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2.  A new topology of the HK97-like fold revealed in Bordetella bacteriophage by cryoEM at 3.5 Å resolution 
eLife  2013;2:e01299.
Bacteriophage BPP-1 infects and kills Bordetella species that cause whooping cough. Its diversity-generating retroelement (DGR) provides a naturally occurring phage-display system, but engineering efforts are hampered without atomic structures. Here, we report a cryo electron microscopy structure of the BPP-1 head at 3.5 Å resolution. Our atomic model shows two of the three protein folds representing major viral lineages: jellyroll for its cement protein (CP) and HK97-like (‘Johnson’) for its major capsid protein (MCP). Strikingly, the fold topology of MCP is permuted non-circularly from the Johnson fold topology previously seen in viral and cellular proteins. We illustrate that the new topology is likely the only feasible alternative of the old topology. β-sheet augmentation and electrostatic interactions contribute to the formation of non-covalent chainmail in BPP-1, unlike covalent inter-protein linkages of the HK97 chainmail. Despite these complex interactions, the termini of both CP and MCP are ideally positioned for DGR-based phage-display engineering.
eLife digest
Whooping cough is a respiratory illness caused by bacteria in the Bordetella genus. Among the general public, Bordetella species have become a hot topic in recent years due to the re-emergence of whooping cough in the United States and elsewhere. Scientists, meanwhile have become interested in a virus called BPP-1 that can kill the Bordetella species.
BPP-1 is a double-stranded DNA virus, and such viruses have long been of interest to scientists because they are the most abundant organisms on Earth. These viruses are also noteworthy because their shells (also known as capsids) are capable of withstanding the very high pressures (up to about 40 atmospheres) that are created by packing so much DNA into the very small volume inside the capsid.
BPP-1 is of particular interest because it is capable of making large-scale changes to its own DNA in order to adapt to changes in its hosts and environment. Of all the organism that do not contain nuclei within their cells (collectively known as prokaryotes), BPP-1 is the only one that is capable of making such changes to its DNA. However, efforts to exploit the properties of BPP-1 for bioengineering applications have been hampered because its detailed structure is not known. Now Zhang et al. have used cryo electron microscopy to study the structure of BPP-1 at the atomic level.
Most viruses belong to one of three major lineages, with each lineage having a characteristic fold in its capsid proteins. Zhang et al. found that BPP-1 contains two of these folds, which suggests that it is a hybrid of two of these lineages. This is the first time that such a structure has been observed. Moreover, Zhang et al. found that one of the folds has an unusual topology that has not been seen before. The atomic structure reveals how double-stranded DNA viruses use a variety of non-covalent interactions and a type of protein ‘chainmail’ to form a highly stable capsid that is capable of withstanding very high pressures.
In addition to enabling applications in bioengineering, the new structure might also provide insights into the evolution of prokaryotes.
PMCID: PMC3863775  PMID: 24347545
CryoEM; atomic resolution; non-covalent chainmail; phage-display; fold topology; Viruses
3.  The architecture of Tetrahymena telomerase holoenzyme 
Nature  2013;496(7444):10.1038/nature12062.
Telomerase adds telomeric repeats to chromosome ends using an internal RNA template and specialized telomerase reverse transcriptase (TERT), thereby maintaining genome integrity. Little is known about the physical relationships among protein and RNA subunits within a biologically functional holoenzyme. Here we describe the architecture of Tetrahymena thermophila telomerase holoenzyme determined by electron microscopy. Six of the 7 proteins and the TERT-binding regions of telomerase RNA (TER) have been localized by affinity labeling. Fitting with high-resolution structures reveals the organization of TERT, TER, and p65 in the RNP catalytic core. p50 has an unanticipated role as a hub between the RNP catalytic core, p75-p19-p45 subcomplex, and the DNA-binding Teb1. A complete in vitro holoenzyme reconstitution assigns function to these interactions in processive telomeric repeat synthesis. These studies provide the first view of the extensive network of subunit associations necessary for telomerase holoenzyme assembly and physiological function.
PMCID: PMC3817743  PMID: 23552895
4.  Limiting factors in atomic resolution cryo electron microscopy: No simple tricks 
Journal of structural biology  2011;175(3):253-263.
To bring cryo electron microscopy (cryoEM) of large biological complexes to atomic resolution, several factors – in both cryoEM image acquisition and 3D reconstruction – that may be neglected at low resolution become significantly limiting. Here we present thorough analyses of four limiting factors: (a) electron-beam tilt, (b) inaccurate determination of defocus values, (c) focus gradient through particles, and (d) particularly for large particles, dynamic (multiple) scattering of electrons. We also propose strategies to cope with these factors: (a) the divergence and direction tilt components of electron-beam tilt could be reduced by maintaining parallel illumination and by using a coma-free alignment procedure, respectively. Moreover, the effect of all beam tilt components, including spiral tilt, could be eliminated by use of a spherical aberration corrector. (b) More accurate measurement of defocus value could be obtained by imaging areas adjacent to the target area at high electron dose and by measuring the image shift induced by tilting the electron beam. (c) Each known Fourier coefficient in the Fourier transform of a cryoEM image is the sum of two Fourier coefficients of the 3D structure, one on each of two curved ‘characteristic surfaces’ in 3D Fourier space. We describe a simple model-based iterative method that could recover these two Fourier coefficients on the two characteristic surfaces. (d) The effect of dynamic scattering could be corrected by deconvolution of a transfer function. These analyses and our proposed strategies offer useful guidance for future experimental designs targeting atomic resolution cryoEM reconstruction.
PMCID: PMC3710782  PMID: 21627992
cryoEM; Atomic resolution; Beam tilt; Dynamic scattering; Defocus gradient
5.  CryoEM structure of the mature dengue virus at 3.5-Å resolution 
Regulated by pH, membrane-anchored proteins E and M play a series of roles during dengue virus maturation and membrane fusion. Our atomic model of the whole virion from cryo electron microscopy at 3.5Å resolution reveals that in the mature virus at neutral extracellular pH, the N-terminal 20-amino acid segment of M (involving three pH-sensing histidines) latches and thereby prevents spring-loaded E fusion protein from prematurely exposing its fusion peptide. This M latch was fastened at an earlier stage, during maturation at acid pH in the trans-Golgi network. At a later stage, to initiate infection in response to acid pH in the late endosome, M releases the latch and exposes the fusion peptide. Thus, M serves as a multistep chaperone of E to control the conformational changes accompanying maturation and infection. These pH-sensitive interactions could serve as targets for drug discovery.
PMCID: PMC3593067  PMID: 23241927
cryo electron microscopy; flavivirus; bio-threat agent; enveloped viruses; chaperone; viral maturation
6.  IRE1 Phosphatase PP2Ce Regulates Adaptive ER Stress Response in the Postpartum Mammary Gland 
PLoS ONE  2014;9(11):e111606.
We recently reported that the PPM1l gene encodes an endoplasmic reticulum (ER) membrane targeted protein phosphatase (named PP2Ce) with highly specific activity towards Inositol-requiring protein-1 (IRE1) and regulates the functional outcome of ER stress. In the present report, we found that the PP2Ce protein is highly expressed in lactating epithelium of the mammary gland. Loss of PP2Ce in vivo impairs physiological unfolded protein response (UPR) and induces stress kinase activation, resulting in loss of milk production and induction of epithelial apoptosis in the lactating mammary gland. This study provides the first in vivo evidence that PP2Ce is an essential regulator of normal lactation, possibly involving IRE1 signaling and ER stress regulation in mammary epithelium.
PMCID: PMC4219728  PMID: 25369058
7.  Association of Herpes Simplex Virus pUL31 with Capsid Vertices and Components of the Capsid Vertex-Specific Complex 
Journal of Virology  2014;88(7):3815-3825.
pUL34 and pUL31 of herpes simplex virus (HSV) comprise the nuclear egress complex (NEC) and are required for budding at the inner nuclear membrane. pUL31 also associates with capsids, suggesting it bridges the capsid and pUL34 in the nuclear membrane to initiate budding. Previous studies showed that capsid association of pUL31 was precluded in the absence of the C terminus of pUL25, which along with pUL17 comprises the capsid vertex-specific complex, or CVSC. The present studies show that the final 20 amino acids of pUL25 are required for pUL31 capsid association. Unexpectedly, in the complete absence of pUL25, or when pUL25 capsid binding was precluded by deletion of its first 50 amino acids, pUL31 still associated with capsids. Under these conditions, pUL31 was shown to coimmunoprecipitate weakly with pUL17. Based on these data, we hypothesize that the final 20 amino acids of pUL25 are required for pUL31 to associate with capsids. In the absence of pUL25 from the capsid, regions of capsid-associated pUL17 are bound by pUL31. Immunogold electron microscopy revealed that pUL31 could associate with multiple sites on a single capsid in the nucleus of infected cells. Electron tomography revealed that immunogold particles specific to pUL31 protein bind to densities at the vertices of the capsid, a location consistent with that of the CVSC. These data suggest that pUL31 loads onto CVSCs in the nucleus to eventually bind pUL34 located within the nuclear membrane to initiate capsid budding.
IMPORTANCE This study is important because it localizes pUL31, a component previously known to be required for HSV capsids to bud through the inner nuclear membrane, to the vertex-specific complex of HSV capsids, which comprises the unique long region 25 (UL25) and UL17 gene products. It also shows this interaction is dependent on the C terminus of UL25. This information is vital for understanding how capsids bud through the inner nuclear membrane.
PMCID: PMC3993549  PMID: 24453362
8.  Vaults Engineered for Hydrophobic Drug Delivery 
The vault nanoparticle is one of the largest known ribonucleoprotein complexes in the sub-100 nm range. Highly conserved and almost ubiquitously expressed in eukaryotes, vaults form a large nanocapsule with a barrel-shaped morphology surrounding a large hollow interior. These properties make vaults an ideal candidate for development into a drug delivery vehicle. In this study, we report the first example of using vaults towards this goal. We engineered recombinant vaults to encapsulate the highly insoluble and toxic hydrophobic compound All-trans Retinoic Acid (ATRA) using a vault binding lipoprotein complex that forms a lipid bilayer nanodisk. These recombinant vaults offer protection to the encapsulated ATRA from external elements. Furthermore, a cryo-electron tomography (cryo-ET) reconstruction shows the vault binding lipoprotein complex sequestered within the vault lumen. Finally, these ATRA loaded vaults have enhanced cytotoxicity against the hepatocellular carcinoma cell line HepG2. The ability to package therapeutic compounds into the vault is an important achievement toward their development into a viable and versatile platform for drug delivery.
PMCID: PMC4182016  PMID: 21506266
Vaults; Nanoparticles; Nanodisk; All-trans Retinoic Acid; Drug Delivery Systems
9.  Low Cost, High Performance GPU Computing Solution for Atomic Resolution CryoEM Single-Particle Reconstruction 
Journal of Structural Biology  2010;172(3):400-406.
Recent advancements in cryo-electron microscopy (cryoEM) have made it technically possible to determine the three-dimensional (3D) structures of macromolecular complexes at atomic resolution. However, processing the large amount of data needed for atomic resolution reconstructions requires either accessing to very expensive computer clusters or waiting for weeks of continuous computation in a personal computer (PC). In this paper, we present a practical computational solution to this 3D reconstruction problem through the optimal utilization of the processing capabilities of both commodity graphics hardware [i.e., general purpose graphics processing unit (GPGPU)]. Our solution, which is implemented in a new program, called eLite3D, has a number of advanced features of general interests. We construct interleaved schemes to prevent the data race condition intrinsic in merging of 2D data into a 3D volume. The speedup of eLite3D is up to 100 times over other commonly used 3D reconstruction programs with the same accuracy, thus allowing completion of atomic resolution 3D reconstructions of large complexes in a PC in 1–2 hours other than days or weeks. Our result provides a practical solution to atomic resolution cryoEM (asymmetric or symmetric) reconstruction and offers useful guidelines for developing GPGPU applications in general.
PMCID: PMC3382114  PMID: 20493949
atomic resolution; cryoEM; 3D reconstruction; GPGPU; parallel processing; CUDA
11.  Assembly and Architecture of the EBV B Cell Entry Triggering Complex 
PLoS Pathogens  2014;10(8):e1004309.
Epstein-Barr Virus (EBV) is an enveloped double-stranded DNA virus of the gammaherpesvirinae sub-family that predominantly infects humans through epithelial cells and B cells. Three EBV glycoproteins, gH, gL and gp42, form a complex that targets EBV infection of B cells. Human leukocyte antigen (HLA) class II molecules expressed on B cells serve as the receptor for gp42, triggering membrane fusion and virus entry. The mechanistic role of gHgL in herpesvirus entry has been largely unresolved, but it is thought to regulate the activation of the virally-encoded gB protein, which acts as the primary fusogen. Here we study the assembly and function of the reconstituted B cell entry complex comprised of gHgL, gp42 and HLA class II. The structure from negative-stain electron microscopy provides a detailed snapshot of an intermediate state in EBV entry and highlights the potential for the triggering complex to bring the two membrane bilayers into proximity. Furthermore, gHgL interacts with a previously identified, functionally important hydrophobic pocket on gp42, defining the overall architecture of the complex and playing a critical role in membrane fusion activation. We propose a macroscopic model of the initiating events in EBV B cell fusion centered on the formation of the triggering complex in the context of both viral and host membranes. This model suggests how the triggering complex may bridge the two membrane bilayers, orienting critical regions of the N- and C- terminal ends of gHgL to promote the activation of gB and efficient membrane fusion.
Author Summary
The various steps by which lipid-enveloped viruses enter or ‘fuse’ with a host cell require the coordination of receptor recognition, viral protein activation and large protein conformational changes that can drive membrane bilayer fusion. Here we report biochemical, structural and functional experiments on the protein complex that triggers activation of the EBV fusion protein (gB) and entry of Epstein-Barr Virus (EBV) into B cells of the immune system. Three viral glycoproteins (gH, gL and gp42) form a well-defined complex with host receptor (HLA). We isolated the complex biochemically and studied its assembly by BLI biosensor and electron microscopy methods. Previous crystal structures revealed a hydrophobic pocket (HP) on the gp42 surface that when mutated disrupts fusion with B cells, but the critical binding ligand remained unknown. Our experiments show that the gp42 HP interacts with gHgL and that mutations of the predicted HP contact residues on gHgL are detrimental for fusion. Constraints imposed by the triggering complex architecture relative to its predicted membrane anchors highlight a close approach and potential deformation of both viral and host membranes affected by HLA receptor binding as a prerequisite to viral entry.
PMCID: PMC4140853  PMID: 25144748
12.  CryoEM Model of the Bullet-Shaped Vesicular Stomatitis Virus 
Science (New York, N.Y.)  2010;327(5966):689-693.
Vesicular stomatitis virus (VSV) is a bullet-shaped rhabdovirus and a model system of negative-strand RNA viruses. Based on direct visualization by cryo-electron microscopy, we show that each virion contains two nested, left-handed helices, an outer helix of matrix protein M and an inner helix of nucleoprotein N and RNA. M has a hub domain with four contact sites that link to neighboring M and N subunits, providing rigidity by clamping adjacent turns of the nucleocapsid. Side-by-side interactions between neighboring N subunits are critical for the nucleocapsid to form a bullet shape, and structure-based mutagenesis results support this description. Together, our data suggest a mechanism of VSV assembly in which the nucleocapsid spirals from the tip to become the helical trunk, both subsequently framed and rigidified by the M layer.
PMCID: PMC2892700  PMID: 20133572
13.  3.88 Å structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy 
Nature  2008;453(7193):415-419.
Cytoplasmic polyhedrosis virus (CPV) is unique within the Reoviridae family in having a turreted single-layer capsid contained within polyhedrin inclusion bodies, yet being fully capable of cell entry and endogenous RNA transcription1–4. Biochemical data have shown that the amino-terminal 79 residues of the CPV turret protein (TP) is sufficient to bring CPV or engineered proteins into the polyhedrin matrix for micro-encapsulation5,6. Here we report the three-dimensional structure of CPV at 3.88Å resolution using single-particle cryo-electron microscopy. Our map clearly shows the turns and deep grooves of α-helices, the strand separation in β-sheets, and densities for loops and many bulky side chains; thus permitting atomic model-building effort from cryoelectron microscopy maps. We observed a helix-to-β-hairpin conformational change between the two conformational states of the capsid shell protein in the region directly interacting with genomic RNA. We have also discovered a messenger RNA release hole coupled with the mRNA capping machinery unique to CPV. Furthermore, we have identified the polyhedrin-binding domain, a structure that has potential in nanobiotechnology applications.
PMCID: PMC2746981  PMID: 18449192
14.  Towards atomic resolution structural determination by single-particle cryo-electron microscopy 
Recent advances in cryo-electron microscopy and single-particle reconstruction (collectively referred to as “cryoEM”) have made it possible to determine the three-dimensional (3D) structures of several macromolecular complexes at near-atomic resolution (~3.8 – 4.5 Å). These achievements were accomplished by overcoming challenges in sample handling, instrumentation, image processing, and model building. At near-atomic resolution, many detailed structural features can be resolved, such as the turns and deep grooves of helices, strand separation in β sheets, and densities for loops and bulky amino acid side chains. Such structural data of the cytoplasmic polyhedrosis virus (CPV), the Epsilon 15 bacteriophage and the GroEL complex have provided valuable constraints for atomic model building using integrative tools, thus significantly enhancing the value of the cryoEM structures. The CPV structure revealed a drastic conformational change from a helix to a β hairpin associated with RNA packaging and replication, coupling of RNA processing and release, and the long sought-after polyhedrin-binding domain. These latest advances in single-particle cryoEM provide exciting opportunities for the 3D structural determination of viruses and macromolecular complexes that are either too large or too heterogeneous to be investigated by conventional X-ray crystallography or nuclear magnetic resonance (NMR) methods.
PMCID: PMC2714865  PMID: 18403197
15.  Cryo-electron tomography of Kaposi’s sarcoma-associated herpesvirus capsids reveals dynamic scaffolding structures essential to capsid assembly and maturation 
Journal of structural biology  2007;161(3):419-427.
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a recently discovered DNA tumor virus that belongs to the γ-herpesvirus subfamily. Though numerous studies on KSHV and other herpesviruses, in general, have revealed much about their multilayered organization and capsid structure, the herpesvirus capsid assembly and maturation pathway remains poorly understood. Structural variability or irregularity of the capsid internal scaffolding core and the lack of adequate tools to study such structures have presented major hurdles to earlier investigations employing more traditional cryo-electron microscopy (cryoEM) single particle reconstruction. In this study, we used cryo-electron tomography (cryoET) to obtain three-dimensional reconstructions of individual KSHV capsids, allowing direct visualization of the capsid internal structures and systematic comparison of the scaffolding cores for the first time. We show that B-capsids are not a structurally homogenous group; rather, they represent an ensemble of “B-capsid-like” particles whose inner scaffolding is highly variable, possibly representing different intermediates existing during the KSHV capsid assembly and maturation. This information, taken together with previous observations, has allowed us to propose a detail pathway of herpesvirus capsid assembly and maturation.
PMCID: PMC2692512  PMID: 18164626
cryo-electron tomography; tumor herpesvirus; Kaposi’s sarcoma-associated herpesvirus; assembly; scaffolding; portal
16.  Tetrahymena Telomerase Holoenzyme Assembly, Activation, and Inhibition by Domains of the p50 Central Hub 
Molecular and Cellular Biology  2013;33(19):3962-3971.
The eukaryotic reverse transcriptase, telomerase, adds tandem telomeric repeats to chromosome ends to promote genome stability. The fully assembled telomerase holoenzyme contains a ribonucleoprotein (RNP) catalytic core and additional proteins that modulate the ability of the RNP catalytic core to elongate telomeres. Electron microscopy (EM) structures of Tetrahymena telomerase holoenzyme revealed a central location of the relatively uncharacterized p50 subunit. Here we have investigated the biochemical and structural basis for p50 function. We have shown that the p50-bound RNP catalytic core has a relatively low rate of tandem repeat synthesis but high processivity of repeat addition, indicative of high stability of enzyme-product interaction. The rate of tandem repeat synthesis is enhanced by p50-dependent recruitment of the holoenzyme single-stranded DNA binding subunit, Teb1. An N-terminal p50 domain is sufficient to stimulate tandem repeat synthesis and bridge the RNP catalytic core, Teb1, and the p75 subunit of the holoenzyme subcomplex p75/p19/p45. In cells, the N-terminal p50 domain assembles a complete holoenzyme that is functional for telomere maintenance, albeit at shortened telomere lengths. Also, in EM structures of holoenzymes, only the N-terminal domain of p50 is visible. Our findings provide new insights about subunit and domain interactions and functions within the Tetrahymena telomerase holoenzyme.
PMCID: PMC3811867  PMID: 23918804
17.  Lateral Clustering of TLR3:dsRNA Signaling Units Revealed by TLR3ecd:3Fabs Quaternary Structure 
Journal of molecular biology  2012;421(1):112-124.
Toll-like receptor 3 (TLR3) recognizes dsRNA and initiates an innate immune response through the formation of a signaling unit (SU) composed of one double-stranded RNA (dsRNA) and two TLR3 molecules. We report the crystal structure of human TLR3 ectodomain (TLR3ecd) in a quaternary complex with three neutralizing Fab fragments. Fab15 binds an epitope that overlaps the C-terminal dsRNA binding site and, in biochemical assays, blocks the interaction of TLR3ecd with dsRNA, thus directly antagonizing TLR3 signaling through inhibition of SU formation. In contrast, Fab12 and Fab1068 bind TLR3ecd at sites distinct from the N- and C-terminal regions that interact with dsRNA and do not inhibit minimal SU formation with short dsRNA. Molecular modeling based on the co-structure rationalizes these observations by showing that both Fab12 and Fab1068 prevent lateral clustering of SUs along the length of the dsRNA ligand. This model is further supported by cell-based assay results using dsRNA ligands of lengths that support single and multiple SUs. Thus, their antagonism of TLR3 signaling indicates that lateral clustering of SUs is required for TLR3 signal transduction.
PMCID: PMC3920545  PMID: 22579623
Toll-like receptor 3; TLR3; innate immunity; lateral TLR3 clustering; quaternary complex
18.  The Smallest Capsid Protein Mediates Binding of the Essential Tegument Protein pp150 to Stabilize DNA-Containing Capsids in Human Cytomegalovirus 
PLoS Pathogens  2013;9(8):e1003525.
Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes birth defects in newborns and life-threatening complications in immunocompromised individuals. Among all human herpesviruses, HCMV contains a much larger dsDNA genome within a similarly-sized capsid compared to the others, and it was proposed to require pp150, a tegument protein only found in cytomegaloviruses, to stabilize its genome-containing capsid. However, little is known about how pp150 interacts with the underlying capsid. Moreover, the smallest capsid protein (SCP), while dispensable in herpes simplex virus type 1, was shown to play essential, yet undefined, role in HCMV infection. Here, by cryo electron microscopy (cryoEM), we determine three-dimensional structures of HCMV capsid (no pp150) and virion (with pp150) at sub-nanometer resolution. Comparison of these two structures reveals that each pp150 tegument density is composed of two helix bundles connected by a long central helix. Correlation between the resolved helices and sequence-based secondary structure prediction maps the tegument density to the N-terminal half of pp150. The structures also show that SCP mediates interactions between the capsid and pp150 at the upper helix bundle of pp150. Consistent with this structural observation, ribozyme inhibition of SCP expression in HCMV-infected cells impairs the formation of DNA-containing viral particles and reduces viral yield by 10,000 fold. By cryoEM reconstruction of the resulting “SCP-deficient” viral particles, we further demonstrate that SCP is required for pp150 functionally binding to the capsid. Together, our structural and biochemical results point to a mechanism whereby SCP recruits pp150 to stabilize genome-containing capsid for the production of infectious HCMV virion.
Author Summary
Human cytomegalovirus (HCMV) causes birth defects in newborns and life-threatening complications in immunocompromised individuals, such as AIDS patients and organ transplant recipients. The smallest capsid protein (SCP) – only 8 kDa molecular mass as compared to the 155 kDa major capsid protein – has been demonstrated to be essential for HCMV growth, but is dispensable in herpes simplex virus type 1. These seemingly contradictory observations have been a paradox. Here, we solve this paradox by high resolution cryo electron microscopy (cryoEM), in conjunction with functional studies using ribozyme inhibition. Our structural comparisons of HCMV virion and capsid reveal molecular interactions at the secondary structure level and suggest that SCP might contribute to capsid binding of pp150, an essential, cytomegalovirus-specific tegument protein. SCP-deficient particles generated by ribozyme inhibition of SCP-expression in HCMV-infected cells show no pp150 tegument density, demonstrating that SCP is required for the functional binding of pp150 to the capsid. Our results suggest that SCP recruits pp150 to stabilize the HCMV nucleocapsid to enable encapsidation of the genome, which is more densely packaged in HCMV than in other herpesviruses. Overall, this study not only resolves the above paradox, but also illustrates the passive acquisition of a new, essential function by SCP in the production of infectious HCMV virions.
PMCID: PMC3744435  PMID: 23966856
19.  Seeing engineered loops in a gene delivery vehicle by cryoEM 
Structure (London, England : 1993)  2012;20(8):1286-1288.
In this issue of Structure, Lerch and colleagues present a 4.5 Å cryo electron microscopy (cryoEM) structure of a variant of an adeno-associated virus that has been genetically engineered for liver gene therapy. The identification of two structurally distinct loops flanking the highly conserved jellyroll β barrel highlights the potentials of high-resolution cryoEM.
PMCID: PMC3422559  PMID: 22884103
20.  PPM1l encodes an inositol requiring-protein 1 (IRE1) specific phosphatase that regulates the functional outcome of the ER stress response★ 
Molecular Metabolism  2013;2(4):405-416.
The protein phosphatase 1-like gene (PPM1l) was identified as causal gene for obesity and metabolic abnormalities in mice. However, the underlying mechanisms were unknown. In this report, we find PPM1l encodes an endoplasmic reticulum (ER) membrane targeted protein phosphatase (PP2Ce) and has specific activity to basal and ER stress induced auto-phosphorylation of Inositol-REquiring protein-1 (IRE1). PP2Ce inactivation resulted in elevated IRE1 phosphorylation and higher expression of XBP-1, CHOP, and BiP at basal. However, ER stress stimulated XBP-1 and BiP induction was blunted while CHOP induction was further enhanced in PP2Ce null cells. PP2Ce protein levels are significantly induced during adipogenesis in vitro and are necessary for normal adipocyte maturation. Finally, we provide evidence that common genetic variation of PPM11 gene is significantly associated with human lipid profile. Therefore, PPM1l mediated IRE1 regulation and downstream ER stress signaling is a plausible molecular basis for its role in metabolic regulation and disorder.
Graphical abstract
PMCID: PMC3854994  PMID: 24327956
IRE1; PPM1l; Protein phosphatase; ER stress; Adipogenesis
21.  Correcting for the Ewald Sphere in High-Resolution Single-Particle Reconstructions 
Methods in enzymology  2010;482:369-380.
To avoid the challenges of crystallization and the size limitations of NMR, it has long been hoped that single-particle cryo-electron microscopy (cryo-EM) would eventually yield atomically interpretable reconstructions. For the most favorable class of specimens (large icosahedral viruses), one of the key obstacles is curvature of the Ewald sphere, which leads to a breakdown of the Projection Theorem used by conventional three-dimensional (3D) reconstruction programs. Here, we review the basic problem and our implementation of the “paraboloid” reconstruction method, which overcomes the limitation by averaging information from images recorded from different points of view.
PMCID: PMC3716286  PMID: 20888969
22.  Structural, mass and elemental analyses of storage granules in methanogenic archaeal cells 
Environmental microbiology  2011;13(9):2587-2599.
Storage granules are an important component of metabolism in many organisms spanning the bacterial, eukaryal and archaeal domains, but systematic analysis of their organization inside cells is lacking. In this study, we identify and characterize granulelike inclusion bodies in a methanogenic archaeon, Methanospirillum hungatei, an anaerobic microorganism that plays an important role in nutrient recycling in the ecosystem. Using cryo electron microscopy, we show that granules in mature M. hungatei are amorphous in structure with a uniform size. Energy dispersive X-ray spectroscopy analysis establishes that each granule is a polyphosphate body (PPB) that consists of high concentrations of phosphorous and oxygen, and increased levels of iron and magnesium. By scanning transmission electron tomography, we further estimate that the mass density within a PPB is a little less than metal titanium at room temperature and is about four times higher than that of the surrounding cytoplasm. Finally, three-dimensional cryo electron tomography reveals that PPBs are positioned off-centre in their radial locations relative to the cylindrical axis of the cell, and almost uniformly placed near cell ends. This positioning ability points to a genetic program that spatially and temporally directs the accumulation of polyphosphate into a storage granule, perhaps for energy-consuming activities, such as cell maintenance, division or motility.
PMCID: PMC3700383  PMID: 21854518
Single-particle cryo electron microscopy (cryoEM) is a technique for determining three-dimensional (3D) structures from projection images of molecular complexes preserved in their “native,” noncrystalline state. Recently, atomic or near-atomic resolution structures of several viruses and protein assemblies have been determined by single-particle cryoEM, allowing ab initio atomic model building by following the amino acid side chains or nucleic acid bases identifiable in their cryoEM density maps. In particular, these cryoEM structures have revealed extended arms contributing to molecular interactions that are otherwise not resolved by the conventional structural method of X-ray crystallography at similar resolutions. High-resolution cryoEM requires careful consideration of a number of factors, including proper sample preparation to ensure structural homogeneity, optimal configuration of electron imaging conditions to record high-resolution cryoEM images, accurate determination of image parameters to correct image distortions, efficient refinement and computation to reconstruct a 3D density map, and finally appropriate choice of modeling tools to construct atomic models for functional interpretation. This progress illustrates the power of cryoEM and ushers it into the arsenal of structural biology, alongside conventional techniques of X-ray crystallography and NMR, as a major tool (and sometimes the preferred one) for the studies of molecular interactions in supramolecular assemblies or machines.
PMCID: PMC3698602  PMID: 21501817
24.  Biochemical and Structural Characterization of the Capsid-Bound Tegument Proteins of Human Cytomegalovirus 
Journal of structural biology  2011;174(3):451-460.
Human cytomegalovirus (HCMV) is the most genetically and structurally complex human herpesvirus and is composed of an envelope, a tegument, and a dsDNA-containing capsid. HCMV tegument plays essential roles in HCMV infection and assembly. Using cryo-electron tomography (cryoET), here we show that HCMV tegument compartment can be divided into two sub-compartments: an inner and an outer tegument. The inner tegument consists of densely-packed proteins surrounding the capsid. The outer tegument contains those components that are loosely packed in the space between the inner tegument and the pleomorphic glycoprotein-containing envelope. To systematically characterize the inner tegument proteins interacting with the capsid, we used chemical treatment to strip off the entire envelope and most tegument proteins to obtain a tegumented capsid with inner tegument proteins. SDS-polyacrylamide gel electrophoresis analyses show that only two tegument proteins, UL32-encoded pp150 and UL48-encoded high molecular weight protein (HMWP), remains unchanged in their abundance in the tegumented capsids as compared to their abundance in the intact particles. 3D reconstructions by single particle cryo-electron microscopy (cryoEM) reveal that the net-like layer of icosahedrally-ordered tegument densities are also the same in the tegumented capsid and in the intact particles. CryoET reconstruction of the tegumented capsid labeled with an anti-pp150 antibody is consistent with the biochemical and cryoEM data in localizing pp150 within the ordered tegument. Taken together, these results suggest that pp150, a betaherpesvirus-specific tegument protein, is a constituent of the net-like layer of icosahedrally-ordered capsid-bound tegument densities, a structure lacking similarities in alpha and gammaherpesviruses.
PMCID: PMC3684277  PMID: 21459145
human cytomegalovirus; tegument proteins; single-particle analysis; cryo-electron microscopy; cryo-electron tomography; chemical treatment; pp150
25.  Single Particle Electron Microscopy Analysis of the Bovine Anion Exchanger 1 Reveals a Flexible Linker Connecting the Cytoplasmic and Membrane Domains 
PLoS ONE  2013;8(2):e55408.
Anion exchanger 1 (AE1) is the major erythrocyte membrane protein that mediates chloride/bicarbonate exchange across the erythrocyte membrane facilitating CO2 transport by the blood, and anchors the plasma membrane to the spectrin-based cytoskeleton. This multi-protein cytoskeletal complex plays an important role in erythrocyte elasticity and membrane stability. An in-frame AE1 deletion of nine amino acids in the cytoplasmic domain in a proximity to the membrane domain results in a marked increase in membrane rigidity and ovalocytic red cells in the disease Southeast Asian Ovalocytosis (SAO). We hypothesized that AE1 has a flexible region connecting the cytoplasmic and membrane domains, which is partially deleted in SAO, thus causing the loss of erythrocyte elasticity. To explore this hypothesis, we developed a new non-denaturing method of AE1 purification from bovine erythrocyte membranes. A three-dimensional (3D) structure of bovine AE1 at 2.4 nm resolution was obtained by negative staining electron microscopy, orthogonal tilt reconstruction and single particle analysis. The cytoplasmic and membrane domains are connected by two parallel linkers. Image classification demonstrated substantial flexibility in the linker region. We propose a mechanism whereby flexibility of the linker region plays a critical role in regulating red cell elasticity.
PMCID: PMC3564912  PMID: 23393575

Results 1-25 (55)