each stage in the process as well as strategies that have been developed to overcome these issues. Finally, we discuss future directions and opportunities for the study of sub-100 kDa membrane proteins by cryo-EM.
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Mar 2023
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Maud
Dumoux
,
Thomas
Glen
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Jake L. R.
Smith
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Elaine M. L.
Ho
,
Luis Ma
Perdigão
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Avery
Pennington
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Sven
Klumpe
,
Neville B. Y.
Yee
,
David A.
Farmer
,
Pui Y. A.
Lai
,
William
Bowles
,
Ron
Kelley
,
Jürgen M
Plitzko
,
Liang
Wu
,
Mark
Basham
,
Daniel K.
Clare
,
C. Alistair
Siebert
,
Michele C.
Darrow
,
James H.
Naismith
,
Michael
Grange
Open Access
Abstract: Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of sub-cellular structures on the mesoscale (10 nm to 10 µm). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents' hydration shells for in-situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining, and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM (cryo-pFIB/SEM). We evaluated the choice of plasma ion source and imaging regimes to produce high quality SEM images of a range of different biological samples. Using an automated workflow we produced three dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20 to 50 nm. Additionally, a tag-free localisation tool for regions of interest is needed to drive the application of in-situ structural biology towards tissue. The combination of serial FIB/SEM with plasma-based ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 µm) to produce lamellae for cryogenic electron tomography.
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Feb 2023
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I03-Macromolecular Crystallography
Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
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Halina
Mikolajek
,
Miriam
Weckener
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Z. Faidon
Brotzakis
,
Jiandong
Huo
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Evmorfia V.
Dalietou
,
Audrey
Le Bas
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Pietro
Sormanni
,
Peter J.
Harrison
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Philip N.
Ward
,
Steven
Truong
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Lucile
Moynie
,
Daniel K.
Clare
,
Maud
Dumoux
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Joshua
Dormon
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Chelsea
Norman
,
Naveed
Hussain
,
Vinod
Vogirala
,
Raymond J.
Owens
,
Michele
Vendruscolo
,
James
Naismith
Diamond Proposal Number(s):
[27031, 27051, 29666]
Open Access
Abstract: Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure–activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein–nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.
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Jul 2022
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Open Access
Abstract: The specimen preparation process is a key determinant in the success of any cryo electron microscopy (cryoEM) structural study and until recently had remained largely unchanged from the initial designs of Jacques Dubochet and others in the 1980s. The process has transformed structural biology, but it is largely manual and can require extensive optimisation for each protein sample. The chameleon instrument with its self-wicking grids and fast-plunge freezing represents a shift towards a robust, automated, and highly controllable future for specimen preparation. However, these new technologies require new workflows and an understanding of their limitations and strengths. As early adopters of the chameleon technology, we report on our experiences and lessons learned through case studies. We use these to make recommendations for the benefit of future users of the chameleon system and the field of cryoEM specimen preparation generally.
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Jun 2022
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Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[26464, 28151]
Open Access
Abstract: Developments in cryo-EM have allowed atomic or near-atomic resolution structure determination to become routine in single particle analysis (SPA). However, near-atomic resolution structures determined using cryo-electron tomography and sub-tomogram averaging (cryo-ET STA) are much less routine. In this paper, we show that by collecting cryo-ET STA data using the same conditions as SPA, with both Correlated Double Sampling (CDS) and super-resolution mode, allowed apoferritin to be reconstructed out to the physical Nyquist frequency of the images. Even with just two tilt series, STA yields an apoferritin map at 2.9 Å resolution. These results highlight the exciting potential of cryo-ET STA in the future of protein structure determination. While processing SPA data recorded in super-resolution mode may yield structures surpassing the physical Nyquist limit, processing cryo-ET STA data in super-resolution mode gave no additional resolution benefit. We further show that collecting SPA data in super-resolution mode, with CDS activated, reduces the estimated B-factor, leading to a reduction in the number of particles required to reach a target resolution without compromising data size on disk and area imaged in SerialEM. However, collecting SPA data in CDS does reduce throughput, given that a similar resolution structure, with a slightly larger B-factor, is achievable with optimised parameters for speed in EPU (without CDS).
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Apr 2022
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[26464]
Abstract: Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below subnanometer resolution and has led to unprecedented insights into the inner working of molecular machines in their native environment, as well as their functional relevant conformations and spatial distribution within biological cells or tissues. Given the tremendous potential of cryo-electron tomography STA in in situ structural cell biology, we previously developed emClarity, a graphics processing unit-accelerated image-processing software that offers STA and classification of macromolecular complexes at high resolution. However, the workflow remains challenging, especially for newcomers to the field. In this protocol, we describe a detailed workflow, processing and parameters associated with each step, from initial tomography tilt-series data to the final 3D density map, with several features unique to emClarity. We use four different samples, including human immunodeficiency virus type 1 Gag assemblies, ribosome and apoferritin, to illustrate the procedure and results of STA and classification. Following the processing steps described in this protocol, along with a comprehensive tutorial and guidelines for troubleshooting and parameter optimization, one can obtain density maps up to 2.8 Å resolution from six tilt series by cryo-electron tomography STA.
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Jan 2022
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