Self-assembly of coherently dynamic, auxetic, two-dimensional protein crystals.

Two-dimensional (2D) crystalline materials possess unique structural, mechanical and electronic properties that make them highly attractive in many applications. Although there have been advances in preparing 2D materials that consist of one or a few atomic or molecular layers, bottom-up assembly of 2D crystalline materials remains a challenge and an active area of development. More challenging is the design of dynamic 2D lattices that can undergo large-scale motions without loss of crystallinity. Dynamic behaviour in porous three-dimensional (3D) crystalline solids has been exploited for stimuli-responsive functions and adaptive behaviour. As in such 3D materials, integrating flexibility and adaptiveness into crystalline 2D lattices would greatly broaden the functional scope of 2D materials. Here we report the self-assembly of unsupported, 2D protein lattices with precise spatial arrangements and patterns using a readily accessible design strategy. Three single- or double-point mutants of the C4-symmetric protein RhuA were designed to assemble via different modes of intermolecular interactions (single-disulfide, double-disulfide and metal-coordination) into crystalline 2D arrays. Owing to the flexibility of the single-disulfide interactions, the lattices of one of the variants ((C98)RhuA) are essentially defect-free and undergo substantial, but fully correlated, changes in molecular arrangement, yielding coherently dynamic 2D molecular lattices. (C98)RhuA lattices display a Poisson's ratio of -1-the lowest thermodynamically possible value for an isotropic material-making them auxetic.

mitoXplorer, a visual data mining platform to systematically analyze and visualize mitochondrial expression dynamics and mutations.

Mitochondria participate in metabolism and signaling. They adapt to the requirements of various cell types. Publicly available expression data permit to study expression dynamics of genes with mitochondrial function (mito-genes) in various cell types, conditions and organisms. Yet, we lack an easy way of extracting these data for mito-genes. Here, we introduce the visual data mining platform mitoXplorer, which integrates expression and mutation data of mito-genes with a manually curated mitochondrial interactome containing ∼1200 genes grouped in 38 mitochondrial processes. User-friendly analysis and visualization tools allow to mine mitochondrial expression dynamics and mutations across various datasets from four model species including human. To test the predictive power of mitoXplorer, we quantify mito-gene expression dynamics in trisomy 21 cells, as mitochondrial defects are frequent in trisomy 21. We uncover remarkable differences in the regulation of the mitochondrial transcriptome and proteome in one of the trisomy 21 cell lines, caused by dysregulation of the mitochondrial ribosome and resulting in severe defects in oxidative phosphorylation. With the newly developed Fiji plugin mitoMorph, we identify mild changes in mitochondrial morphology in trisomy 21. Taken together, mitoXplorer (http://mitoxplorer.ibdm.univ-mrs.fr) is a user-friendly, web-based and freely accessible software, aiding experimental scientists to quantify mitochondrial expression dynamics.

Cryo-EM Elucidation of the Structure of Bacteriophage P22 Virions after Genome Release.

Genome ejection proteins are required to facilitate transport of bacteriophage P22 double-stranded DNA safely through membranes of Salmonella. The structures and locations of all proteins in the context of the mature virion are known, with the exception of three ejection proteins. Furthermore, the changes that occur to the proteins residing in the mature virion upon DNA release are not fully understood. We used cryogenic electron microscopy to obtain what is, to our knowledge, the first asymmetric reconstruction of mature bacteriophage P22 after double-stranded DNA has been extruded from the capsid-a state representative of one step during viral infection. Results of icosahedral and asymmetric reconstructions at estimated resolutions of 7.8 and 12.5 Å resolutions, respectively, are presented. The reconstruction shows tube-like protein density extending from the center of the tail assembly. The portal protein does not revert to the more contracted, procapsid state, but instead maintains an extended and splayed barrel structure. These structural details contribute to our understanding of the molecular mechanism of P22 phage infection and also set the foundation for future exploitation serving engineering purposes.

Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging.

UNLABELLED: The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the ßA strand region under the icosahedral 2-fold axis rather than antiparallel to the ßB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency. IMPORTANCE: The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ∼1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production.

Three-dimensional structure of a protozoal double-stranded RNA virus that infects the enteric pathogen Giardia lamblia.

UNLABELLED: Giardia lamblia virus (GLV) is a small, nonenveloped, nonsegmented double-stranded RNA (dsRNA) virus infecting Giardia lamblia, the most common protozoan pathogen of the human intestine and a major agent of waterborne diarrheal disease worldwide. GLV (genus Giardiavirus) is a member of family Totiviridae, along with several other groups of protozoal or fungal viruses, including Leishmania RNA viruses and Trichomonas vaginalis viruses. Interestingly, GLV is more closely related than other Totiviridae members to a group of recently discovered metazoan viruses that includes penaeid shrimp infectious myonecrosis virus (IMNV). Moreover, GLV is the only known protozoal dsRNA virus that can transmit efficiently by extracellular means, also like IMNV. In this study, we used transmission electron cryomicroscopy and icosahedral image reconstruction to examine the GLV virion at an estimated resolution of 6.0 Å. Its outermost diameter is 485 Å, making it the largest totivirus capsid analyzed to date. Structural comparisons of GLV and other totiviruses highlighted a related "T=2" capsid organization and a conserved helix-rich fold in the capsid subunits. In agreement with its unique capacity as a protozoal dsRNA virus to survive and transmit through extracellular environments, GLV was found to be more thermoresistant than Trichomonas vaginalis virus 1, but no specific protein machinery to mediate cell entry, such as the fiber complexes in IMNV, could be localized. These and other structural and biochemical findings provide a basis for future work to dissect the cell entry mechanism of GLV into a "primitive" (early-branching) eukaryotic host and an important enteric pathogen of humans. IMPORTANCE: Numerous pathogenic bacteria, including Corynebacterium diphtheriae, Salmonella enterica, and Vibrio cholerae, are infected with lysogenic bacteriophages that contribute significantly to bacterial virulence. In line with this phenomenon, several pathogenic protozoa, including Giardia lamblia, Leishmania species, and Trichomonas vaginalis are persistently infected with dsRNA viruses, and growing evidence indicates that at least some of these protozoal viruses can likewise enhance the pathogenicity of their hosts. Understanding of these protozoal viruses, however, lags far behind that of many bacteriophages. Here, we investigated the dsRNA virus that infects the widespread enteric parasite Giardia lamblia. Using electron cryomicroscopy and icosahedral image reconstruction, we determined the virion structure of Giardia lamblia virus, obtaining new information relating to its assembly, stability, functions in cell entry and transcription, and similarities and differences with other dsRNA viruses. The results of our study set the stage for further mechanistic work on the roles of these viruses in protozoal virulence.

OmpA and OmpC are critical host factors for bacteriophage Sf6 entry in Shigella.

Despite being essential for successful infection, the molecular cues involved in host recognition and genome transfer of viruses are not completely understood. Bacterial outer membrane proteins A and C co-purify in lipid vesicles with bacteriophage Sf6, implicating both outer membrane proteins as potential host receptors. We determined that outer membrane proteins A and C mediate Sf6 infection by dramatically increasing its rate and efficiency. We performed a combination of in vivo studies with three omp null mutants of Shigella flexneri, including classic phage plaque assays and time-lapse fluorescence microscopy to monitor genome ejection at the single virion level. Cryo-electron tomography of phage 'infecting' outer membrane vesicles shows the tail needle contacting and indenting the outer membrane. Lastly, in vitro ejection studies reveal that lipopolysaccharide and outer membrane proteins are both required for Sf6 genome release. We conclude that Sf6 phage entry utilizes either outer membrane proteins A or C, with outer membrane protein A being the preferred receptor.

Three-dimensional structure of victorivirus HvV190S suggests coat proteins in most totiviruses share a conserved core.

Double-stranded (ds)RNA fungal viruses are currently assigned to six different families. Those from the family Totiviridae are characterized by nonsegmented genomes and single-layer capsids, 300-450 Å in diameter. Helminthosporium victoriae virus 190S (HvV190S), prototype of recently recognized genus Victorivirus, infects the filamentous fungus Helminthosporium victoriae (telomorph: Cochliobolus victoriae), which is the causal agent of Victoria blight of oats. The HvV190S genome is 5179 bp long and encompasses two large, slightly overlapping open reading frames that encode the coat protein (CP, 772 aa) and the RNA-dependent RNA polymerase (RdRp, 835 aa). To our present knowledge, victoriviruses uniquely express their RdRps via a coupled termination-reinitiation mechanism that differs from the well-characterized Saccharomyces cerevisiae virus L-A (ScV-L-A, prototype of genus Totivirus), in which the RdRp is expressed as a CP/RdRp fusion protein due to ribosomal frameshifting. Here, we used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structures of HvV190S virions and two types of virus-like particles (capsids lacking dsRNA and capsids lacking both dsRNA and RdRp) at estimated resolutions of 7.1, 7.5, and 7.6 Å, respectively. The HvV190S capsid is thin and smooth, and contains 120 copies of CP arranged in a "T = 2" icosahedral lattice characteristic of ScV-L-A and other dsRNA viruses. For aid in our interpretations, we developed and used an iterative segmentation procedure to define the boundaries of the two, chemically identical CP subunits in each asymmetric unit. Both subunits have a similar fold, but one that differs from ScV-L-A in many details except for a core α-helical region that is further predicted to be conserved among many other totiviruses. In particular, we predict the structures of other victoriviruses to be highly similar to HvV190S and the structures of most if not all totiviruses including, Leishmania RNA virus 1, to be similar as well.

Visualization of a missing link in retrovirus capsid assembly.

For a retrovirus such as HIV to be infectious, a properly formed capsid is needed; however, unusually among viruses, retrovirus capsids are highly variable in structure. According to the fullerene conjecture, they are composed of hexamers and pentamers of capsid protein (CA), with the shape of a capsid varying according to how the twelve pentamers are distributed and its size depending on the number of hexamers. Hexamers have been studied in planar and tubular arrays, but the predicted pentamers have not been observed. Here we report cryo-electron microscopic analyses of two in-vitro-assembled capsids of Rous sarcoma virus. Both are icosahedrally symmetric: one is composed of 12 pentamers, and the other of 12 pentamers and 20 hexamers. Fitting of atomic models of the two CA domains into the reconstructions shows three distinct inter-subunit interactions. These observations substantiate the fullerene conjecture, show how pentamers are accommodated at vertices, support the inference that nucleation is a crucial morphologic determinant, and imply that electrostatic interactions govern the differential assembly of pentamers and hexamers.

Single particle analysis integrated with microscopy: a high-throughput approach for reconstructing icosahedral particles.

In cryo-electron microscopy and single particle analysis, data acquisition and image processing are generally carried out in sequential steps and computation of a three-dimensional reconstruction only begins once all the micrographs have been acquired. We are developing an integrated system for processing images of icosahedral particles during microscopy to provide reconstructed density maps in real-time at the highest possible resolution. The system is designed as a combination of pipelines to run in parallel on a computer cluster and analyzes micrographs as they are acquired, handling automatically all the processing steps from defocus estimation and particle picking to origin/orientation determination. An ab initio model is determined independently from the first micrographs collected, and new models are generated as more particles become available. As a proof of concept, we simulated data acquisition sessions using three sets of micrographs of good to excellent quality that were previously recorded from different icosahedral viruses. Results show that the processing of single micrographs can keep pace with an acquisition rate of about two images per minute. The reconstructed density map improves steadily during the image acquisition phase and its quality at the end of data collection is only moderately inferior to that obtained by expert users who processed semi-automatically all the micrographs after the acquisition. The current prototype demonstrates the advantages of integrating three-dimensional image processing with microscopy, which include an ability to monitor acquisition in terms of the final structure and to predict how much data and microscope resources are needed to achieve a desired resolution.

One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions.

The resolution of density maps from single particle analysis is usually measured in terms of the highest spatial frequency to which consistent information has been obtained. This calculation represents an average over the entire reconstructed volume. In practice, however, substantial local variations in resolution may occur, either from intrinsic properties of the specimen or for technical reasons such as a non-isotropic distribution of viewing orientations. To address this issue, we propose the use of a space-frequency representation, the short-space Fourier transform, to assess the quality of a density map, voxel-by-voxel, i.e. by local resolution mapping. In this approach, the experimental volume is divided into small subvolumes and the resolution determined for each of them. It is illustrated in applications both to model data and to experimental density maps. Regions with lower-than-average resolution may be mobile components or ones with incomplete occupancy or result from multiple conformational states. To improve the interpretability of reconstructions, we propose an adaptive filtering approach that reconciles the resolution to which individual features are calculated with the results of the local resolution map.

Three-dimensional reconstruction of icosahedral particles from single micrographs in real time at the microscope.

Single particle analysis is a valuable tool in cryo-electron microscopy for determining the structure of biological complexes. However, the conformational state and the preparation of the sample are factors that play a critical role in the ultimate attainable resolution. In some cases extensive analysis at the microscope of a sample under different conditions is required to derive the optimal acquisition conditions. Currently this analysis is limited to raw micrographs, thus conveying only limited information on the structure of the complex. We are developing a computing system that generates a three-dimensional reconstruction from a single micrograph acquired under cryogenic and low dose conditions, and containing particles with icosahedral symmetry. The system provides the microscopist with immediate structural information from a sample while it is in the microscope and during the preliminary acquisition stage. The system is designed to run without user intervention on a multi-processor computing resource and integrates all the processing steps required for the analysis. Tests performed on experimental data sets show that the probability of obtaining a reliable reconstruction from one micrograph is primarily determined by the quality of the sample, with success rates close to 100% when sample conditions are optimal, and decreasing to about 60% when conditions are sub-optimal. The time required to generate a reconstruction depends significantly on the diameter of the particles, and in most instances takes about 1min. The proposed approach can provide valuable three-dimensional information, albeit at low resolution, on conformational states, epitope binding, and stoichiometry of icosahedral multi-protein complexes.

Structural changes in Influenza virus at low pH characterized by cryo-electron tomography.

Influenza virus enters host cells by endocytosis. The low pH of endosomes triggers conformational changes in hemagglutinin (HA) that mediate fusion of the viral and endosomal membranes. We have used cryo-electron tomography to visualize influenza A virus at pH 4.9, a condition known to induce fusogenicity. After 30 min, when all virions are in the postfusion state, dramatic changes in morphology are apparent: elongated particles are no longer observed, larger particles representing fused virions appear, the HA spikes become conspicuously disorganized, a layer of M1 matrix protein is no longer resolved on most virions, and the ribonucleoprotein complexes (RNPs) coagulate on the interior surface of the virion. To probe for intermediate states, preparations were imaged after 5 min at pH 4.9. These virions could be classified according to their glycoprotein arrays (organized or disorganized) and whether or not they have a resolved M1 layer. Employing subtomogram averaging, we found, in addition to the neutral-pH state of HA, two intermediate conformations that appear to reflect an outwards movement of the fusion peptide and rearrangement of the HA1 subunits, respectively. These changes are reversible. The tomograms also document pH-induced changes affecting the M1 layer that appear to render the envelope more pliable and hence conducive to fusion. However, it appears desirable for productive infection that fusion should proceed before the RNPs become coagulated with matrix protein, as eventually happens at low pH.

Visualization of the two-step fusion process of the retrovirus avian sarcoma/leukosis virus by cryo-electron tomography.

Retrovirus infection starts with the binding of envelope glycoproteins to host cell receptors. Subsequently, conformational changes in the glycoproteins trigger fusion of the viral and cellular membranes. Some retroviruses, such as avian sarcoma/leukosis virus (ASLV), employ a two-step mechanism in which receptor binding precedes low-pH activation and fusion. We used cryo-electron tomography to study virion/receptor/liposome complexes that simulate the interactions of ASLV virions with cells. Binding the soluble receptor at neutral pH resulted in virions capable of binding liposomes tightly enough to alter their curvature. At virion-liposome interfaces, the glycoproteins are ∼3-fold more concentrated than elsewhere in the viral envelope, indicating specific recruitment to these sites. Subtomogram averaging showed that the oblate globular domain in the prehairpin intermediate (presumably the receptor-binding domain) is connected to both the target and the viral membrane by 2.5-nm-long stalks and is partially disordered, compared with its native conformation. Upon lowering the pH, fusion took place. Fusion is a stochastic process that, once initiated, must be rapid, as only final (postfusion) products were observed. These fusion products showed glycoprotein spikes on their surface, with their interiors occupied by patches of dense material but without capsids, implying their disassembly. In addition, some of the products presented a density layer underlying and resolved from the viral membrane, which may represent detachment of the matrix protein to facilitate the fusion process.

The UL36 tegument protein of herpes simplex virus 1 has a composite binding site at the capsid vertices.

Herpesviruses have an icosahedral nucleocapsid surrounded by an amorphous tegument and a lipoprotein envelope. The tegument comprises at least 20 proteins destined for delivery into the host cell. As the tegument does not have a regular structure, the question arises of how its proteins are recruited. The herpes simplex virus 1 (HSV-1) tegument is known to contact the capsid at its vertices, and two proteins, UL36 and UL37, have been identified as candidates for this interaction. We show that the interaction is mediated exclusively by UL36. HSV-1 nucleocapsids extracted from virions shed their UL37 upon incubation at 37°C. Cryo-electron microscopy (cryo-EM) analysis of capsids with and without UL37 reveals the same penton-capping density in both cases. As no other tegument proteins are retained in significant amounts, it follows that this density feature (∼100 kDa) represents the ordered portion of UL36 (336 kDa). It binds between neighboring UL19 protrusions and to an adjacent UL17 molecule. These observations support the hypothesis that UL36 plays a major role in the tegumentation of the virion, providing a flexible scaffold to which other tegument proteins, including UL37, bind. They also indicate how sequential conformational changes in the maturing nucleocapsid control the ordered binding, first of UL25/UL17 and then of UL36.

Cryo-EM study of Hepatitis B virus core antigen capsids decorated with antibodies from a human patient.

The capsid (core antigen, HBcAg) is one of three major antigens present in patients infected with Hepatitis B virus. The capsids are icosahedral particles, whose most prominent features are spikes that extend 25 Å out from the contiguous "floor". At the spike tip are two copies of the "immunodominant loop". Previously, the epitopes of seven murine monoclonal antibodies have been identified by cryo-EM analysis of Fab-labeled capsids. All but one are conformational and all but one map around the spike tip. The exception, which is also the tightest-binder, straddles an inter-molecular interface on the floor. Seeking to relate these observations to the immunological response of infected humans, we isolated anti-cAg antibodies from a patient, prepared Fabs, and analyzed their binding to capsids. A priori, one possibility was that many different Fabs would give an undifferentiated continuum of Fab-related density. In fact, the density observed was highly differentiated and could be reproduced by modeling with just five Fabs, three binding to the spike and two to the floor. These results show that epitopes on the floor, far (~30 Å) from the immunodominant loop, are clinically relevant and that murine anti-cAg antibodies afford a good model for the human system.

Procapsid assembly, maturation, nuclear exit: dynamic steps in the production of infectious herpesvirions.

Herpesviruses, a family of animal viruses with large (125-250 kbp) linear DNA genomes, are highly diversified in terms of host range; nevertheless, their virions conform to a common architecture. The genome is confined at high density within a thick-walled icosahedral capsid with the uncommon (among viruses, generally) but unvarying triangulation number T = 16. The envelope is a membrane in which some 11 different viral glycoproteins are implanted. Between the capsid and the envelope is a capacious compartment called the tegument that accommodates ∼20-40 different viral proteins (depending on which virus) destined for delivery into a host cell. A strong body of evidence supports the hypothesis that herpesvirus capsids and those of tailed bacteriophages stem from a distant common ancestor, whereas their radically different infection apparatuses - envelope on one hand and tail on the other - reflect subsequent coevolution with divergent hosts. Here we review the molecular components of herpesvirus capsids and the mechanisms that regulate their assembly, with particular reference to the archetypal alphaherpesvirus, herpes simplex virus type 1; assess their duality with the capsids of tailed bacteriophages; and discuss the mechanism whereby, once DNA packaging has been completed, herpesvirus nucleocapsids exit from the nucleus to embark on later stages of the replication cycle.

Suppression of a morphogenic mutant in Rous sarcoma virus capsid protein by a second-site mutation: a cryoelectron tomography study.

Retrovirus assembly is driven by polymerization of the Gag polyprotein as nascent virions bud from host cells. Gag is then processed proteolytically, releasing the capsid protein (CA) to assemble de novo inside maturing virions. CA has N-terminal and C-terminal domains (NTDs and CTDs, respectively) whose folds are conserved, although their sequences are divergent except in the 20-residue major homology region (MHR) in the CTD. The MHR is thought to play an important role in assembly, and some mutations affecting it, including the F167Y substitution, are lethal. A temperature-sensitive second-site suppressor mutation in the NTD, A38V, restores infectivity. We have used cryoelectron tomography to investigate the morphotypes of this double mutant. Virions produced at the nonpermissive temperature do not assemble capsids, although Gag is processed normally; moreover, they are more variable in size than the wild type and have fewer glycoprotein spikes. At the permissive temperature, virions are similar in size and spike content as in the wild type and capsid assembly is restored, albeit with altered polymorphisms. The mutation F167Y-A38V (referred to as FY/AV in this paper) produces fewer tubular capsids than wild type and more irregular polyhedra, which tend to be larger than in the wild type, containing approximately 30% more CA subunits. It follows that FY/AV CA assembles more efficiently in situ than in the wild type and has a lower critical concentration, reflecting altered nucleation properties. However, its infectivity is lower than that of the wild type, due to a 4-fold-lower budding efficiency. We conclude that the wild-type CA protein sequence represents an evolutionary compromise between competing requirements for optimization of Gag assembly (of the immature virion) and CA assembly (in the maturing virion).

Computational resources for cryo-electron tomography in Bsoft.

The Bsoft package [Heymann, J.B., Belnap, D.M., 2007. Bsoft: image processing and molecular modeling for electron microscopy. J. Struct. Biol. 157, 3-18] has been enhanced by adding utilities for processing electron tomographic (ET) data; in particular, cryo-ET data characterized by low contrast and high noise. To handle the high computational load efficiently, a workflow was developed, based on the database-like parameter handling in Bsoft, aimed at minimizing user interaction and facilitating automation. To the same end, scripting elements distribute the processing among multiple processors on the same or different computers. The resolution of a tomogram depends on the precision of projection alignment, which is usually based on pinpointing fiducial markers (electron-dense gold particles). Alignment requires accurate specification of the tilt axis, and our protocol includes a procedure for determining it to adequate accuracy. Refinement of projection alignment provides information that allows assessment of its precision, as well as projection quality control. We implemented a reciprocal space algorithm that affords an alternative to back-projection or real space algorithms for calculating tomograms. Resources are also included that allow resolution assessment by cross-validation (NLOO2D); denoising and interpretation; and the extraction, mutual alignment, and averaging of tomographic sub-volumes.

Direct induction of microtubule branching by microtubule nucleation factor SSNA1.

Microtubules are central elements of the eukaryotic cytoskeleton that often function as part of branched networks. Current models for branching include nucleation of new microtubules from severed microtubule seeds or from γ-tubulin recruited to the side of a pre-existing microtubule. Here, we found that microtubules can be directly remodelled into branched structures by the microtubule-remodelling factor SSNA1 (also known as NA14 or DIP13). The branching activity of SSNA1 relies on its ability to self-assemble into fibrils in a head-to-tail fashion. SSNA1 fibrils guide protofilaments of a microtubule to split apart to form daughter microtubules. We further found that SSNA1 localizes at axon branching sites and has a key role in neuronal development. SSNA1 mutants that abolish microtubule branching in vitro also fail to promote axon development and branching when overexpressed in neurons. We have, therefore, discovered a mechanism for microtubule branching and implicated its role in neuronal development.