Iterative Helical Real Space Reconstruction Research Articles

Structural Analysis of Human Fascin-1: Essential Protein for Actin Filaments Bundling.

Fascin, a major actin cross-linking protein, is expressed in most vertebrate epithelial tissues. It organizes actin filaments into well-ordered bundles that are responsible for the extension of dynamic membrane protrusions, including microspikes, filopodia, and invadopodia from cell surfaces, which are involved in cell migration and invasion as critical components of cancer metastasis. However, it is not well-understood how fascin-1 induces actin binding/bundling and where fascin-1 localizes along the actin filaments, thus facilitating actin bundle formation. In the present study, we attempted to clarify these problems by using biochemical and electron microscopic analyses using various fascin-1 constructs. Three dimensional structures of actin/fascin-1 complex were obtained by electron microscopy (EM) with iterative helical real-space reconstruction (IHRSR) and tomography. We revealed that the N-terminal region containing the Actin-Binding Site 2 (ABS2) of fascin-1 is responsible for actin bundling and the C-terminal region is important for the dimerization of fascin-1. We also found that the dimerization of fascin-1 through intermolecular interactions of the C-terminal region is essential for actin bundling. Since fascin is an important factor in cancer development, it is expected that the findings of present study will provide useful information for development of therapeutic strategies for cancer.

Structural Studies of Alpha-Synuclein Amyloid Filaments

Amyloid filaments of the alpha-synuclein (alpha-Syn) protein are considered the hallmark of Parkinson's disease (PD) and no biomarkers for early diagnosis have yet been developed. Thioflavin T (ThT) is the most frequently used dye to detect amyloids. We are using cryo-EM of ThT-bound alpha-Syn fibrils of PD familial variants (e.g., A30P, E46K and, A53T) to understand its mechanism of binding. The heterogeneity of amyloid filaments has been one of the greatest obstacles to structural studies. We used the fluorescence of ThT to follow the kinetics of alpha-Syn fibril formation at different ionic strengths to control fibril length and sample homogeneity for cryo-EM studies. The results show we can modulate secondary nucleation events involved in filament growth (i.e., the fragmentation rate) using sodium chloride concentrations below 1mM. Controlled proteinase-K digestion coupled with electron-spray ionization mass spectrometry of the fibril variants revealed distinctly different filament structures. Additionally, the power spectra of A30P and A53T filaments showed the 1/4.7 Å-1 layer line typical of cross-beta amyloids but the two have distinctly different helical organization. The A53T power spectrum revealed that the 1/4.7 Å-1 layer line is not a meridional but arises from a 1-start helix. This shows that the two strands in the filament are staggered, with a rise per subunit of 4.7 Å within each protofilament. We have confirmed this symmetry using the Iterative Helical Real Space Reconstruction (IHRSR) method to generate near-atomic resolution density maps of these amyloid filaments. Understanding the molecular mechanism in which ThT binds to amyloids represents a first step for structure-guided design of small molecules that may act as biomarkers for early PD detection.

Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function.

The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, "free" and "blocked", formed an asymmetric structure named the "interacting-heads motif" (IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca2+-activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and post-tetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.

Endophilin-Dynamin Complex Assembly - A General Mechanism of Membrane Remodeling Control

Endophilin belongs to a group of proteins containing membrane binding and bending BAR domains. Neuronal-specific endophilin A1 mediates membrane bending in association with dynamin 1-catalyzed membrane fission. In a previous study, we show that endophilin A1 co-localizes with dynamin 1 on necks of clathrin-coated pits in nerve terminals and assembles into a complex with dynamin 1 on tubulated liposomes in vitro. Using cryo-EM and iterative helical real space reconstruction (IHRSR) we have generated a preliminary 3D density map of the endophilin A1–dynamin 1 complex.

An Obsevation of Native Skeletal Thin Filament Structure at High Calcium Condition by Electron Cryomicroscopy

It is hampering our understanding on the molecular mechanism of muscle contraction thin filament-linked modulation lack of electron cryomicryoscopy data supporting the steric model (Nature 368,64-67,1994). Recently well-oriented sols of flamentous (F)-actin formed by Ca2+-actin and length controlled by gelsolin give detailed X-ray fibre diffraction patterns led to the flat-actin conformation resolved in F-actin (Nature 457,441-550,2009). von der Eckon et al (Nature,519,114-119,2015) defined a three-dimensional structure of F-actin at a resolution of 3.7A and in synthetic complex with tropomyosin (no troponin complex) at a resolution of 6.5A using electron cryomicroscopy. Here is the first report about a well-defined structure of native skeletal thin filament at high calcium condition by electron cryomicroscopy with Iterative Helical Real-Space Reconstruction (IHRSR): tropomyosin locates C-state on actin, spreads over one of myosin subfragment-1 (S1) strongly binding sites between subdomain_I and_III of the actin (JMB,266,8-14,1997); one intra-strand contact with strong 3D-reconstructed density and more than one inter-strand contacts are resolved among neighboring actin monomers (n, n+1, n+2), mainly formed through actin hydrophobic plug 256-271 linked to various regions of the opposite actin subunits, different from those of S1 decorated F-actin and the published F-actin and F-actin-Tropomyosin complex, in the native skeletal thin filament suggesting actin conformation diversity—globular-actin conformation dominating in high calcium condition, the flat conformation coexisting, and the two identified actin conformations are dynamic in thin filaments during the modulation of skeletal muscle contraction.

The Structure of the Actin-Smooth Muscle MYOSIN II Complex in the Rigor State

The actin-based motility of myosin utilizes catalysis of ATP to drive relative sliding of actin and myosin. Attachment to F-actin triggers conformational changes in the myosin head that accelerates release of catalytic products coupled to changes in actin affinity and motion of a structural domain called the lever arm. The earliest detailed model based on cryoEM and X-ray crystallography postulated that higher F-actin affinity and lever arm movement were coupled to closure of a feature of the myosin head dubbed the actin-binding cleft. Several studies since then using crystallography of myosin-V and cryoEM of myosin-I and -II have provided details of this model. Compared with non-muscle myosins, there are comparatively few catalytic intermediates available for myosin II. Details of the myosin II interaction with actin may differ from those for non-muscle myosins due in part to different lengths of important surface loops. Here we report on a 0.8 nm reconstruction of actin decorated with the smooth muscle myosin-II motor domain (MD) in the rigor state obtained by cryoEM and Iterative Helical Real Space Reconstruction. Quasiatomic models for both F-actin and the MD were obtained independently three times using molecular dynamics flexible fitting. SD3 and SD4 of F-actin are nearly identical while SD1 and SD2 show the most difference. The MD density is less well defined compared to the actin filament due to incomplete saturation. A significant density that could be assigned to loop1 is present. Loop2 and the myopathy loop show significant contacts with actin but these contacts appear variable between the three independent fittings. The comparison between nucleotide free acto-MD structure and the prepower-stroke crystal structure of the smooth muscle myosin MD suggests significant conformational changes occurred that may relate to the weak to strong transition.

Resolving the Structural Basis of Factor VIII Activation

Activated Factor VIII (FVIIIa) is the essential co-factor for the serine protease Factor IXa (FIXa) in the membrane-bound Tenase complex. In the blood coagulation process, the interaction between FVIIIa and FIXa in presence of Ca2+ and phospholipids amplifies the proteolytic activity of Factor IXa more than 100.000 times. The Tenase complex catalyzes the activation of Factor X and initiates a cascade of multi-protein complex formation which amplifies the Thrombin production and secures the formation of a blood clot.Here we report for the first time the 3D-structure of the membrane-bound FVIIIa form, which is the most physiologically relevant in relation to the delicate equilibrium of hemostasis and thrombosis.The structure of the membrane bound light chain (domains A3-C1-C2) of human FVIII helically organized onto lipid bilayer nanotubes (LNT) clarified that the domains adopt a different organization than in the crystal structure which represents the molecule in solution. These differences of the membrane-bound FVIII structure have to be confirmed for the full length FVIII protein and the active form. To achieve this, high resolution Cryo-EM data of porcine FVIII and FVIIIa helically organized on LNT at closest to physiological conditions were collected. A newly developed process of iterated rounds of 2D refinement was applied to select only highly organized homogenous helical segments for an effective iterative helical real-space reconstruction (IHRSR). The calculated porcine FVIII and FVIIIa membrane-bound structures at subnanometer resolution were combined with flexible docking to define the changes in the FVIIIa interfaces important for its function.Understanding the structural differences and similarities between the membrane-bound activated FVIII and the non-activated FVIII will make an indispensable contribution to the field of coagulation in determining unique targets for the design of specific therapeutic drugs against Hemophilia A and thrombosis.

Variable Internal Flexibility Characterizes the Helical Capsid Formed by Agrobacterium VirE2 Protein on Single-Stranded DNA

Agrobacterium is known for gene transfer to plants. In addition to a linear ssDNA oligonucleotide, Agrobacterium tumefaciens secretes an abundant ssDNA-binding effector, VirE2. In many ways VirE2 adapts the conjugation mechanism to transform the eukaryotic host. The crystal structure of VirE2 shows two compact domains joined by a flexible linker. Bound to ssDNA, VirE2 forms an ordered solenoidal shell, or capsid known as the T-complex. Here, we present a three-dimensional reconstruction of the VirE2-ssDNA complex using cryo-electron microscopy and iterative helical real-space reconstruction. High-resolution refinement was not possible due to inherent heterogeneity in the protein structure. By a combination of computational modeling, chemical modifications, mass spectroscopy, and electron paramagnetic resonance, we found that the N-terminal domain is tightly constrained by both tangential and longitudinal links, while the C terminus is weakly constrained. The quaternary structure is thus rigidly assembled while remaining locally flexible. This flexibility may be important in accommodating substrates without sequence specificity.

Functional attributes of the Saccharomyces cerevisiae meiotic recombinase Dmc1

The role of Dmc1 as a meiosis-specific general recombinase was first demonstrated in Saccharomyces cerevisiae. Progress in understanding the biochemical mechanism of ScDmc1 has been hampered by its tendency to form inactive aggregates. We have found that the inclusion of ATP during protein purification prevents Dmc1 aggregation. ScDmc1 so prepared is capable of forming D-loops and responsive to its accessory factors Rad54 and Rdh54. Negative staining electron microscopy and iterative helical real-space reconstruction revealed that the ScDmc1-ssDNA nucleoprotein filament harbors 6.5 protomers per turn with a pitch of ∼106Å. The ScDmc1 purification procedure and companion molecular analyses should facilitate future studies on this recombinase.

Electron Microscopy Reconstruction of Helical Assemblies

The cryo-electron microscopy reconstruction technique has been developed rapidly in recent years and becoming an important approach for structural studies of macromolecular complexes,and it includes three different methods,cryo-electron tomography,electron crystallography,and single particle analysis(SPA).Among them,the SPA technique is now becoming a powerful tool to determine near-atomic resolution structures of macromolecular complexes.However,for those biological macromolecular assemblies with helical symmetry,such as tobacco mosaic virus(TMV),microtubules,microfilament,human immunodeficiency virus 1 capsid protein and etc.,it is not easy to obtain their three dimensional structures by using SPA because there are lots of math to deal with.In this paper,the authors described the helical reconstruction technique in details,including the basic mathematical description of helical assemblies,helical diffraction and its indexing,the relationship between Fourier transform of helical points array and that of real helical assemblies,and how to determine the helical parameters.Based on the above introduction,the authors reviewed two main helical reconstruction algorithms,the Fourier-Bessel reconstruction and the iterative helical real space reconstruction(IHRSR).At the end,they selected the helical assembly of Par-3 NTD(Par3 is a kind of protein related to cell polarity control and Par-3 NTD is its N-terminal domain) as an example to show a detailed helical reconstruction protocol using IHRSR algorithm.

The myosin interacting-heads motif is present in the relaxed thick filament of the striated muscle of scorpion

Electron microscopy (EM) studies of 2D crystals of smooth muscle myosin molecules have shown that in the inactive state the two heads of a myosin molecule interact asymmetrically forming a myosin interacting-heads motif. This suggested that inactivation of the two heads occurs by blocking of the actin-binding site of one (free head) and the ATP hydrolysis site of the other (blocked head). This motif has been found by EM of isolated negatively stained myosin molecules of unregulated (vertebrate skeletal and cardiac muscle) and regulated (invertebrate striated and vertebrate smooth muscle) myosins, and nonmuscle myosin. The same motif has also been found in 3D-reconstructions of frozen-hydrated (tarantula, Limulus, scallop) and negatively stained (scallop, vertebrate cardiac) isolated thick filaments. We are carrying out studies of isolated thick filaments from other species to assess how general this myosin interacting-heads motif is. Here, using EM, we have visualized isolated, negatively stained thick filaments from scorpion striated muscle. We modified the iterative helical real space reconstruction (IHRSR) method to include filament tilt, and band-pass filtered the aligned segments before averaging, achieving a 3.3 nm resolution 3D-reconstruction. This reconstruction revealed the presence of the myosin interacting-heads motif (adding to evidence that is widely spread), together with 12 subfilaments in the filament backbone. This demonstrates that conventional negative staining and imaging can be used to detect the presence of the myosin interacting-heads motif in helically ordered thick filaments from different species and muscle types, thus avoiding the use of less accessible cryo-EM and low electron-dose procedures.

Subnanometer Structure of the Actin/Myosin/Tropomyosin Complex

Actins and myosins play a central role in life as they form the molecular motors that transform chemical energy into movement. Without them, we would be confined to a very static existence.To fully understand how the interplay between actin and myosin leads to the conversion of the energy stored in ATP into mechanical energy, one needs structural information. Therefore, we have set out to determine the complex structure of the three major muscle proteins (actin, myosin and tropomyosin) using cryo electron microscopy and iterative helical real-space reconstruction. Using state of the art instruments and reconstruction algorithms we have determined a sub-nanometer resolution electron density map of the complex. With the help of electron-density guided flexible fitting we have subsequently refined the structure to obtain a quasi-atomic resolution structure of the full complex which enabled us, for the first time, to visualize interactions between actin and myosin that have been postulated by computational methods beforehand. In addition, we could also identify a novel interaction between myosin and tropomyosin.

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable. In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

Helical image reconstruction of the outward-open human erythrocyte band 3 membrane domain in tubular crystals

The C-terminal membrane domain of erythrocyte band 3 functions as an anion exchanger. Here, we report the three-dimensional (3D) structure of the membrane domain in an inhibitor-stabilized, outward-open conformation at 18A resolution. Unstained, frozen-hydrated tubular crystals containing the membrane domain of band 3 purified from human red blood cells (hB3MD) were examined using cryo-electron microscopy and iterative helical real-space reconstruction (IHRSR). The 3D image reconstruction of the tubular crystals showed the molecular packing of hB3MD dimers with dimensions of 60 x 110 A in the membrane plane and a thickness of 70A across the membrane. Immunoelectron microscopy and carboxyl-terminal digestion demonstrated that the intracellular surface of hB3MD was exposed on the outer surface of the tubular crystal. A 3D density map revealed that hB3MD consists of at least two subdomains and that the outward-open form is characterized by a large hollow area on the extracellular surface and continuous density on the intracellular surface.

Structural Convergence between Cryo-EM and NMR Reveals Intersubunit Interactions Critical for HIV-1 Capsid Function

Mature HIV-1 particles contain conical-shaped capsids that enclose the viral RNA genome and perform essential functions in the virus life cycle. Previous structural analysis of two- and three-dimensional arrays of the capsid protein (CA) hexamer revealed three interfaces. Here, we present a cryoEM study of a tubular assembly of CA and a high-resolution NMR structure of the CA C-terminal domain (CTD) dimer. In the solution dimer structure, the monomers exhibit different relative orientations compared to previous X-ray structures. The solution structure fits well into the EM density map, suggesting that the dimer interface is retained in the assembled CA. We also identified a CTD-CTD interface at the local three-fold axis in the cryoEM map and confirmed its functional importance by mutagenesis. In the tubular assembly, CA intermolecular interfaces vary slightly, accommodating the asymmetry present in tubes. This provides the necessary plasticity to allow for controlled virus capsid dis/assembly.

Histone Octamer Helical Tubes Suggest that an Internucleosomal Four-Helix Bundle Stabilizes the Chromatin Fiber

A major question in chromatin involves the exact organization of nucleosomes within the 30-nm chromatin fiber and its structural determinants of assembly. Here we investigate the structure of histone octamer helical tubes via the method of iterative helical real-space reconstruction. Accurate placement of the x-ray structure of the histone octamer within the reconstructed density yields a pseudoatomic model for the entire helix, and allows precise identification of molecular interactions between neighboring octamers. One such interaction that would not be obscured by DNA in the nucleosome consists of a twofold symmetric four-helix bundle formed between pairs of H2B-α3 and H2B-αC helices of neighboring octamers. We believe that this interface can act as an internucleosomal four-helix bundle within the context of the chromatin fiber. The potential relevance of this interface in the folding of the 30-nm chromatin fiber is discussed.

The cyanide hydratase from Neurospora crassa forms a helix which has a dimeric repeat

The fungal cyanide hydratases form a functionally specialized subset of the nitrilases which catalyze the hydrolysis of cyanide to formamide with high specificity. These hold great promise for the bioremediation of cyanide wastes. The low resolution (3.0 nm) three-dimensional reconstruction of negatively stained recombinant cyanide hydratase fibers from the saprophytic fungus Neurospora crassa by iterative helical real space reconstruction reveals that enzyme fibers display left-handed D(1) S(5.4) symmetry with a helical rise of 1.36 nm. This arrangement differs from previously characterized microbial nitrilases which demonstrate a structure built along similar principles but with a reduced helical twist. The cyanide hydratase assembly is stabilized by two dyadic interactions between dimers across the one-start helical groove. Docking of a homology-derived atomic model into the experimentally determined negative stain envelope suggests the location of charged residues which may form salt bridges and stabilize the helix.

Reversible Self-Assembly of Weibel-Palade Body-Like Tubules from Recombinant N-Terminal Domains of von Willebrand Factor.

Weibel-Palade bodies (WPBs) are elongated secretory granules of endothelial cells that are packed with tubules composed of von Willebrand factor (VWF), a multimeric protein required for hemostasis. Disruption of tubular packing prevents the orderly secretion of VWF multimers and blocks the subsequent binding of platelets. The cigar-like shape and tubular cross section of WPBs are conserved in all vertebrates, but little is known about how VWF specifies this packing arrangement. Starting from recombinant 82 kDa VWF propeptide (domains D1D2) and 114 kDa disulfide-bonded D'D3 dimer, we now have assembled tubules reversibly in vitro with the same dimensions as VWF tubules in WPBs. Assembly was induced at pH 6.2, reversed at pH 7.4, and required Ca2+. Recombinant D'D3 dimers did not self-associate at pH 7.4 or pH 6.2, with or without Ca2+. Without Ca2+, VWF propeptide did not bind to D'D3 dimers. At pH 7.4, with Ca2+, VWF propeptide formed noncovalent 160 kDa dimers and, when mixed with D'D3 dimers, assembled a 280 kDa complex of two propeptides and one D'D3 dimer as shown by gel filtration chromatography and multi-angle light scattering. Lowering the pH to 6.2 caused the formation of >3 MDa aggregates with the same stoichiometry, which dissociated upon adding EDTA or raising the pH to 7.4. Quick-freeze deep-etch EM showed that the large aggregates are hollow right-handed tubular helices. The iterative helical real space reconstruction method was used to make 3D reconstructions of the tubules at 22 Å resolution from negative stain EM images (Figure, left). Tubules consist of a right-handed helix with axial rise of 26.2 Å and twist of 85.6 degrees per subunit, or 4.2 subunits per 11 nm turn. The dimensions (outside diameter 25 nm, inside diameter 12 nm) are similar to those of tubules in WPBs in thin sections of endothelial cells by transmission EM (Figure, right and its insert). Each subunit contains one D'D3 dimer flanked by two D1D2 propeptides (Figure, center). Each D'D3 dimer makes a total of six contacts with D1D2 domains. Each D1D2 propeptide makes three contacts with D'D3 and just one end-to-end homotypic contact. The spatial arrangement of these building blocks and inter-domain contacts in tubules suggest a model by which decreasing pH along the secretory pathway coordinates the formation of intersubunit disulfide bonds with the tubular packaging of VWF multimers. Within the WPB, Ca2+-dependent and pH-dependent binding of D1D2 to D'D3 domains stabilizes the packing of VWF multimers into tubules, which behave as constrained springs. Upon secretion, the increased pH weakens these constraints and permits the helical tubules to unfurl into flowing blood without tangling. [Display omitted]

On the structures of filamentous bacteriophage Ff (fd, f1, M13)

The filamentous bacteriophage (Inovirus) strain Ff (fd, f1, M13) is widely used in molecular biophysics as a simple model system. A low resolution molecular model of the fd protein coat has been reported, derived from iterative helical real space reconstruction of cryo-electron micrographs (cryoEM). This model is significantly different from the model previously derived from X-ray fibre diffraction and solid-state NMR. We show that the cryoEM model agrees neither with solid-state NMR data nor with X-ray fibre diffraction data of fd, and has some puzzling structural features, for instance nanometre holes through the protein coat. We refine the cryoEM model against the X-ray data, and find that the model after refinement closely approximates the model derived directly from X-ray fibre diffraction and solid-state NMR data. We suggest possible reasons for the differences between the models derived from cryoEM and X-ray diffraction.