EM Reconstruction Research Articles

INCF Program on Multiscale Modeling Workshop: From Cellular/Network Models to Tissue Simulation

The INCF Program on Multiscale Modeling (MSM) aims to promote the development of software infrastructure to facilitate neural modeling across many different scales. A new focus of the Program could be to support the transition from single neuron (network) modeling to neural tissue simulation. The aim would be to develop model description languages and the software infrastructure required to leverage existing molecular, neuron, and network models to build neural tissue models that accommodate both virtual tissue and reconstructed tissue modeling within a single framework. The aim of this workshop was to assess the current state of the field and available software infrastructure for tissue modeling from within the field of neuroscience and other fields in order to broadly define an outline of the development needed. The INCF will take the recommendations of this workshop to determine the need for subsequent actions and follow-up workshops, in order to direct future activities within the MSM Program, including the formation of new Program task force(s) as needed. In addition, The INCF aims to integrate these efforts with other INCF Programs such as those currently in progress in the Digital Brain Atlasing Program, Standards for Data Sharing Program and the Program on Ontologies of Neural Structures. The participants of the workshop suggested that the INCF could establish a working group to investigate how to describe spatial structures at the micro-scale, including the investigation of annotated mesh formats, mesh outputs from EM reconstruction, coordinate transforms, and markup schemes (Digital Atlasing, SBML). Participants noted that a thematic workshop on topic of ‘How to model when you do not know the parameters’ would be useful, with computational biologists included as participants. The participants also recommended that the INCF should announce an open call in the form of a competition for interested parties who want to commit to commenting on the state of the field for some of the use cases recommended during the discussion. The candidates should respond with a plan which will be reviewed against a set of criteria set forth during the workshop. The INCF should then hold followup workshops around the use cases with the output of these workshops being a set of recommendations for actions by the INCF. One identified use case linking different levels involves advancing the understanding of the relationship of sub cellular processes and cellular level electrical behavior. Since the workshop, a proposal has been developed further by the community for a workshop on the topic ‘Methods in bridging subcellular and cellular level models’.

Volume EM Reconstruction of Spinal Cord Reveals Wiring Specificity in Speed-Related Motor Circuits

Spinal interneurons coordinate the activity of motoneurons to generate the spatiotemporal patterns of muscle contractions required for vertebrate locomotion. It is controversial to what degree the orderly, gradual recruitment of motoneurons is determined by biophysical differences among them rather than by specific connections from presynaptic interneurons to subsets of motoneurons. To answer this question, we mapped all connections from two types of interneurons onto all motoneurons in a larval zebrafish spinal cord hemisegment, using serial block-face electron microscopy (SBEM). We found specific synaptic connectivity from dorsal but not from ventral excitatory ipsilateral interneurons, with large motoneurons, active only when strong force is required, receiving specific inputs from dorsally located interneurons, active only during fast swims. By contrast, the connectivity between inhibitory commissural interneurons and motoneurons lacks any discernible pattern. The wiring pattern is consistent with a recruitment mechanism that depends to a considerable extent on specific connectivity.

P-C5 Broadly neutralizing nanobodies selected from dromedary immune libraries with subtype C SOSIP Env glycoproteins: Optimization and preclinical development

Nanobodies or VHH are the smallest naturally occurring antibody fragments derived from heavy chain only antibodies from Camelidae. Due to their physicochemical properties (high stability, high affinity and target specificity, extended CDR3 loops and their small size allowing to enter into protein cavities), nanobodies are very suited for preventive and therapeutic applications. We recently selected nanobodies with broad neutralizing capacity against primary HIV-1 strains of different subtypes from phage immune libraries generated from dromedaries immunized with HIV-1 subtype C gp140 SOSIP Env glycoproteins (Koch et al., 2017, in press). Two nanobodies with complementary neutralization pattern neutralized 19 out of 21 pseudoviruses in the standard TZM-bl assay. Epitope mapping data by competition ELISAs as well as negative-stain EM reconstructions with trimeric SOSIPs identified the CD4 binding site as the major target. A new selection performed on a next-generation optC SOSIP664 plus sCD4 with libraries generated at a late timepoint (7 months after the initial 7 weeks immunization cycle) identified 2 new nanobodies, which are currently being analyzed for neutralization. We further proved functionality of the nanobodies at acidic pH (as found in the vagina) and identified nanobody combinations resulting in increased breadth of neutralization in vitro. In view of preventive applications at vaginal sites of HIV-1 transmission, we are expressing the best nanobodies in a membrane-bound form and as a secreted version from lactobacilli (L. rhamnosus), which colonize the human vagina. Finally, nanobodies will be analyzed in a humanized mouse model of HIV-1 infection for their HIV-neutralizing capacity in vivo.

Set Reconstruction on the Hypercube

Given an action of a group $G$ on a set $S$, the $k$-deck of a subset $T$ of $S$ is the multiset of all subsets of $T$ of size $k$, each given up to translation by $G$. For a given subset $T$, the {\em reconstruction number} of $T$ is the minimum $k$ such that the $k$-deck uniquely identifies $T$ up to translation by $G$, and the {\em reconstruction number} of the action $G:S$ is the maximum reconstruction number of any subset of $S$. The concept of reconstruction number extends naturally to multisubsets $T$ of $S$ and in~\cite{CPC:257539}, the author calculated the multiset-reconstruction number of all finite abelian groups. In particular, it was shown that the multiset-reconstruction number of $\mathbb{Z}_2^n$ was $n+1$. This provides an upper bound of $n+1$ to the reconstruction number of $\mathbb{Z}_2^n$. The author also showed a lower bound of $\lfloor{\frac{n+1}2}\rfloor$ in the same paper. The purpose of this note is to close the gap. The reconstruction number of $\mathbb{Z}_2^n$ is $\lfloor{n+1-\log_2(n+1-\log_2(n))}\rfloor.$

MR-Guided Kernel EM Reconstruction for Reduced Dose PET Imaging.

PET image reconstruction is highly susceptible to the impact of Poisson noise, and if shorter acquisition times or reduced injected doses are used, the noisy PET data become even more limiting. The recent development of kernel expectation maximisation (KEM) is a simple way to reduce noise in PET images, and we show in this work that impressive dose reduction can be achieved when the kernel method is used with MR-derived kernels. The kernel method is shown to surpass maximum likelihood expectation maximisation (MLEM) for the reconstruction of low-count datasets (corresponding to those obtained at reduced injected doses) producing visibly clearer reconstructions for unsmoothed and smoothed images, at all count levels. The kernel EM reconstruction of 10% of the data had comparable whole brain voxel-level error measures to the MLEM reconstruction of 100% of the data (for simulated data, at 100 iterations). For regional metrics, the kernel method at reduced dose levels attained a reduced coefficient of variation and more accurate mean values compared to MLEM. However, the advances provided by the kernel method are at the expense of possible over-smoothing of features unique to the PET data. Further assessment on clinical data is required to determine the level of dose reduction that can be routinely achieved using the kernel method, whilst maintaining the diagnostic utility of the scan.

MR-Guided Kernel EM Reconstruction for Reduced Dose PET Imaging

MR-Guided Kernel EM Reconstruction for Reduced Dose PET Imaging

Cryo-EM structure of the SAGA and NuA4 coactivator subunit Tra1 at 3.7 angstrom resolution.

Coactivator complexes SAGA and NuA4 stimulate transcription by post-translationally modifying chromatin. Both complexes contain the Tra1 subunit, a highly conserved 3744-residue protein from the Phosphoinositide 3-Kinase-related kinase (PIKK) family and a direct target for multiple sequence-specific activators. We present the Cryo-EM structure of Saccharomyces cerevsisae Tra1 to 3.7 Å resolution, revealing an extensive network of alpha-helical solenoids organized into a diamond ring conformation and is strikingly reminiscent of DNA-PKcs, suggesting a direct role for Tra1 in DNA repair. The structure was fitted into an existing SAGA EM reconstruction and reveals limited contact surfaces to Tra1, hence it does not act as a molecular scaffold within SAGA. Mutations that affect activator targeting are distributed across the Tra1 structure, but also cluster within the N-terminal Finger region, indicating the presence of an activator interaction site. The structure of Tra1 is a key milestone in deciphering the mechanism of multiple coactivator complexes.

Structural characterization of the NAP; the major adhesion complex of the human pathogen Mycoplasma genitalium.

Mycoplasma genitalium, the causative agent of non-gonococcal urethritis and pelvic inflammatory disease in humans, is a small eubacterium that lacks a peptidoglycan cell wall. On the surface of its plasma membrane is the major surface adhesion complex, known as NAP that is essential for adhesion and gliding motility of the organism. Here, we have performed cryo-electron tomography of intact cells and detergent permeabilized M. genitalium cell aggregates, providing sub-tomogram averages of free and cell-attached NAPs respectively, revealing a tetrameric complex with two-fold rotational (C2) symmetry. Each NAP has two pairs of globular lobes (named α and β lobes), arranged as a dimer of heterodimers with each lobe connected by a stalk to the cell membrane. The β lobes are larger than the α lobes by 20%. Classification of NAPs showed that the complex can tilt with respect to the cell membrane. A protein complex containing exclusively the proteins P140 and P110, was purified from M. genitalium and was structurally characterized by negative-stain single particle EM reconstruction. The close structural similarity found between intact NAPs and the isolated P140/P110 complexes, shows that dimers of P140/P110 heterodimers are the only components of the extracellular region of intact NAPs in M. genitalium.

On EM Reconstruction of a Mutli Channel Shielded DMBT Tandem Applicator for Cervical Cancer Brachytherapy: A Feasibility Study

On EM Reconstruction of a Mutli Channel Shielded DMBT Tandem Applicator for Cervical Cancer Brachytherapy: A Feasibility Study

Trends in the Electron Microscopy Data Bank (EMDB).

Recent technological advances, such as the introduction of the direct electron detector, have transformed the field of cryo-EM and the landscape of molecular and cellular structural biology. This study analyses these trends from the vantage point of the Electron Microscopy Data Bank (EMDB), the public archive for three-dimensional EM reconstructions. Over 1000 entries were released in 2016, representing almost a quarter of the total number of entries (4431). Structures at better than 6 Å resolution now represent one of the fastest-growing categories, while the share of annually released tomography-related structures is approaching 20%. The use of direct electron detectors is growing very rapidly: they were used for 70% of the structures released in 2016, in contrast to none before 2011. Microscopes from FEI have an overwhelming lead in terms of usage, and the use of the RELION software package continues to grow rapidly after having attained a leading position in the field. China is rapidly emerging as a major player in the field, supplementing the US, Germany and the UK as the big four. Similarly, Tsinghua University ranks only second to the MRC Laboratory for Molecular Biology in terms of involvement in publications associated with cryo-EM structures at better than 4 Å resolution. Overall, the numbers point to a rapid democratization of the field, with more countries and institutes becoming involved.

BioEM: GPU-accelerated computing of Bayesian inference of electron microscopy images

In cryo-electron microscopy (EM), molecular structures are determined from large numbers of projection images of individual particles. To harness the full power of this single-molecule information, we use the Bayesian inference of EM (BioEM) formalism. By ranking structural models using posterior probabilities calculated for individual images, BioEM in principle addresses the challenge of working with highly dynamic or heterogeneous systems not easily handled in traditional EM reconstruction. However, the calculation of these posteriors for large numbers of particles and models is computationally demanding. Here we present highly parallelized, GPU-accelerated computer software that performs this task efficiently. Our flexible formulation employs CUDA, OpenMP, and MPI parallelization combined with both CPU and GPU computing. The resulting BioEM software scales nearly ideally both on pure CPU and on CPU+GPU architectures, thus enabling Bayesian analysis of tens of thousands of images in a reasonable time. The general mathematical framework and robust algorithms are not limited to cryo-electron microscopy but can be generalized for electron tomography and other imaging experiments. Program summaryProgram Title: BioEM.Program Files doi:http://dx.doi.org/10.17632/d2jjs2wdhv.1Licensing provisions: GNU GPL v3.Programming language: C++, CUDA.Nature of problem: Analysis of electron microscopy images.Solution method: GPU-accelerated Bayesian inference with numerical grid sampling.External routines/libraries: Boost 1.5, FFTW 3, MPI.

Simultaneous reconstruction and segmentation for dynamic SPECT imaging

This work deals with the reconstruction of dynamic images that incorporate characteristic dynamics in certain subregions, as arising for the kinetics of many tracers in emission tomography (SPECT, PET). We make use of a basis function approach for the unknown tracer concentration by assuming that the region of interest can be divided into subregions with spatially constant concentration curves. Applying a regularised variational framework reminiscent of the Chan-Vese model for image segmentation we simultaneously reconstruct both the labelling functions of the subregions as well as the subconcentrations within each region. Our particular focus is on applications in SPECT with the Poisson noise model, resulting in a Kullback–Leibler data fidelity in the variational approach. We present a detailed analysis of the proposed variational model and prove existence of minimisers as well as error estimates. The latter apply to a more general class of problems and generalise existing results in literature since we deal with a nonlinear forward operator and a nonquadratic data fidelity. A computational algorithm based on alternating minimisation and splitting techniques is developed for the solution of the problem and tested on appropriately designed synthetic data sets. For those we compare the results to those of standard EM reconstructions and investigate the effects of Poisson noise in the data.

Mapping Epitopes of Pan‐Ebolavirus Antibodies From Human Survivors

Members of the Ebolavirus genus including Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV) and Budibugyo ebolavirus (BDBV) cause viral hemorrhagic fever and have high case fatality rates. The current outbreak in Western Africa is the largest on record and has infected over 28,000 individuals, yet no currently approved vaccines or therapeutics exist. Previous reports have revealed that ZMapp™, a cocktail of three antibodies that target the glycoprotein (GP) of EBOV, can revert an active infection in nonhuman primates. A group of recently identified antibodies from human survivors of BDBV and EBOV infections were shown to neutralize their respective virus. Some of the BDBV antibodies were also shown to be cross‐reactive against SUDV and EBOV; however, the basis of cross‐reactivity remains unknown. Here we report single‐particle negative stain EM reconstructions of each antibody in complex with GP. The cross‐reactive antibodies have overlapping epitopes and bind at the top of GP in a subdomain termed the glycan cap which contains multiple N‐linked glycosylation sites. Interestingly, many BDBV‐specific and EBOV‐specific antibodies bind in very close proximity. Therefore, the glycan cap contains structural motifs that influence species cross‐reactivity and neutralization potential of targeting antibodies. Higher resolution structures of these antibodies will be valuable for understanding the details of this important site of vulnerability.Support or Funding InformationThis work was supported by NIAID Center for Excellence in Translational Research Grant U19AI109762 (to E.O.S., and A.B.W.). J.A.D. was supported by The Scripps Research Institute Summer Undergraduate Research Fellowship Program.

Refinement of atomic models in high resolution EM reconstructions using Flex-EM and local assessment

As the resolutions of Three Dimensional Electron Microscopic reconstructions of biological macromolecules are being improved, there is a need for better fitting and refinement methods at high resolutions and robust approaches for model assessment. Flex-EM/MODELLER has been used for flexible fitting of atomic models in intermediate-to-low resolution density maps of different biological systems. Here, we demonstrate the suitability of the method to successfully refine structures at higher resolutions (2.5–4.5Å) using both simulated and experimental data, including a newly processed map of Apo-GroEL. A hierarchical refinement protocol was adopted where the rigid body definitions are relaxed and atom displacement steps are reduced progressively at successive stages of refinement. For the assessment of local fit, we used the SMOC (segment-based Manders’ overlap coefficient) score, while the model quality was checked using the Qmean score. Comparison of SMOC profiles at different stages of refinement helped in detecting regions that are poorly fitted. We also show how initial model errors can have significant impact on the goodness-of-fit. Finally, we discuss the implementation of Flex-EM in the CCP-EM software suite.

Web-based volume slicer for 3D electron-microscopy data from EMDB.

We describe the functionality and design of the Volume slicer – a web-based slice viewer for EMDB entries. This tool uniquely provides the facility to view slices from 3D EM reconstructions along the three orthogonal axes and to rapidly switch between them and navigate through the volume. We have employed multiple rounds of user-experience testing with members of the EM community to ensure that the interface is easy and intuitive to use and the information provided is relevant. The impetus to develop the Volume slicer has been calls from the EM community to provide web-based interactive visualisation of 2D slice data. This would be useful for quick initial checks of the quality of a reconstruction. Again in response to calls from the community, we plan to further develop the Volume slicer into a fully-fledged Volume browser that provides integrated visualisation of EMDB and PDB entries from the molecular to the cellular scale.

Altered brain energetics induces mitochondrial fission arrest in Alzheimer's Disease.

Altered brain metabolism is associated with progression of Alzheimer’s Disease (AD). Mitochondria respond to bioenergetic changes by continuous fission and fusion. To account for three dimensional architecture of the brain tissue and organelles, we applied 3-dimensional electron microscopy (3D EM) reconstruction to visualize mitochondrial structure in the brain tissue from patients and mouse models of AD. We identified a previously unknown mitochondrial fission arrest phenotype that results in elongated interconnected organelles, “mitochondria-on-a-string” (MOAS). Our data suggest that MOAS formation may occur at the final stages of fission process and was not associated with altered translocation of activated dynamin related protein 1 (Drp1) to mitochondria but with reduced GTPase activity. Since MOAS formation was also observed in the brain tissue of wild-type mice in response to hypoxia or during chronological aging, fission arrest may represent fundamental compensatory adaptation to bioenergetic stress providing protection against mitophagy that may preserve residual mitochondrial function. The discovery of novel mitochondrial phenotype that occurs in the brain tissue in response to energetic stress accurately detected only using 3D EM reconstruction argues for a major role of mitochondrial dynamics in regulating neuronal survival.

Nanoconnectomic upper bound on the variability of synaptic plasticity.

Information in a computer is quantified by the number of bits that can be stored and recovered. An important question about the brain is how much information can be stored at a synapse through synaptic plasticity, which depends on the history of probabilistic synaptic activity. The strong correlation between size and efficacy of a synapse allowed us to estimate the variability of synaptic plasticity. In an EM reconstruction of hippocampal neuropil we found single axons making two or more synaptic contacts onto the same dendrites, having shared histories of presynaptic and postsynaptic activity. The spine heads and neck diameters, but not neck lengths, of these pairs were nearly identical in size. We found that there is a minimum of 26 distinguishable synaptic strengths, corresponding to storing 4.7 bits of information at each synapse. Because of stochastic variability of synaptic activation the observed precision requires averaging activity over several minutes.

Structure and mechanism of the Rubisco-assembly chaperone Raf1.

Biogenesis of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits, requires assembly chaperones. Here we analyzed the role of Rubisco accumulation factor1 (Raf1), a dimer of ∼40-kDa subunits. We find that Raf1 from Synechococcus elongatus acts downstream of chaperonin-assisted RbcL folding by stabilizing RbcL antiparallel dimers for assembly into RbcL8 complexes with four Raf1 dimers bound. Raf1 displacement by RbcS results in holoenzyme formation. Crystal structures show that Raf1 from Arabidopsis thaliana consists of a β-sheet dimerization domain and a flexibly linked α-helical domain. Chemical cross-linking and EM reconstruction indicate that the β-domains bind along the equator of each RbcL2 unit, and the α-helical domains embrace the top and bottom edges of RbcL2. Raf1 fulfills a role similar to that of the assembly chaperone RbcX, thus suggesting that functionally redundant factors ensure efficient Rubisco biogenesis.

DNA Induces Conformational Changes in a Recombinant Human Minichromosome Maintenance Complex

ATP-dependent DNA unwinding activity has been demonstrated for recombinant archaeal homohexameric minichromosome maintenance (MCM) complexes and their yeast heterohexameric counterparts, but in higher eukaryotes such as Drosophila, MCM-associated DNA helicase activity has been observed only in the context of a co-purified Cdc45-MCM-GINS complex. Here, we describe the production of the recombinant human MCM (hMCM) complex in Escherichia coli. This protein displays ATP hydrolysis activity and is capable of unwinding duplex DNA. Using single-particle asymmetric EM reconstruction, we demonstrate that recombinant hMCM forms a hexamer that undergoes a conformational change when bound to DNA. Recombinant hMCM produced without post-translational modifications is functional in vitro and provides an important tool for biochemical reconstitution of the human replicative helicase.

Zebrafish Myofilaments and their Assemblies are Good Structural Models for Studying Disease Mutations

Zebrafish have become a prominent animal model of human disease because of their genetic tractability and rapid development (Lieschke & Currie, 2007). Several muscle diseases have been studied using zebrafish, including muscular dystrophy (Johnson et al., 2013), hypertrophic and dilated cardiomyopathy, nemaline myopathy (Sehnert et al., 2002; Asnani & Peterson, 2014; Telfer et al., 2012) and distal arthrogryposis (Ha et al., 2013). Here we show that zebrafish myofilaments and their assemblies have molecular structures similar to those of higher vertebrates, demonstrating their usefulness in elucidating structural changes due to disease mutations. EM and 3D reconstruction have shown that striated muscle contraction is regulated by Ca2+-induced movement of tropomyosin on thin filaments, which uncovers myosin-binding sites on actin. Using negative staining EM and single particle reconstruction we find that tropomyosin is well resolved in zebrafish native thin filaments and undergoes the same Ca2+-induced movement as seen in other species. Zebrafish thin filaments are therefore good models for studying the impact of thin filament mutations (actin, troponin, tropomyosin, nebulin) on thin filament structure and regulation. Thick filaments are also readily isolated from zebrafish muscle and their 3D reconstruction is similar to that of mammalian filaments (González-Solá et al., 2014). We have isolated intact filament assemblies (myofibrils and A-segments) from zebrafish and find that these also closely resemble those of other vertebrates, including mammals, clearly revealing MyBP-C and M-line periodic organization. These are therefore good models for elucidating the structural impact of mutations in myosin, MyBP-C and M-line proteins. We conclude that the structures of zebrafish skeletal muscle thick and thin filaments and their native assemblies closely resemble those of higher vertebrates, making them excellent models for studying the molecular impact of disease-causing mutations on filament structure.