Interaction of human erythrocyte catalase with air-water interface in cryoEM.

One of the key goals in single-particle cryo-microscopy is to obtain a uniform distribution of particle orientations, so that the three-dimensional structure has isotropic resolution in Fourier space. A common problem arises from the interaction of protein molecules with the air-water interface that exists on both surfaces of the thin film of liquid that is formed prior to plunge-freezing into liquid ethane. Some proteins and other macromolecular complexes are disrupted by interaction with the air-water interface. Other proteins or macromolecules either become concentrated through their interaction with the interface or are excluded because they bind strongly to some other part of the grid or the filter paper used in blotting. In this paper, the interaction of human erythrocyte catalase with the air-water interface is investigated and minimized by the addition of certain detergents. Detergents can form an amphipathic monolayer at the air-water interface that creates a barrier and leaves the molecules free to adopt a variety of orientations, thus facilitating the 3D structure determination. These results suggest that further characterization and development of detergents for cryo-microscopy plunge-freezing would be useful.

Assessment of pharyngeal motor function using a novel velopharyngeal squeeze maneuver and a novel endoscopic pharyngeal contraction grade scale in patients with dysphagia after radiotherapy for nasopharyngeal carcinoma.

BACKGROUND: To investigate a novel velopharyngeal squeeze maneuver (VPSM) and novel endoscopic pharyngeal contraction grade (EPCG) scale for the evaluation of pharyngeal motor function. METHODS: During endoscopic examination of 77 post-irradiated nasopharyngeal carcinoma patients and control subjects, VPSM was rated and lateral pharyngeal wall movement graded with EPCG scale during swallowing. Pharyngeal constriction ratio (PCR) measured by videofluoroscopy was used for correlation. RESULTS: VPSM and EPCG scale showed almost perfect intra-rater and inter-rater reliability (Kappa: >0.90). VPSM was present in 61% of patients suggesting good pharyngeal motor function. VPSM was predictive of EPCG scale (Wald statistic = 29.99, p < 0.001). EPCG scale also correlated strongly with PCR (r: 0.812) and was predictive for aspiration (odds ratio: 22.14 [95% CI 5.01-97.89, p < 0.001]). CONCLUSIONS: VPSM and EPCG scale are two novel tools to assess pharyngeal motor function, and both correlate well with pharyngeal contractility and aspiration.

Engineering Crystal Packing in RNA-Protein Complexes II: A Historical Perspective from the Structural Studies of the Spliceosome.

Cryo-electron microscopy has greatly advanced our understanding of how the spliceosome cycles through different conformational states to conduct the chemical reactions that remove introns from pre-mRNA transcripts. The Cryo-EM structures were built upon decades of crystallographic studies of various spliceosomal RNA-protein complexes. In this review we give an overview of the crystal structures solved in the Nagai group, utilizing many of the strategies to design crystal packing as described in the accompanying paper.

Short Sleep Duration Is Weakly Associated with Carotid Intima-Media Thickness in Adolescents.

OBJECTIVE: To investigate the relationship between sleep duration and carotid intima-media thickness (CIMT) in adolescents. We hypothesized that short sleep duration was associated with an increased CIMT. STUDY DESIGN: This was a cross-sectional study. Healthy participants aged 10-18 years were recruited from a school-based cohort established to examine the prevalence of obstructive sleep apnea in Hong Kong. All participants completed a prospective 7-day sleep diary, underwent anthropometric measurements, overnight polysomnography, and CIMT assessment. Overweight participants or those with an obstructive apnea hypopnea index of ≥5 were excluded from analysis. Regression analysis was used to assess the association between CIMT and sleep duration and other possible correlates. RESULTS: One hundred forty-two participants completed the assessments. Male participants tended to have shorter sleep duration than females (P = .012). There were no differences in age, body mass index, Tanner developmental stage, or parental history of hypertension between groups of different sleep durations. There was a weak but significant association between short sleep duration and CIMT (r = -0.273; P < .001). CONCLUSION: Sleep duration was found to have a weakly negative association with CIMT. Further research is needed to determine whether adult adverse cardiovascular events may originate in childhood owing to short sleep duration.

Reversible swelling of SBMV is associated with reversible disordering.

The structures of the compact and swollen southern bean mosaic virus (SBMV) particles have been compared by X-ray diffraction and proton magnetic resonance (PMR). Small-angle X-ray scattering showed that removal of divalent cations at alkaline pH causes the particle diameter to increase from 289Å in the native SBMV by 12% in solution and by 9% in microcrystals. The swelling is fully reversible upon re-addition of Ca2+ and Mg2+ ions, as shown by the X-ray patterns at 6Å resolution and by the 270MHz PMR spectra. Beyond 30Å resolution, X-ray patterns from the compact SBMV in solution and in microcrystals show fine fringes of ∼1/225Å-1 width extending to 6Å resolution, whereas patterns from the swollen SBMV in solution and in microcrystals show only broader fringes of ∼1/90Å-1 width, Model calculations demonstrate that the fine fringes from compact SBMV arise from regular packing of the protein subunits on the icosahedral surface lattice; the smearing of fine fringes in the swollen virus pattern can be simulated by uncorrelated displacements of pentamers and hexamers of protein subunits, with a standard deviation of 6Å from their mean locations. The PMR spectrum of compact SBMV is poorly resolved, whereas PMR spectrum of swollen SBMV shows sharp resonances in the methyl proton region. The line-narrowing for a fraction of the aliphatic protons upon swelling cannot be accounted for by rotational relaxation of the particle of 6×106MW, but must be attributed to internal motion in small regions of the protein subunits.

Re-refinement of the spliceosomal U4 snRNP core-domain structure.

The core domain of small nuclear ribonucleoprotein (snRNP), comprised of a ring of seven paralogous proteins bound around a single-stranded RNA sequence, functions as the assembly nucleus in the maturation of U1, U2, U4 and U5 spliceosomal snRNPs. The structure of the human U4 snRNP core domain was initially solved at 3.6 Å resolution by experimental phasing using data with tetartohedral twinning. Molecular replacement from this model followed by density modification using untwinned data recently led to a structure of the minimal U1 snRNP at 3.3 Å resolution. With the latter structure providing a search model for molecular replacement, the U4 core-domain structure has now been re-refined. The U4 Sm site-sequence AAUUUUU has been shown to bind to the seven Sm proteins SmF-SmE-SmG-SmD3-SmB-SmD1-SmD2 in an identical manner as the U1 Sm-site sequence AAUUUGU, except in SmD1 where the bound U replaces G. The progression from the initial to the re-refined structure exemplifies a tortuous route to accuracy: where well diffracting crystals of complex assemblies are initially unavailable, the early model errors are rectified by exploiting preliminary interpretations in further experiments involving homologous structures. New insights are obtained from the more accurate model.

Endothelial cilia are essential for developmental vascular integrity in zebrafish.

The cilium is a signaling platform of the vertebrate cell. It has a critical role in polycystic kidney disease and nephronophthisis. Cilia have been detected on endothelial cells, but the function of these organelles in the vasculature remains incompletely defined. In this study, using genetic and chemical genetic tools in the model organism zebrafish, we reveal an essential role of cilia in developmental vascular integrity. Embryos expressing mutant intraflagellar transport genes, which are essential and specific for cilia biogenesis, displayed increased risk of developmental intracranial hemorrhage, whereas the morphology of the vasculature remained normal. Moreover, cilia were present on endothelial cells in the developing zebrafish vasculature. We further show that the involvement of cilia in vascular integrity is endothelial autonomous, because endothelial-specific re-expression of intraflagellar transport genes in respective mutants rescued the intracranial hemorrhage phenotype. Finally, whereas inhibition of Hedgehog signaling increased the risk of intracranial hemorrhage in ciliary mutants, activation of the pathway rescued this phenotype. In contrast, embryos expressing an inactivating mutation in pkd2, one of two autosomal dominant cystic kidney disease genes, did not show increased risk of developmental intracranial hemorrhage. These results suggest that Hedgehog signaling is a major mechanism for this novel role of endothelial cilia in establishing vascular integrity.

Structural basis of Brr2-Prp8 interactions and implications for U5 snRNP biogenesis and the spliceosome active site.

The U5 small nuclear ribonucleoprotein particle (snRNP) helicase Brr2 disrupts the U4/U6 small nuclear RNA (snRNA) duplex and allows U6 snRNA to engage in an intricate RNA network at the active center of the spliceosome. Here, we present the structure of yeast Brr2 in complex with the Jab1/MPN domain of Prp8, which stimulates Brr2 activity. Contrary to previous reports, our crystal structure and mutagenesis data show that the Jab1/MPN domain binds exclusively to the N-terminal helicase cassette. The residues in the Jab1/MPN domain, whose mutations in human Prp8 cause the degenerative eye disease retinitis pigmentosa, are found at or near the interface with Brr2, clarifying its molecular pathology. In the cytoplasm, Prp8 forms a precursor complex with U5 snRNA, seven Smproteins, Snu114, and Aar2, but after nuclear import, Brr2 replaces Aar2 to form mature U5 snRNP. Our structure explains why Aar2 and Brr2 are mutually exclusive and provides important insights into the assembly of U5 snRNP.

Target-of-rapamycin complex 1 (Torc1) signaling modulates cilia size and function through protein synthesis regulation.

The cilium serves as a cellular antenna by coordinating upstream environmental cues with numerous downstream signaling processes that are indispensable for the function of the cell. This role is supported by the revelation that defects of the cilium underlie an emerging class of human disorders, termed "ciliopathies." Although mounting interest in the cilium has demonstrated the essential role that the organelle plays in vertebrate development, homeostasis, and disease pathogenesis, the mechanisms regulating cilia morphology and function remain unclear. Here, we show that the target-of-rapamycin (TOR) growth pathway modulates cilia size and function during zebrafish development. Knockdown of tuberous sclerosis complex 1a (tsc1a), which encodes an upstream inhibitor of TOR complex 1 (Torc1), increases cilia length. In contrast, treatment of embryos with rapamycin, an inhibitor of Torc1, shortens cilia length. Overexpression of ribosomal protein S6 kinase 1 (S6k1), which encodes a downstream substrate of Torc1, lengthens cilia. Furthermore, we provide evidence that TOR-mediated cilia assembly is evolutionarily conserved and that protein synthesis is essential for this regulation. Finally, we demonstrate that TOR signaling and cilia length are pivotal for a variety of downstream ciliary functions, such as cilia motility, fluid flow generation, and the establishment of left-right body asymmetry. Our findings reveal a unique role for the TOR pathway in regulating cilia size through protein synthesis and suggest that appropriate and defined lengths are necessary for proper function of the cilium.

Qilin is essential for cilia assembly and normal kidney development in zebrafish.

Defects in the cilium, a once thought vestigial organelle, have recently been implicated in many human diseases, including a number of cystic kidney diseases such as polycystic kidney disease (PKD), Bardet Bieldl Syndrome, and Meckel-Gruber Syndrome. In a forward genetic screen, qilin was identified as a novel gene important in the pathogenesis of kidney cysts in zebrafish. In this paper we characterized qilin(hi3959A) mutant's phenotypes in detail, investigated cilia formation in this mutant and performed structural and functional analysis of the Qilin protein. Results reveal Qilin's essential role in cilia assembly and maintenance in multiple organs, including the kidney, the lateral line organ, and the outer segment of the photoreceptor cell. In addition, rescue experiments suggest that defective pronephric cilia correlate with the formation of kidney cysts in qilin(hi3959A) mutants. Further, genetic analysis suggests that qilin interacts with multiple intraflagellar transport (IFT) complex B genes, which is supported by the striking phenotypic similarities between qilin(hi3959A) and IFT complex B mutants. Finally, through deletion analysis we provide evidence that the well-conserved N-terminus and the coiled-coil domain of Qilin are both essential and sufficient for its function. Taken all the observations together, we propose that Qilin acts in a similar role as IFT complex B proteins in cilia assembly, maintenance and kidney development in zebrafish.

Analysis of cilia structure and function in zebrafish.

The cilium, a previously little studied cell surface protrusion, has emerged as an important organelle in vertebrate cells. This tiny structure is essential for normal embryonic development, including the formation of left-right asymmetry, limb morphogenesis, and the differentiation of sensory cells. In the adult, cilia also function in a variety of processes, such as the survival of photoreceptor cells, and the homeostasis in several tissues, including the epithelia of nephric ducts. Human ciliary malfunction is associated with situs inversus, kidney cysts, polydactyly, blindness, mental retardation, obesity, and many other abnormalities. The genetic accessibility and optical transparency of the zebrafish make it an excellent vertebrate model system to study cilia biology. In this chapter, we describe the morphology and distribution of cilia in zebrafish embryonic and larval organs. We also provide essential protocols to analyze cilia formation and function.

Structure of the spliceosomal U4 snRNP core domain and its implication for snRNP biogenesis.

The spliceosome is a dynamic macromolecular machine that assembles on pre-messenger RNA substrates and catalyses the excision of non-coding intervening sequences (introns). Four of the five major components of the spliceosome, U1, U2, U4 and U5 small nuclear ribonucleoproteins (snRNPs), contain seven Sm proteins (SmB/B', SmD1, SmD2, SmD3, SmE, SmF and SmG) in common. Following export of the U1, U2, U4 and U5 snRNAs to the cytoplasm, the seven Sm proteins, chaperoned by the survival of motor neurons (SMN) complex, assemble around a single-stranded, U-rich sequence called the Sm site in each small nuclear RNA (snRNA), to form the core domain of the respective snRNP particle. Core domain formation is a prerequisite for re-import into the nucleus, where these snRNPs mature via addition of their particle-specific proteins. Here we present a crystal structure of the U4 snRNP core domain at 3.6 Å resolution, detailing how the Sm site heptad (AUUUUUG) binds inside the central hole of the heptameric ring of Sm proteins, interacting one-to-one with SmE-SmG-SmD3-SmB-SmD1-SmD2-SmF. An irregular backbone conformation of the Sm site sequence combined with the asymmetric structure of the heteromeric protein ring allows each base to interact in a distinct manner with four key residues at equivalent positions in the L3 and L5 loops of the Sm fold. A comparison of this structure with the U1 snRNP at 5.5 Å resolution reveals snRNA-dependent structural changes outside the Sm fold, which may facilitate the binding of particle-specific proteins that are crucial to biogenesis of spliceosomal snRNPs.

Recognition of a signal peptide by the signal recognition particle.

Targeting of proteins to appropriate subcellular compartments is a crucial process in all living cells. Secretory and membrane proteins usually contain an amino-terminal signal peptide, which is recognized by the signal recognition particle (SRP) when nascent polypeptide chains emerge from the ribosome. The SRP-ribosome nascent chain complex is then targeted through its GTP-dependent interaction with SRP receptor to the protein-conducting channel on endoplasmic reticulum membrane in eukaryotes or plasma membrane in bacteria. A universally conserved component of SRP (refs 1, 2), SRP54 or its bacterial homologue, fifty-four homologue (Ffh), binds the signal peptides, which have a highly divergent sequence divisible into a positively charged n-region, an h-region commonly containing 8-20 hydrophobic residues and a polar c-region. No structure has been reported that exemplifies SRP54 binding of any signal sequence. Here we have produced a fusion protein between Sulfolobus solfataricus SRP54 (Ffh) and a signal peptide connected via a flexible linker. This fusion protein oligomerizes in solution through interaction between the SRP54 and signal peptide moieties belonging to different chains, and it is functional, as demonstrated by its ability to bind SRP RNA and SRP receptor FtsY. We present the crystal structure at 3.5 A resolution of an SRP54-signal peptide complex in the dimer, which reveals how a signal sequence is recognized by SRP54.

Cilia in cell signaling and human disorders.

One of the most widespread cellular organelles in nature is cilium, which is found in many unicellular and multicellular organisms. Formerly thought to be a mostly vestigial organelle, the cilium has been discovered in the past several decades to play critical motile and sensory roles involved in normal organogenesis during development. The role of cilia has also been implicated in an ever increasing array of seemingly unrelated human diseases, including blindness, kidney cysts, neural tube defects and obesity. In this article we review some of the recent developments in research on cilia, and how defects in ciliogenesis and function can give rise to developmental disorders and disease.

Interpreting a low resolution map of human U1 snRNP using anomalous scatterers.

We recently determined the crystal structure of the functional core of human U1 snRNP, consisting of nine proteins and one RNA, based on a 5.5 A resolution electron density map. At 5-7 A resolution, alpha helices and beta sheets appear as rods and slabs, respectively, hence it is not possible to determine protein fold de novo. Using inverse beam geometry, accurate anomalous signals were obtained from weakly diffracting and radiation sensitive P1 crystals. We were able to locate anomalous scatterers with positional errors below 2 A. This enabled us not only to place protein domains of known structure accurately into the map but also to trace an extended polypeptide chain, of previously undetermined structure, using selenomethionine derivatives of single methionine mutants spaced along the sequence. This method of Se-Met scanning, in combination with structure prediction, is a powerful tool for building a protein of unknown fold into a low resolution electron density map.

Crystal structure of human spliceosomal U1 snRNP at 5.5 A resolution.

Human spliceosomal U1 small nuclear ribonucleoprotein particles (snRNPs), which consist of U1 small nuclear RNA and ten proteins, recognize the 5' splice site within precursor messenger RNAs and initiate the assembly of the spliceosome for intron excision. An electron density map of the functional core of U1 snRNP at 5.5 A resolution has enabled us to build the RNA and, in conjunction with site-specific labelling of individual proteins, to place the seven Sm proteins, U1-C and U1-70K into the map. Here we present the detailed structure of a spliceosomal snRNP, revealing a hierarchical network of intricate interactions between subunits. A striking feature is the amino (N)-terminal polypeptide of U1-70K, which extends over a distance of 180 A from its RNA binding domain, wraps around the core domain consisting of the seven Sm proteins and finally contacts U1-C, which is crucial for 5'-splice-site recognition. The structure of U1 snRNP provides insights into U1 snRNP assembly and suggests a possible mechanism of 5'-splice-site recognition.

Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications.

Cry4Ba, isolated from Bacillus thuringiensis subsp. israelensis, is specifically toxic to the larvae of Aedes and Anopheles mosquitoes. The structure of activated Cry4Ba toxin has been determined by multiple isomorphous replacement with anomalous scattering and refined to R(cryst) = 20.5% and R(free)= 21.8% at 1.75 Angstroms resolution. It resembles previously reported Cry toxin structures but shows the following distinctions. In domain I the helix bundle contains only the long and amphipathic helices alpha3-alpha7. The N-terminal helices alpha1-alpha2b, absent due to proteolysis during crystallisation, appear inessential to toxicity. In domain II the beta-sheet prism presents short apical loops without the beta-ribbon extension of inner strands, thus placing the receptor combining sites close to the sheets. In domain III the beta-sandwich contains a helical extension from the C-terminal strand beta23, which interacts with a beta-hairpin excursion from the edge of the outer sheet. The structure provides a rational explanation of recent mutagenesis and biophysical data on this toxin. Furthermore, added to earlier structures from the Cry toxin family, Cry4Ba completes a minimal structural database covering the Coleoptera, Lepidoptera, Diptera and Lepidoptera/Diptera specificity classes. A multiple structure alignment found that the Diptera-specific Cry4Ba is structurally more closely similar to the Lepidoptera-specific Cry1Aa than the Coleoptera-specific Cry3Aa, but most distantly related to Lepidoptera/Diptera-specific Cry2Aa. The structures are most divergent in domain II, supporting the suggestion that this domain has a major role in specificity determination. They are most similar in the alpha3-alpha7 major fragment of domain I, which contains the alpha4-alpha5 hairpin crucial to pore formation. The collective knowledge of Cry toxin structure and mutagenesis data will lead to a more critical understanding of the structural basis for receptor binding and pore formation, as well as allowing the scope of diversity to be better appreciated.

Structure of bovine rhodopsin in a trigonal crystal form.

We have determined the structure of bovine rhodopsin at 2.65 A resolution using untwinned native crystals in the space group P3(1), by molecular replacement from the 2.8 A model (1F88) solved in space group P4(1). The new structure reveals mechanistically important details unresolved previously, which are considered in the membrane context by docking the structure into a cryo-electron microscopy map of 2D crystals. Kinks in the transmembrane helices facilitate inter-helical polar interactions. Ordered water molecules extend the hydrogen bonding networks, linking Trp265 in the retinal binding pocket to the NPxxY motif near the cytoplasmic boundary, and the Glu113 counterion for the protonated Schiff base to the extracellular surface. Glu113 forms a complex with a water molecule hydrogen bonded between its main chain and side-chain oxygen atoms. This can be expected to stabilise the salt-bridge with the protonated Schiff base linking the 11-cis-retinal to Lys296. The cytoplasmic ends of helices H5 and H6 have been extended by one turn. The G-protein interaction sites mapped to the cytoplasmic ends of H5 and H6 and a spiral extension of H5 are elevated above the bilayer. There is a surface cavity next to the conserved Glu134-Arg135 ion pair. The cytoplasmic loops have the highest temperature factors in the structure, indicative of their flexibility when not interacting with G protein or regulatory proteins. An ordered detergent molecule is seen wrapped around the kink in H6, stabilising the structure around the potential hinge in H6. These findings provide further explanation for the stability of the dark state structure. They support a mechanism for the activation, initiated by photo-isomerisation of the chromophore to its all-trans form, that involves pivoting movements of kinked helices, which, while maintaining hydrophobic contacts in the membrane interior, can be coupled to amplified translation of the helix ends near the membrane surfaces.

Crystals of native and modified bovine rhodopsins and their heavy atom derivatives.

Rhodopsin, the pigment protein responsible for dim-light vision, is a G protein-coupled receptor that converts light absorption into the activation of a G protein, transducin, to initiate the visual response. We have crystallised detergent-solubilised bovine rhodopsin in the native form and after chemical modifications as needles 10-40 microm in cross-section. The crystals belong to the trigonal space group P3(1), with two molecules of rhodopsin per asymmetric unit, related by a non-crystallographic 2-fold axis parallel with the crystallographic screw axis along c (needle axis). The unit cell dimensions are a=103.8 A, c=76.6 A for native rhodopsin, but vary over a wide range after heavy atom derivatisation, with a between 101.5 A and 113.9 A, and c between 76.6 A and 79.2 A. Rhodopsin molecules are packed with the bundle of transmembrane helices tilted from the c-axis by about 100 degrees . The two molecules in the asymmetric unit form contacts along the entire length of their transmembrane helices 5 in an antiparallel orientation, and they are stacked along the needle axis according to the 3-fold screw symmetry. Hence hydrophobic contacts are prominent at protein interfaces both along and normal to the needle axis. The best crystals of native rhodopsin in this crystal form diffracted X-rays from a microfocused synchrotron source to 2.55 A maximum resolution. We describe steps taken to extend the diffraction limit from about 10 A to 2.6 A.

The three-dimensional structure of bovine rhodopsin determined by electron cryomicroscopy.

G-protein-coupled receptors are integral membrane proteins that respond to environmental signals and initiate signal transduction pathways, which activate cellular processes. Rhodopsin, a well known member of the G-protein-coupled receptor family, is located in the disk membranes of the rod outer segment, where it is responsible for the visualization of dim light. Rhodopsin is the most extensively studied G-protein-coupled receptor, and knowledge about its structure serves as a template for other related receptors. We have gained detailed structural knowledge from the crystal structure (1), which was solved by x-ray crystallography in 2000 using three-dimensional crystals. Here we report a three-dimensional density map of bovine rhodopsin determined by electron cryomicroscopy of two-dimensional crystals with p22(1)2(1) symmetry. The usage of relatively small and disordered crystals made the process of structure determination challenging. Special attention was paid to the extraction of amplitudes and phases, since usable raw data were limited to a maximum tilt of 45 degrees. In the refinement process, an improved unbending procedure was applied. This led to a final resolution of 5.5 A in the membrane plane and approximately 13 A perpendicular to it, making our electron density map the most accurate map of a G-protein-coupled receptor currently available by electron microscopy. Most important is the information we gain about the center of the membrane plane and the orientation of the molecule relative to the bilayer. This information cannot be retrieved from the three-dimensional crystals. In our electron density map, all seven transmembrane helices were identified, and their arrangement is in agreement with the arrangement known from the crystal structure (1). In the retinal binding pocket, a density peak adjacent to helix 3 suggests the position of the beta-ionine ring of the chromophore, and in its vicinity several of the bigger amino acids can be identified.