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The skeletal muscle Ca 2+ release channel (RyR1), a homotetramer, regulates the release of Ca 2+ from the sarcoplasmic reticulum to initiate muscle contraction. In this work, we have delineated the RyR1 monomer boundaries in a subnanometer-resolution electron cryomicroscopy (cryo-EM) density map. In the cytoplasmic region of each RyR1 monomer, 36 α-helices and 7 β-sheets can be resolved. A β-sheet was also identified close to the membrane-spanning region that resembles the cytoplasmic pore structures of inward rectifier K + channels. Three structural folds, generated for amino acids 12–565 using comparative modeling and cryo-EM density fitting, localize close to regions implicated in communication with the voltage sensor in the transverse tubules. Eleven of the 15 disease-related residues for these domains are mapped to the surface of these models. Four disease-related residues are found in a basin at the interfaces of these regions, creating a pocket in which the immunophilin FKBP12 can fit. Taken together, these results provide a structural context for both channel gating and the consequences of certain malignant hyperthermia and central core disease-associated mutations in RyR1.

Medical imaging has produced a large number of volumetric images capturing biological structures in 3D. Computer-based understanding of these structures can often benefit from the knowledge of shape components, particularly rod-like and plate-like parts, in such volumes. Previously, skeletons have been a common tool for identifying these shape components in a solid object. However, obtaining skeletons of a grayscale volume poses new challenges due to the lack of a clear boundary between object and background. In this paper, we present a new skeletonization algorithm on grayscale volumes typical to medical imaging (e.g., MRI, CT and EM scans), for the purpose of identifying shape components. Our algorithm does not require an explicit segmentation of the volume into object and background, and is capable of producing skeletal curves and surfaces that lie centered at rod-shaped and plate-shaped parts in the grayscale volume. Our method is demonstrated on both synthetic and medical data.

A half-century after the determination of the first three-dimensional crystal structure of a protein, more than 40,000 structures ranging from single polypeptides to large assemblies have been reported. The challenge for crystallographers, however, remains the growing of a diffracting crystal. Here we report the 4.5-A resolution structure of a 22-MDa macromolecular assembly, the capsid of the infectious epsilon15 (epsilon15) particle, by single-particle electron cryomicroscopy. From this density map we constructed a complete backbone trace of its major capsid protein, gene product 7 (gp7). The structure reveals a similar protein architecture to that of other tailed double-stranded DNA viruses, even in the absence of detectable sequence similarity. However, the connectivity of the secondary structure elements (topology) in gp7 is unique. Protruding densities are observed around the two-fold axes that cannot be accounted for by gp7. A subsequent proteomic analysis of the whole virus identifies these densities as gp10, a 12-kDa protein. Its structure, location and high binding affinity to the capsid indicate that the gp10 dimer functions as a molecular staple between neighbouring capsomeres to ensure the particle's stability. Beyond epsilon15, this method potentially offers a new approach for modelling the backbone conformations of the protein subunits in other macromolecular assemblies at near-native solution states.

* National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030** Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390GroEL with its co­chaperonin, GroES, is the prototypical type­I chaperonin, assisting in the protein folding process through an ATP­driven cycle

SummaryAn increasing number of structural studies of large macromolecular complexes, both in X-ray crystallography and cryo-electron microscopy, have resulted in intermediate-resolution (5–10 Å) density maps. Despite being limited in resolution, significant structural and functional information may be extractable from these maps. To aid in the analysis and annotation of these complexes, we have developed SSEhunter, a tool for the quantitative detection of α helices and β sheets. Based on density skeletonization, local geometry calculations, and a template-based search, SSEhunter has been tested and validated on a variety of simulated and authentic subnanometer-resolution density maps. The result is a robust, user-friendly approach that allows users to quickly visualize, assess, and annotate intermediate-resolution density maps. Beyond secondary structure element identification, the skeletonization algorithm in SSEhunter provides secondary structure topology, which is potentially useful in leading to structural models of individual molecular components directly from the density.

Background Antigen escape tumor cell variants prevail in tumors recurring after treatment with chimeric antigen receptor (CAR) T cells with a single specificity. Recurrent tumors preserve alternative non-targeted tumor associated antigens. Hypothesis A bispecific CAR will mitigate antigen escape enhancing the antitumor activity of T cells. Methods and results HER2 and IL13Rα2 are currently targeted in Phase I glioblastoma (GBM) trials using CAR T cells. We created a bispecific CAR molecule with a HER2-specific scFv joined in tandem to an IL13Rα2-binding moiety in the CAR exodomain (Tandem CAR) and a CD28.ζ signaling endodomain. We used computational modeling to interrogate this design. GBM patients' Tandem CAR T cells showed distinct binding to soluble HER2 and IL13Rα2 and killed primary autologous GBM cells. Three-dimensional reconstitution and quantification of confocal images of the Tandem CAR T cell/tumor interface revealed enhanced bifunctional immunological synapses compared to conventional CARs. Further, Tandem CAR T cells exhibited significantly enhanced inexhaustible activation dynamics when compared to conventional HER2 or IL13Rα2 CAR T cells and better controlled established GBM in an orthotopic murine model by offsetting both HER2 and IL13Rα2 escape. Conclusion Tandem chimeric antigen receptors enhance T cell activation and mitigate antigen escape through bifunctional immunological synapse formation in GBM.

Efforts in structural biology have targeted the systematic determination of all protein structures through experimental determination or modeling. In recent years, 3-D electron cryomicroscopy (cryoEM) has assumed an increasingly important role in determining the structures of these large macromolecular assemblies to intermediate resolutions (6–10 Å). While these structures provide a snapshot of the assembly and its components in well-defined functional states, the resolution limits the ability to build accurate structural models. In contrast, sequence-based modeling techniques are capable of producing relatively robust structural models for isolated proteins or domains. In this work, we developed and applied a hybrid modeling approach, utilizing cryoEM density and ab initio modeling to produce a structural model for the core domain of a herpesvirus structural protein, VP26. Specifically, this method, first tested on simulated data, utilizes the cryoEM density map as a geometrical constraint in identifying the most native-like models from a gallery of models generated by ab initio modeling. The resulting model for the core domain of VP26, based on the 8.5-Å resolution herpes simplex virus type 1 (HSV-1) capsid cryoEM structure and mutational data, exhibited a novel fold. Additionally, the core domain of VP26 appeared to have a complementary interface to the known upper-domain structure of VP5, its cognate binding partner. While this new model provides for a better understanding of the assembly and interactions of VP26 in HSV-1, the approach itself may have broader applications in modeling the components of large macromolecular assemblies., Synopsis Efforts in structural genomics have targeted the systematic determination of all protein structures primarily using X-ray crystallography and nuclear magnetic resonance. These initiatives have typically focused on domains, single-protein and in some cases small complexes, and as such macromolecular machines are relatively underrepresented. However, in recent years, electron cryomicroscopy (cryoEM) has assumed an increasingly important role in determining the structure of large macromolecular machines in their biologically active states to intermediate resolutions (5–10 Å). Concurrently, modeling techniques, such as comparative and ab initio modeling, have played an increasingly important role in structure determination of small proteins not amenable to other structural techniques. In this work, Baker and colleagues have leveraged ab initio modeling and cryoEM to assess and identify structural models for the macromolecular components within a large complex. Specifically, the cryoEM density can be used to select the most native-like models from a large gallery of potential models. Applied to the smallest herpesvirus capsid protein, VP26 (12 kDa), it was possible to determine its core domain structure (residues 42–105), which helped to elucidate interactions among the structural protein in the virion. Beyond VP26, these techniques potentially provide a new pathway for accurate structure determination of proteins in their biological and functional states.

Comparative analysis of capsid protein structures in the eukaryote-infecting herpesviruses ( Herpesviridae ) and the prokaryote-infecting tailed DNA bacteriophages ( Caudovirales ) revealed a characteristic fold that is restricted to these two virus lineages and is indicative of common ancestry. This fold not only serves as a major architectural element in capsid stability but also enables the conformational flexibility observed during viral assembly and maturation. On the basis of this and other emerging relationships, it seems increasingly likely that the very diverse collection of extant viruses may have arisen from a relatively small number of primordial progenitors.

We developed a method for structure characterization of assembly components by iterative comparative protein structure modeling and fitting into cryo-electron microscopy (cryoEM) density maps. Specifically, we calculate a comparative model of a given component by considering many alternative alignments between the target sequence and a related template structure while optimizing the fit of a model into the corresponding density map. The method relies on the previously developed Moulder protocol that iterates over alignment, model building, and model assessment. The protocol was benchmarked using 20 varied target-template pairs of known structures with less than 30% sequence identity and corresponding simulated density maps at resolutions from 5A to 25A. Relative to the models based on the best existing sequence profile alignment methods, the percentage of C(alpha) atoms that are within 5A of the corresponding C(alpha) atoms in the superposed native structure increases on average from 52% to 66%, which is half-way between the starting models and the models from the best possible alignments (82%). The test also reveals that despite the improvements in the accuracy of the fitness function, this function is still the bottleneck in reducing the remaining errors. To demonstrate the usefulness of the protocol, we applied it to the upper domain of the P8 capsid protein of rice dwarf virus that has been studied by cryoEM at 6.8A. The C(alpha) root-mean-square deviation of the model based on the remotely related template, bluetongue virus VP7, improved from 8.7A to 6.0A, while the best possible model has a C(alpha) RMSD value of 5.3A. Moreover, the resulting model fits better into the cryoEM density map than the initial template structure. The method is being implemented in our program MODELLER for protein structure modeling by satisfaction of spatial restraints and will be applicable to the rapidly increasing number of cryoEM density maps of macromolecular assemblies.

We explore structural characterization of protein assemblies by a combination of electron cryo-microscopy (cryoEM) and comparative protein structure modeling. Specifically, our method finds an optimal atomic model of a given assembly subunit and its position within an assembly by fitting alternative comparative models into a cryoEM map. The alternative models are calculated by MODELLER [J. Mol. Biol. 234 (1993) 313] from different sequence alignments between the modeled protein and its template structures. The fitting of these models into a cryoEM density map is performed either by FOLDHUNTER [J. Mol. Biol. 308 (2001) 1033] or by a new density fitting module of MODELLER (Mod-EM). Identification of the most accurate model is based on the correlation between the model accuracy and the quality of fit into the cryoEM density map. To quantify this correlation, we created a benchmark consisting of eight proteins of different structural folds with corresponding density maps simulated at five resolutions from 5 to 15 angstroms, with three noise levels each. Each of the proteins in the set was modeled based on 300 different alignments to their remotely related templates (12-32% sequence identity), spanning the range from entirely inaccurate to essentially accurate alignments. The benchmark revealed that one of the most accurate models can usually be identified by the quality of its fit into the cryoEM density map, even for noisy maps at 15 angstroms resolution. Therefore, a cryoEM density map can be helpful in improving the accuracy of a comparative model. Moreover, a pseudo-atomic model of a component in an assembly may be built better with comparative models of the native subunit sequences than with experimentally determined structures of their homologs.

Due to the sensitivity of biological sample to the radiation damage, the low dose imaging conditions used for electron microscopy result in extremely noisy images. The processes of digitization, image alignment, and 3D reconstruction also introduce additional sources of noise in the final 3D structure. In this paper, we investigate the effectiveness of a bilateral denoising filter in various biological electron microscopy applications. In contrast to the conventional low pass filters, which inevitably smooth out both noise and structural features simultaneously, we found that bilateral filter holds a distinct advantage in being capable of effectively suppressing noise without blurring the high resolution details. In as much, we have applied this technique to individual micrographs, entire 3D reconstructions, segmented proteins, and tomographic reconstructions.

Background Alzheimer disease (AD) is characterized by memory and visuospatial deficits with relative sparing of personality. Mutations in 3 genes (presenilin 1 and 2 and amyloid precursor protein) are associated with presenile AD. Presenilin 1 gene mutations have not been described in African Americans. Methods We studied an African American family with autosomal dominant rapidly progressive dementia and psychosis occurring early in the fifth decade of life. We performed neurologic evaluations, psychometrics, and neuroimaging. We sequenced the amyloid precursor protein gene, presenilin 1 and 2, and tau in affected and unaffected family members. One patient underwent a brain biopsy and subsequent autopsy. Results Personality change, auditory and visual hallucinations, delusions, memory impairment, word-finding difficulties, and subsequent rigidity, dystonia, myoclonus, and mutism developed in 2 brothers. Neuropsychometric testing in one was consistent with frontotemporal dementia or atypical AD. Neuroimaging studies showed diffuse cortical involvement. A clinical diagnosis of familial non-Alzheimer dementia was made. However, results of temporal lobe biopsy in one revealed amyloid neuritic plaques, and autopsy results confirmed the diagnosis of AD. Gene sequencing revealed a presenilin 1 point mutation (M139V) cosegregating with the disease. A tau polymorphism in exon 7 (A178T) was found in an affected brother and unaffected relatives. Conclusions We report the first documented presenilin mutation in African American patients presenting with early personality change, psychosis, and memory loss with preserved praxis. The M139V mutation can present differently between kindreds, with some features suggestive of a frontal lobe syndrome. The M139V mutation can lead to atypical AD, and genetic background may have a role in determining the phenotype of genetically defined AD.

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

To the Editor: We read with great interest the article by Janssen et al.1 regarding clinical features seen in nine apparently separately ascertained kindreds with frontotemporal dementia and parkinsonism (FTDP-17) harboring the same intronic 10+16 tau mutation. They attempted to correlate variations in phenotype observed in their families with APOE e4 allele status. …

Advances in electron cryomicroscopy (cryo-EM) have made possible the structural determination of large biological machines in the resolution range of 6-9 angstroms. Rice dwarf virus and the acrosomal bundle represent two distinct types of machines amenable to cryo-EM investigations at subnanometer resolutions. However, calculating the density map is only the first step, and much analysis remains to extract structural insights and the mechanism of action in these machines. This paper will review the computational and visualization methodologies necessary for analysis (structure mining) of the computed cryo-EM maps of these machines. These steps include component segmentation, averaging based on local symmetry among components, density connectivity trace, incorporation of bioinformatics analysis, and fitting of high-resolution component data, if available. The consequences of these analyses can not only identify accurately some of the secondary structure elements of the molecular components in machines but also suggest structural mechanisms related to their biological functions.

In this work, we employ single-particle electron cryo-microscopy (cryo-EM) to reconstruct GroEL to approximately 4 A resolution with both D7 and C7 symmetry. Using a newly developed skeletonization algorithm and secondary structure element identification in combination with sequence-based secondary structure prediction, we demonstrate that it is possible to achieve a de novo Calpha trace directly from a cryo-EM reconstruction. The topology of our backbone trace is completely accurate, though subtle alterations illustrate significant differences from existing crystal structures. In the map with C7 symmetry, the seven monomers in each ring are identical; however, the subunits have a subtly different structure in each ring, particularly in the equatorial domain. These differences include an asymmetric salt bridge, density in the nucleotide-binding pocket of only one ring, and small shifts in alpha helix positions. This asymmetric conformation is different from previous asymmetric structures, including GroES-bound GroEL, and may represent a "primed state" in the chaperonin pathway.

TRiC is a eukaryotic chaperonin essential for de novo folding of ∼10% newly synthesized cytosolic proteins, many of which cannot be folded by other cellular chaperones. Unlike prokaryotic and archael chaperonins, each of its two rings consists of eight unique, but similar subunits. Using single particle cryo-EM, we determined the mammalian TRiC structure without any symmetry imposition at 4.7 A resolution, which is the highest resolution asymmetric cryo-EM reconstruction to date. An analysis of this map allowed us to elucidate the relative orientation of the two rings and the two-fold symmetry axis location between them. A subsequent two-fold symmetrized map yielded a 4.0 A structure, in which a large fraction of side chains and structural elements including loops and insertions appear as visible densities. These features permitted unambiguous identification of all eight individual subunits, despite their similarity. A Cα backbone model of the entire TRiC complex was subsequently refined from initial homology models against the cryo-EM density based on our subunit identification. A refined all-atom model for a single subunit showed ∼95% of the dihedral angles in the allowable regions of the Ramachandran plot. Our model reveals that the cavity walls of TRiC exhibit an overall positively charged surface property, the opposite of GroEL. The interior surface chemical properties likely play an important role for TRiC's unique substrate specificity.

The protein folding machine Methanococcus maripaludis chaperonin (Mm-cpn) is a type II archael chaperonin that has a built-in lid. It is a 16-subunit homo-oligomer of ∼1 MDa arranged in a two back-to-back rings that is structurally very similar to the mammalian chaperonin such as TRiC. The substrate folding is accompanied by a conformational change triggered by nucleotide binding and hydrolysis.Using single particle cryo-EM and image reconstruction, we solve both the wild type and lidless mutant Mm-cpn in open and closed states respectively at resolutions between 10 and 4.3 A. The open state is a nucleotide-free state while the closed state corresponds to the transition state of ATP hydrolysis. Cα backbone models of these four 3-D reconstructions have been hand traced or flexibly fitted depending on their resolutions. The models show clearly the subunits' equatorial domain rotation between the open and closed states, which is unique and different from the well-studied type I chaperonin (GroE) found in E.Coli.

Background Many proteins undergo extensive conformational changes as part of their functionality. Tracing these changes is important for understanding the way these proteins function. Traditional biophysics-based conformational search methods require a large number of calculations and are hard to apply to large-scale conformational motions. Results In this work we investigate the application of a robotics-inspired method, using backbone and limited side chain representation and a coarse grained energy function to trace large-scale conformational motions. We tested the algorithm on four well known medium to large proteins and we show that even with relatively little information we are able to trace low-energy conformational pathways efficiently. The conformational pathways produced by our methods can be further filtered and refined to produce more useful information on the way proteins function under physiological conditions. Conclusions The proposed method effectively captures large-scale conformational changes and produces pathways that are consistent with experimental data and other computational studies. The method represents an important first step towards a larger scale modeling of more complex biological systems.