Cryo-EM structures of cotton wool plaques' amyloid ß and of tau filaments in dominantly inherited Alzheimer disease.

Cotton wool plaques (CWPs) have been described as features of the neuropathologic phenotype of dominantly inherited Alzheimer disease (DIAD) caused by some missense and deletion mutations in the presenilin 1 (PSEN1) gene. CWPs are round, eosinophilic amyloid-ß (Aß) plaques that lack an amyloid core and are recognizable, but not fluorescent, in Thioflavin S (ThS) preparations. Amino-terminally truncated and post-translationally modified Aß peptide species are the main component of CWPs. Tau immunopositive neurites may be present in CWPs. In addition, neurofibrillary tangles coexist with CWPs. Herein, we report the structure of Aß and tau filaments isolated from brain tissue of individuals affected by DIAD caused by the PSEN1 V261I and A431E mutations, with the CWP neuropathologic phenotype. CWPs are predominantly composed of type I Aß filaments present in two novel arrangements, type Ic and type Id; additionally, CWPs contain type I and type Ib Aß filaments. Tau filaments have the AD fold, which has been previously reported in sporadic AD and DIAD. The formation of type Ic and type Id Aß filaments may be the basis for the phenotype of CWPs. Our data are relevant for the development of PET imaging methodologies to best detect CWPs in DIAD.

Cryo-EM structures of prion protein filaments from Gerstmann-Sträussler-Scheinker disease.

Prion protein (PrP) aggregation and formation of PrP amyloid (APrP) are central events in the pathogenesis of prion diseases. In the dominantly inherited prion protein amyloidosis known as Gerstmann-Sträussler-Scheinker (GSS) disease, plaques made of PrP amyloid are present throughout the brain. The c.593t > c mutation in the prion protein gene (PRNP) results in a phenylalanine to serine amino acid substitution at PrP residue 198 (F198S) and causes the most severe amyloidosis among GSS variants. It has been shown that neurodegeneration in this disease is associated with the presence of extracellular APrP plaques and neuronal intracytoplasmic Tau inclusions, that have been shown to contain paired helical filaments identical to those found in Alzheimer disease. Using cryogenic electron microscopy (cryo-EM), we determined for the first time the structures of filaments of human APrP, isolated post-mortem from the brain of two symptomatic PRNP F198S mutation carriers. We report that in GSS (F198S) APrP filaments are composed of dimeric, trimeric and tetrameric left-handed protofilaments with their protomers sharing a common protein fold. The protomers in the cross-ß spines consist of 62 amino acids and span from glycine 80 to phenylalanine 141, adopting a previously unseen spiral fold with a thicker outer layer and a thinner inner layer. Each protomer comprises nine short ß-strands, with the ß1 and ß8 strands, as well as the ß4 and ß9 strands, forming a steric zipper. The data obtained by cryo-EM provide insights into the structural complexity of the PrP filament in a dominantly inherited human PrP amyloidosis. The novel findings highlight the urgency of extending our knowledge of the filaments' structures that may underlie distinct clinical and pathologic phenotypes of human neurodegenerative diseases.

Structure of Tau filaments in Prion protein amyloidoses.

In human neurodegenerative diseases associated with the intracellular aggregation of Tau protein, the ordered cores of Tau filaments adopt distinct folds. Here, we analyze Tau filaments isolated from the brain of individuals affected by Prion-Protein cerebral amyloid angiopathy (PrP-CAA) with a nonsense mutation in the PRNP gene that leads to early termination of translation of PrP (Q160Ter or Q160X), and Gerstmann-Sträussler-Scheinker (GSS) disease, with a missense mutation in the PRNP gene that leads to an amino acid substitution at residue 198 (F198S) of PrP. The clinical and neuropathologic phenotypes associated with these two mutations in PRNP are different; however, the neuropathologic analyses of these two genetic variants have consistently shown the presence of numerous neurofibrillary tangles (NFTs) made of filamentous Tau aggregates in neurons. We report that Tau filaments in PrP-CAA (Q160X) and GSS (F198S) are composed of 3-repeat and 4-repeat Tau isoforms, having a striking similarity to NFTs in Alzheimer disease (AD). In PrP-CAA (Q160X), Tau filaments are made of both paired helical filaments (PHFs) and straight filaments (SFs), while in GSS (F198S), only PHFs were found. Mass spectrometry analyses of Tau filaments extracted from PrP-CAA (Q160X) and GSS (F198S) brains show the presence of post-translational modifications that are comparable to those seen in Tau aggregates from AD. Cryo-EM analysis reveals that the atomic models of the Tau filaments obtained from PrP-CAA (Q160X) and GSS (F198S) are identical to those of the Tau filaments from AD, and are therefore distinct from those of Pick disease, chronic traumatic encephalopathy, and corticobasal degeneration. Our data support the hypothesis that in the presence of extracellular amyloid deposits and regardless of the primary amino acid sequence of the amyloid protein, similar molecular mechanisms are at play in the formation of identical Tau filaments.

Flaviviruses have imperfect icosahedral symmetry.

Flaviviruses assemble initially in an immature, noninfectious state and undergo extensive conformational rearrangements to generate mature virus. Previous cryo-electron microscopy (cryo-EM) structural studies of flaviviruses assumed icosahedral symmetry and showed the concentric organization of the external glycoprotein shell, the lipid membrane, and the internal nucleocapsid core. We show here that when icosahedral symmetry constraints were excluded in calculating the cryo-EM reconstruction of an immature flavivirus, the nucleocapsid core was positioned asymmetrically with respect to the glycoprotein shell. The core was positioned closer to the lipid membrane at the proximal pole, and at the distal pole, the outer glycoprotein spikes and inner membrane leaflet were either perturbed or missing. In contrast, in the asymmetric reconstruction of a mature flavivirus, the core was positioned concentric with the glycoprotein shell. The deviations from icosahedral symmetry demonstrated that the core and glycoproteins have varied interactions, which likely promotes viral assembly and budding.

Sub-3 Šapoferritin structure determined with full range of phase shifts using a single position of volta phase plate.

Volta Phase Plate (VPP) has become an invaluable tool for cryo-EM structural determination of small protein complexes by increasing image contrast. Currently, the standard protocol of VPP usage periodically changes the VPP position to a fresh spot during data collection. Such a protocol was to target the phase shifts to a relatively narrow range (around 90°) based on the observations of increased phase shifts and image blur associated with more images taken with a single VPP position. Here, we report a 2.87 Šresolution structure of apoferritin reconstructed from a dataset collected using only a single position of VPP. The reconstruction resolution and map density features are nearly identical to the reconstruction from the control dataset collected with periodic change of VPP positions. Further experiments have verified that similar results, including a 2.5 Šresolution structure, could be obtained with a full range of phase shifts, different spots of variable phase shift increasing rates, and at different ages of the VPP post-installation. Furthermore, we have found that the phase shifts at low resolutions, probably related to the finite size of the Volta spots, could not be correctly modeled by current CTF model using a constant phase shift at all frequencies. In dataset III, severe beam tilt issue was identified but could be computationally corrected with iterative refinements. The observations in this study may provide new insights into further improvement of both the efficiency and robustness of VPP, and to help turn VPP into a plug-and-play device for high-resolution cryo-EM.

Cryo-EM structure of Escherichia coli σ70 RNA polymerase and promoter DNA complex revealed a role of σ non-conserved region during the open complex formation.

First step of gene expression is transcribing the genetic information stored in DNA to RNA by the transcription machinery including RNA polymerase (RNAP). In Escherichia coli, a primary σ70 factor forms the RNAP holoenzyme to express housekeeping genes. The σ70 contains a large insertion between the conserved regions 1.2 and 2.1, the σ non-conserved region (σNCR), but its function remains to be elucidated. In this study, we determined the cryo-EM structures of the E. coli RNAP σ70 holoenzyme and its complex with promoter DNA (open complex, RPo) at 4.2 and 5.75 Å resolutions, respectively, to reveal native conformations of RNAP and DNA. The RPo structure presented here found an interaction between the σNCR and promoter DNA just upstream of the -10 element, which was not observed in a previously determined E. coli RNAP transcription initiation complex (RPo plus short RNA) structure by X-ray crystallography because of restraint of crystal packing effects. Disruption of the σNCR and DNA interaction by the amino acid substitutions (R157A/R157E) influences the DNA opening around the transcription start site and therefore decreases the transcription activity of RNAP. We propose that the σNCR and DNA interaction is conserved in proteobacteria, and RNAP in other bacteria replaces its role with a transcription factor.

Direct observation of conformational dynamics of the PH domain in phospholipases Cϵ and ß may contribute to subfamily-specific roles in regulation.

Phospholipase C (PLC) enzymes produce second messengers that increase the intracellular Ca2+ concentration and activate protein kinase C (PKC). These enzymes also share a highly conserved arrangement of core domains. However, the contributions of the individual domains to regulation are poorly understood, particularly in isoforms lacking high-resolution information, such as PLCϵ. Here, we used small-angle X-ray scattering (SAXS), EM, and functional assays to gain insights into the molecular architecture of PLCϵ, revealing that its PH domain is conformationally dynamic and essential for activity. We further demonstrate that the PH domain of PLCß exhibits similar dynamics in solution that are substantially different from its conformation observed in multiple previously reported crystal structures. We propose that this conformational heterogeneity contributes to subfamily-specific differences in activity and regulation by extracellular signals.

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions.

Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.

Visualization of uncorrelated, tandem symmetry mismatches in the internal genome packaging apparatus of bacteriophage T7.

Motor-driven packaging of a dsDNA genome into a preformed protein capsid through a unique portal vertex is essential in the life cycle of a large number of dsDNA viruses. We have used single-particle electron cryomicroscopy to study the multilayer structure of the portal vertex of the bacteriophage T7 procapsid, the recipient of T7 DNA in packaging. A focused asymmetric reconstruction method was developed and applied to selectively resolve neighboring pairs of symmetry-mismatched layers of the portal vertex. However, structural features in all layers of the multilayer portal vertex could not be resolved simultaneously. Our results imply that layers with mismatched symmetries can join together in several different relative orientations, and that orientations at different interfaces assort independently to produce structural isomers, a process that we call combinatorial assembly isomerism. This isomerism explains rotational smearing in previously reported asymmetric reconstructions of the portal vertex of T7 and other bacteriophages. Combinatorial assembly isomerism may represent a new regime of structural biology in which globally varying structures assemble from a common set of components. Our reconstructions collectively validate previously proposed symmetries, compositions, and sequential order of T7 portal vertex layers, resolving in tandem the 5-fold gene product 10 (gp10) shell, 12-fold gp8 portal ring, and an internal core stack consisting of 12-fold gp14 adaptor ring, 8-fold bowl-shaped gp15, and 4-fold gp16 tip. We also found a small tilt of the core stack relative to the icosahedral fivefold axis and propose that this tilt assists DNA spooling without tangling during packaging.

The 1.59Å resolution structure of the minor pseudopilin EpsH of Vibrio cholerae reveals a long flexible loop.

The type II secretion complex exports folded proteins from the periplasm to the extracellular milieu. It is used by the pathogenic bacterium Vibrio cholerae to export several proteins, including its major virulence factor, cholera toxin. The pseudopilus is an essential component of the type II secretion system and likely acts as a piston to push the folded proteins across the outer membrane through the secretin pore. The pseudopilus is composed of the major pseudopilin, EpsG, and four minor pseudopilins, EpsH, EpsI, EpsJ and EpsK. We determined the x-ray crystal structure of the head domain of EpsH at 1.59Å resolution using molecular replacement with the previously reported EpsH structure, 2qv8, as the template. Three additional N-terminal amino acids present in our construct prevent an artifactual conformation of residues 160-166, present in one of the two monomers of the 2qv8 structure. Additional crystal contacts stabilize a long flexible loop comprised of residues 104-135 that is more disordered in the 2qv8 structure but is partially observed in our structure in very different positions for the two EpsH monomers in the asymmetric unit. In one of the conformations the loop is highly extended. Modeling suggests the highly charged loop is capable of contacting EpsG and possibly secreted protein substrates, suggesting a role in specificity of pseudopilus assembly or secretion function.

Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged Escherichiacoli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.

A Viral Protein 4-Based Trivalent Nanoparticle Vaccine Elicited High and Broad Immune Responses and Protective Immunity against the Predominant Rotaviruses.

The current live rotavirus (RV) vaccines show reduced effectiveness in developing countries, calling for vaccine strategies with improved efficacy and safety. We generated pseudovirus nanoparticles (PVNPs) that display multiple ectodomains of RV viral protein 4 (VP4), named S-VP4e, as a nonreplicating RV vaccine candidate. The RV spike protein VP4s that bind host receptors and facilitate viral entry are excellent targets for vaccination. In this study, we developed scalable methods to produce three S-VP4e PVNPs, each displaying the VP4e antigens from one of the three predominant P[8], P[4], and P[6] human RVs (HRVs). These PVNPs were recognized by selected neutralizing VP4-specific monoclonal antibodies, bound glycan receptors, attached to permissive HT-29 cells, and underwent cleavage by trypsin between VP8* and VP5*. 3D PVNP models were constructed to understand their structural features. A trivalent PVNP vaccine containing the three S-VP4e PVNPs elicited high and well-balanced VP4e-specific antibody titers in mice directed against the three predominant HRV P types. The resulting antisera neutralized the three HRV prototypes at high titers; greater than 4-fold higher than the neutralizing responses induced by a trivalent vaccine consisting of the S60-VP8* PVNPs. Finally, the trivalent S-VP4e PVNP vaccine provided 90-100% protection against diarrhea caused by HRV challenge. Our data supports the trivalent S-VP4e PVNPs as a promising nonreplicating HRV vaccine candidate for parenteral delivery to circumvent the suboptimal immunization issues of all present live HRV vaccines. The established PVNP-permissive cell and PVNP-glycan binding assays will be instrumental for further investigating HRV-host cell interactions and neutralizing effects of VP4-specific antibodies and antivirals.

The 2.6 Å Structure of a Tulane Virus Variant with Minor Mutations Leading to Receptor Change.

Human noroviruses (HuNoVs) are a major cause of acute gastroenteritis, contributing significantly to annual foodborne illness cases. However, studying these viruses has been challenging due to limitations in tissue culture techniques for over four decades. Tulane virus (TV) has emerged as a crucial surrogate for HuNoVs due to its close resemblance in amino acid composition and the availability of a robust cell culture system. Initially isolated from rhesus macaques in 2008, TV represents a novel Calicivirus belonging to the Recovirus genus. Its significance lies in sharing the same host cell receptor, histo-blood group antigen (HBGA), as HuNoVs. In this study, we introduce, through cryo-electron microscopy (cryo-EM), the structure of a specific TV variant (the 9-6-17 TV) that has notably lost its ability to bind to its receptor, B-type HBGA-a finding confirmed using an enzyme-linked immunosorbent assay (ELISA). These results offer a profound insight into the genetic modifications occurring in TV that are necessary for adaptation to cell culture environments. This research significantly contributes to advancing our understanding of the genetic changes that are pivotal to successful adaptation, shedding light on fundamental aspects of Calicivirus evolution.

Cryo-EM structures of amyloid-ß and tau filaments in Down syndrome.

Adult individuals with Down syndrome (DS) develop Alzheimer disease (AD). Whether there is a difference between AD in DS and AD regarding the structure of amyloid-ß (Aß) and tau filaments is unknown. Here we report the structure of Aß and tau filaments from two DS brains. We found two Aß40 filaments (types IIIa and IIIb) that differ from those previously reported in sporadic AD and two types of Aß42 filaments (I and II) identical to those found in sporadic and familial AD. Tau filaments (paired helical filaments and straight filaments) were identical to those in AD, supporting the notion of a common mechanism through which amyloids trigger aggregation of tau. This knowledge is important for understanding AD in DS and assessing whether adults with DS could be included in AD clinical trials.

Pseudovirus Nanoparticles Displaying Plasmodium Circumsporozoite Proteins Elicited High Titers of Sporozoite-Binding Antibody.

BACKGROUND: malaria caused by Plasmodium parasites remains a public health threat. The circumsporozoite proteins (CSPs) of Plasmodium sporozoite play a key role in Plasmodium infection, serving as an excellent vaccine target. METHODS: using a self-assembled S60 nanoparticle platform, we generated pseudovirus nanoparticles (PVNPs) displaying CSPs, named S-CSPs, for enhanced immunogenicity. RESULTS: purified Hisx6-tagged or tag-free S-CSPs self-assembled into PVNPs that consist of a norovirus S60 inner shell and multiple surface-displayed CSPs. The majority of the PVNPs measured ~27 nm with some size variations, and their three-dimensional structure was modeled. The PVNP-displayed CSPs retained their glycan receptor-binding function. A mouse immunization study showed that PVNPs induced a high antibody response against CSP antigens and the PVNP-immunized mouse sera stained the CSPs of Plasmodium sporozoites at high titer. CONCLUSIONS AND DISCUSSION: the PVNP-displayed CSPs retain their authentic antigenic feature and receptor-binding function. The CSP-specific antibody elicited by the S-CSP PVNPs binds original CSPs and potentially inhibits the attachment of Plasmodium sporozoites to their host cells, a key step for liver invasion by the sporozoites. Thus, S-CSP PVNPs may be an excellent vaccine candidate against malaria caused by Plasmodium parasites.

The αTSR Domain of Plasmodium Circumsporozoite Protein Bound Heparan Sulfates and Elicited High Titers of Sporozoite Binding Antibody After Displayed by Nanoparticles.

Introduction: Malaria is a devastating infectious illness caused by protozoan Plasmodium parasites. The circumsporozoite protein (CSP) on Plasmodium sporozoites binds heparan sulfate proteoglycan (HSPG) receptors for liver invasion, a critical step for prophylactic and therapeutic interventions. Methods: In this study, we characterized the αTSR domain that covers region III and the thrombospondin type-I repeat (TSR) of the CSP using various biochemical, glycobiological, bioengineering, and immunological approaches. Results: We found for the first time that the αTSR bound heparan sulfate (HS) glycans through support by a fused protein, indicating that the αTSR is a key functional domain and thus a vaccine target. When the αTSR was fused to the S domain of norovirus VP1, the fusion protein self-assembled into uniform S60-αTSR nanoparticles. Three-dimensional structure reconstruction revealed that each nanoparticle consists of an S60 nanoparticle core and 60 surface displayed αTSR antigens. The nanoparticle displayed αTSRs retained the binding function to HS glycans, indicating that they maintained authentic conformations. Both tagged and tag-free S60-αTSR nanoparticles were produced via the Escherichia coli system at high yield by scalable approaches. They are highly immunogenic in mice, eliciting high titers of αTSR-specific antibody that bound specifically to the CSPs of Plasmodium falciparum sporozoites at high titer. Discussion and Conclusion: Our data demonstrated that the αTSR is an important functional domain of the CSP. The S60-αTSR nanoparticle displaying multiple αTSR antigens is a promising vaccine candidate potentially against attachment and infection of Plasmodium parasites.

A bioengineered pseudovirus nanoparticle displaying SARS-CoV 2 RBD fully protects mice from mortality and weight loss caused by SARS-CoV 2 challenge.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused considerable morbidity and mortality worldwide. Although authorized COVID-19 vaccines have been shown highly effective, their significantly lower efficacy against heterologous variants, and the rapid decrease of vaccine-elicited immunity raises serious concerns, calling for improved vaccine tactics. To this end, a pseudovirus nanoparticle (PVNP) displaying the receptor binding domains (RBDs) of SARS-CoV-2 spike, named S-RBD, was generated and shown it as a promising COVID-19 vaccine candidate. The S-RBD PVNP was produced using both prokaryotic and eukaryotic systems. A 3D structural model of the S-RBD PVNPs was built based on the known structures of the S60 particle and RBDs, revealing an S60 particle-based icosahedral symmetry with multiple surface-displayed RBDs that retain authentic conformations and receptor-binding functions. The PVNP is highly immunogenic, eliciting high titers of RBD-specific IgG and neutralizing antibodies in mice. The S-RBD PVNP demonstrated exceptional protective efficacy, and fully (100%) protected K18-hACE2 mice from mortality and weight loss after a lethal SARS-CoV-2 challenge, supporting the S-RBD PVNPs as a potent COVID-19 vaccine candidate. By contrast, a PVNP displaying the N-terminal domain (NTD) of SARS-CoV-2 spike exhibited only 50% protective efficacy. Since the RBD antigens of our PVNP vaccine are adjustable as needed to address the emergence of future variants, and various S-RBD PVNPs can be combined as a cocktail vaccine for broad efficacy, these non-replicating PVNPs offer a flexible platform for a safe, effective COVID-19 vaccine with minimal manufacturing cost and time.

Cross-ß helical filaments of Tau and TMEM106B in Gray and White Matter of Multiple System Tauopathy with presenile Dementia.

Background: The Microtubule-Associated Protein Tau (MAPT) is one of the proteins that are central to neurodegenerative diseases. The nature of intracellular tau aggregates is determined by the cell types whether neuronal or glial, the participating tau isoforms, and the structure of the amyloid filament. The transmembrane protein 106B (TMEM106B) has recently emerged as another significant player in neurodegeneration and aging. In the central nervous system, the composition of the gray and white matter differs considerably. The gray matter consists of nerve cell bodies, dendrites, unmyelinated axons, synaptic terminals, astrocytes, oligodendrocytes (satellite cells) and microglia. The white matter differs from the gray for the presence of axonal tracts as the only neuronal component and for the absence of nerve cell bodies, dendrites and synaptic terminals. Cryogenic electron microscopy (cryo-EM) studies have unveiled the structure of tau and TMEM106B, from the cerebral cortex, in several neurodegenerative diseases; however, whether tau and TMEM106B filaments from the gray and white matter share a common fold requires additional investigation. Methods: We isolated tau and TMEM106B from the cerebral cortex and white matter of the frontal lobes of two individuals affected by multiple system tauopathy with presenile dementia (MSTD), a disease caused by the MAPT intron 10 mutation +3. We used immunostaining, biochemical, genetics and cryo-EM methods to characterize tau and TMEM106B. Results: We determined that tau filaments in the gray and the white matter of MSTD individuals can induce tau aggregation and have identical AGD type 2 folds. TMEM106B amyloid filaments were also found in the gray and white matter of MSTD; the filament folds were identical in the two anatomical regions. Conclusions: Our findings show for the first time that in MSTD two types of amyloid filaments extracted from the gray matter have identical folds to those extracted from the white matter. Whether in this genetic disorder there is a relationship in the pathogenesis of the tau and TMEM106B filaments, remains to be determined. Furthermore, additional studies are needed for other proteins and other neurodegenerative diseases to establish whether filaments extracted from the gray and white matter would have identical folds.

High resolution single particle Cryo-EM refinement using JSPR.

JSPR is a single particle cryo-EM image processing and 3D reconstruction software developed in the Jiang laboratory at Purdue University. It began as a few refinement scripts for symmetric and asymmetric reconstructions of icosahedral viruses, but has grown into a comprehensive suite of tools for building ab initio reconstructions, high resolution refinements of viruses, protein complexes of arbitrary symmetries including helical tubes/filaments, and image file handling utilities. In this review, we will present examples achieved using JSPR and demonstrate recently implemented features of JSPR such as multi-aberration "alignments" and automatic optimization of masking for the assessment of map resolution using "true" FSC.