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Wah Chiu received his BA in Physics (1969) and PhD in Biophysics (1975) from the University of California, Berkeley. He is a professor in the Department of Bioengineering, Department of Microbiology and Immunology and the SLAC National Accelerator Laboratory at Stanford University. He is a pioneer in methodology development for electron cryo-microscopy. His work has made multiple transformational contributions in developing single particle electron cryo-microscopy as a tool for the structural determination of molecular machines towards atomic resolution.
For three decades, Dr. Chiu directs a NIH funded 3DEM Resource Center. He has solved many cryo-EM structures including viruses, chaperonins, membrane proteins, ion channels, cytoskeleton protein complexes, protein-DNA complexes, DNA and RNA in collaboration with many scientists around the world. His 3DEM Resource Center continues to establish high standard testing and characterization protocols for cryoEM instrumentation and to develop new image processing and modeling algorithms for cryo-EM structure determination.
Dr. Chiu's research, collaboration and training efforts have been recognized by his elected membership to the Academia Sinica, Taiwan (2008), and the United States National Academy of Sciences (2012), in addition to several honors including the Distinguished Science Award from the Microscopy Society of America (2014) and the Honorary Doctorate of Philosophy from the University of Helsinki, Finland (2014).
Academic Appointments
Professor, Photon Science Directorate
Professor, Bioengineering
Professor, Microbiology & Immunology
Member, Bio-X
Faculty Fellow, Stanford ChEM-H
Member, Wu Tsai Neurosciences Institute
Honors & Awards
Elected Member, United States National Academy of Sciences (2012)
Elected Academician, Academia Sinica, Taiwan (2008)
Distinguished Scientist Award for the Biological Sciences, Microscopy Society of America (2014)
Honorary Doctorate of Philosophy, University of Helsinki, Finland (2014)
Barbara and Corbin J. Robertson Jr. Presidential Award for Excellence in Education, Baylor College of Medicine (2015)
Elected Member, The Academy of Medicine, Engineering, and Science of Texas (2013)
Distinguished Faculty Award, Baylor College of Medicine Alumni Association (2013)
Distinguished Service Professorship, Baylor College of Medicine (2010)
Achievement Award, Society of Chinese Bioscientists in America Houston Chapter (2011)
Presidential Award, American Academy of Nanomedicine (2006)
Research Fellow, Japan Society for the Promotion of Science (1999)
Alexander von Humboldt Research Prize, Alexander von Humboldt Foundation (1996)
Guggenheim Fellow, Guggenheim Foundation (1986)
Presidential Scholar, Electron Microscopy Society of America (1974)
Award of Merit, Oakland City Council (1972)
Boards, Advisory Committees, Professional Organizations
Member, Scientific Advisory Board, Biozentrum, Universität Basel, Basel, Switzerland (2016 - Present)
Member, Scientific Advisory Board, BioXFEL Center, University of Buffalo (2014 - Present)
Member, Scientific Advisory Board, Institute of Biological Chemistry Institute, Academia Sinica, Taiwan (2011 - Present)
Member, Advisory Committee, World-wide Protein Data Bank (wwPDB) (2010 - Present)
Member, Scientific Advisory Board, RCSB Protein Data Bank (2005 - Present)
Member, Panel for SystemsX, Research Council of the Swiss National Science Foundation (2007 - 2016)
Member, Scientific Advisory Board, Division of Structural Biology, St. Jude Children's Research Hospital (2015 - 2016)
Member, Scientific Advisory Board, Max Planck Institute of Biochemistry in Martinsried, Germany (2013 - 2017)
Chair, Expert Panel for Biomedical Engineering & Life Sciences Cluster, Singapore Ministry of Education (2012 - 2015)
Member, Scientific Advisory Board, Michael E. DeBakey VA Medical Center, Houston (2012 - 2017)
Professional Education
Ph.D., University of California, Berkeley, Biophysics (1975)
B.A., University of California, Berkeley, Physics (1969)
Additional Info
Current Research and Scholarly Interests
My research includes methodology improvements in single particle cryo-EM for atomic resolution structure determination of molecules and molecular machines, as well as in cryo-ET of cells and organelles towards subnanometer resolutions. We collaborate with many researchers around the country and outside the USA on understanding biological processes such as protein folding, virus assembly and disassembly, pathogen-host interactions, signal transduction, and transport across cytosol and membranes.
Cryo-EM of Molecular Machines, Stanford University (1/1/2018 - Present)
Use cryo-EM to determine atomic structures of channels, pumps, transporters, chaperonins, protein degradation machines, and viruses in different functional states in conjunction with biochemical and physiological characterizations.
Stanford, CA
Unified Data Resource for 3DEM, Stanford University (1/1/2018)
Unified Data Resource for 3DEM
Stanford, CA
- Helen Berman, Professor, Rutgers University
From structure to therapy for Huntington's disease, Stanford University (1/1/2018)
Stanford, CA
Pathogen-Host interactions studied by cryo-EM and cryo-ET, Stanford University (1/1/2018 - Present)
Our interests span across DNA and RNA viruses infecting from bacteria, archaea and mammals.
Stanford, CA
DNA and RAN Origami Characterization, Stanford University (1/1/2018)
Stanford, CA
Atomic resolution single particle cryoEM, Stanford University
Stanford, CA
2019-20 Courses
Independent Studies (4)
- Directed Investigation
BIOE 392 (Aut, Win, Spr, Sum)
- Directed Study
BIOE 391 (Aut, Win, Spr, Sum)
- Graduate Research
BIOPHYS 300 (Aut, Win, Spr, Sum)
- Graduate Research
MI 399 (Win, Spr, Sum)
- Directed Investigation
Stanford Advisees
Postdoctoral Faculty Sponsor
Chih-Ta Chien, Shanshan Li, Boxue Ma, Guan Chin Su, Rahel Woldeyes, Gong Her Wu
Doctoral Dissertation Advisor (AC)
David Chmielewski, Yanyan Zhao, Weijiang Zhou
Postdoctoral Research Mentor
Guan Chin Su, Rahel Woldeyes
Doctoral (Program)
Grace Zhong
Graduate and Fellowship Programs
All Publications
Cryo-EM structures of Helicobacter pylori vacuolating cytotoxin A oligomeric assemblies at near-atomic resolution
2019; 116 (14): 6800–6805
View details for DOI 10.1073/pnas.1821959116
View details for Web of Science ID 000463069900052
Coupling of ssRNA cleavage with DNase activity in type III-A CRISPR-Csm revealed by cryo-EM and biochemistry
2019; 29 (4): 305–12
View details for DOI 10.1038/s41422-019-0151-x
View details for Web of Science ID 000462861400007
The 3.5-A CryoEM Structure of Nanodisc-Reconstituted Yeast Vacuolar ATPase Vo Proton Channel.
Mol Cell
2018; 69 (6): 993-1004
View details for DOI 10.1016/j.molcel.2018.02.006
Accurate model annotation of a near-atomic resolution cryo-EM map
2017; 114 (12): 3103-3108
Electron cryomicroscopy (cryo-EM) has been used to determine the atomic coordinates (models) from density maps of biological assemblies. These models can be assessed by their overall fit to the experimental data and stereochemical information. However, these models do not annotate the actual density values of the atoms nor their positional uncertainty. Here, we introduce a computational procedure to derive an atomic model from a cryo-EM map with annotated metadata. The accuracy of such a model is validated by a faithful replication of the experimental cryo-EM map computed using the coordinates and associated metadata. The functional interpretation of any structural features in the model and its utilization for future studies can be made in the context of its measure of uncertainty. We applied this protocol to the 3.3-Å map of the mature P22 bacteriophage capsid, a large and complex macromolecular assembly. With this protocol, we identify and annotate previously undescribed molecular interactions between capsid subunits that are crucial to maintain stability in the absence of cementing proteins or cross-linking, as occur in other bacteriophages.
View details for DOI 10.1073/pnas.1621152114
View details for Web of Science ID 000396893600057
View details for PubMedID 28270620
The Chaperonin TRiC/CCT Associates with Prefoldin through a Conserved Electrostatic Interface Essential for Cellular Proteostasis
2019; 177 (3): 751-+
View details for DOI 10.1016/j.cell.2019.03.012
View details for Web of Science ID 000464947700026
Redox Engineering of Cytochrome c using DNA Nanostructure-Based Charged Encapsulation and Spatial Control
2019; 11 (15): 13874–80
View details for DOI 10.1021/acsami.8b07101
View details for Web of Science ID 000465189000005
Structure of Calcarisporiella thermophila Hsp104 Disaggregase that Antagonizes Diverse Proteotoxic Misfolding Events
2019; 27 (3): 449-+
View details for DOI 10.1016/j.str.2018.11.001
View details for Web of Science ID 000460258100007
Structures of TRPV2 in distinct conformations provide insight into role of the pore turret
2019; 26 (1): 40-+
Cation channels of the transient receptor potential (TRP) family serve important physiological roles by opening in response to diverse intra- and extracellular stimuli that regulate their lower or upper gates. Despite extensive studies, the mechanism coupling these gates has remained obscure. Previous structures have failed to resolve extracellular loops, known in the TRPV subfamily as 'pore turrets', which are proximal to the upper gates. We established the importance of the pore turret through activity assays and by solving structures of rat TRPV2, both with and without an intact turret at resolutions of 4.0 Å and 3.6 Å, respectively. These structures resolve the full-length pore turret and reveal fully open and partially open states of TRPV2, both with unoccupied vanilloid pockets. Our results suggest a mechanism by which physiological signals, such as lipid binding, can regulate the lower gate and couple to the upper gate through a pore-turret-facilitated mechanism.
View details for DOI 10.1038/s41594-018-0168-8
View details for Web of Science ID 000454902900006
View details for PubMedID 30598551
View details for PubMedCentralID PMC6458597
Assessment of structural features in Cryo-EM density maps using SSE and side chain Z-scores
2018; 204 (3): 564–71
We introduce a new method for assessing resolvability of structural features in density maps from Cryo-Electron Microscopy (Cryo-EM) using fitted or derived models. It calculates Z-scores for secondary structure elements (SSEs) and side chains. Z-scores capture how much larger the cross-correlation score (CCS) is for atoms in such features at their placed locations compared to the CCS at displaced positions. Z-scores are larger when the structural features are well-resolved, as confirmed by visual analysis. This method was applied to all 66 maps submitted to the 2015/2016 EMDB map challenge. For each map, the fitted model provided by the map committee was used in this assessment. The average Z-scores for each map and fitted model correlate moderately well with reported map resolutions (r2 = 0.45 for SSE Z-scores and r2 = 0.56 for side chain Z-scores). Rankings of the submitted maps based on average Z-scores seem to more closely agree with visual analysis. Z-scores can also be used to pinpoint which parts of a model are well-resolved in a map, and which parts of the model may need further fitting or refinement to make the model better match the density.
View details for DOI 10.1016/j.jsb.2018.08.015
View details for Web of Science ID 000454373000022
View details for PubMedID 30144506
View details for PubMedCentralID PMC6525962
Evaluation system and web infrastructure for the second cryo-EM model challenge
2018; 204 (1): 96–108
An evaluation system and a web infrastructure were developed for the second cryo-EM model challenge. The evaluation system includes tools to validate stereo-chemical plausibility of submitted models, check their fit to the corresponding density maps, estimate their overall and per-residue accuracy, and assess their similarity to reference cryo-EM or X-ray structures as well as other models submitted in this challenge. The web infrastructure provides a convenient interface for analyzing models at different levels of detail. It includes interactively sortable tables of evaluation scores for different subsets of models and different sublevels of structure organization, and a suite of visualization tools facilitating model analysis. The results are publicly accessible at http://model-compare.emdatabank.org.
View details for DOI 10.1016/j.jsb.2018.07.006
View details for Web of Science ID 000444661500013
View details for PubMedID 30017700
View details for PubMedCentralID PMC6205695
Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly
2018; 172 (5): 966-978
View details for DOI 10.1016/j.cell.2018.02.009
- 10.1016/j.jmb.2018.08.013 Visualizing Individual RuBisCO and Its Assembly into Carboxysomes in Marine Cyanobacteria by Cryo-Electron Tomography 2018
- 10.1016/j.chom.2018.07.018 Neutralizing Antibodies Inhibit Chikungunya Virus Budding at the Plasma Membrane 2018; 24 (3): 417-428
Structure of the 30 kDa HIV-1 RNA Dimerization Signal by a Hybrid Cryo-EM, NMR, and Molecular Dynamics Approach
Structure of the 30 kDa HIV-1 RNA Dimerization Signal by a Hybrid Cryo-EM, NMR, and Molecular Dynamics Approach
2018; 26 (3): 490-498
Cryoelectron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy are routinely used to determine structures of macromolecules with molecular weights over 65 and under 25 kDa, respectively. We combined these techniques to study a 30 kDa HIV-1 dimer initiation site RNA ([DIS]2; 47 nt/strand). A 9 Å cryo-EM map clearly shows major groove features of the double helix and a right-handed superhelical twist. Simulated cryo-EM maps generated from time-averaged molecular dynamics trajectories (10 ns) exhibited levels of detail similar to those in the experimental maps, suggesting internal structural flexibility limits the cryo-EM resolution. Simultaneous inclusion of the cryo-EM map and 2H-edited NMR-derived distance restraints during structure refinement generates a structure consistent with both datasets and supporting a flipped-out base within a conserved purine-rich bulge. Our findings demonstrate the power of combining global and local structural information from these techniques for structure determination of modest-sized RNAs.
View details for DOI 10.1016/j.str.2018.01.001
View details for PubMedCentralID PMC5842133
SuRVoS: Super-Region Volume Segmentation workbench
2017; 198 (1): 43-53
Segmentation of biological volumes is a crucial step needed to fully analyse their scientific content. Not having access to convenient tools with which to segment or annotate the data means many biological volumes remain under-utilised. Automatic segmentation of biological volumes is still a very challenging research field, and current methods usually require a large amount of manually-produced training data to deliver a high-quality segmentation. However, the complex appearance of cellular features and the high variance from one sample to another, along with the time-consuming work of manually labelling complete volumes, makes the required training data very scarce or non-existent. Thus, fully automatic approaches are often infeasible for many practical applications. With the aim of unifying the segmentation power of automatic approaches with the user expertise and ability to manually annotate biological samples, we present a new workbench named SuRVoS (Super-Region Volume Segmentation). Within this software, a volume to be segmented is first partitioned into hierarchical segmentation layers (named Super-Regions) and is then interactively segmented with the user's knowledge input in the form of training annotations. SuRVoS first learns from and then extends user inputs to the rest of the volume, while using Super-Regions for quicker and easier segmentation than when using a voxel grid. These benefits are especially noticeable on noisy, low-dose, biological datasets.
View details for DOI 10.1016/j.jsb.2017.02.007
View details for Web of Science ID 000400318400007
View details for PubMedID 28246039
View details for PubMedCentralID PMC5405849
An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump
2017; 6
Bacterial efflux pumps confer multidrug resistance by transporting diverse antibiotics from the cell. In Gram-negative bacteria, some of these pumps form multi-protein assemblies that span the cell envelope. Here, we report the near-atomic resolution cryoEM structures of the Escherichia coli AcrAB-TolC multidrug efflux pump in resting and drug transport states, revealing a quaternary structural switch that allosterically couples and synchronizes initial ligand binding with channel opening. Within the transport-activated state, the channel remains open even though the pump cycles through three distinct conformations. Collectively, our data provide a dynamic mechanism for the assembly and operation of the AcrAB-TolC pump.
View details for DOI 10.7554/eLife.24905
View details for PubMedID 28355133
Visualizing Adsorption of Cyanophage P-SSP7 onto Marine Prochlorococcus
2017; 7
Marine cyanobacteria perform roughly a quarter of global carbon fixation, and cyanophages that infect them liberate some of this carbon during infection and cell lysis. Studies of the cyanobacterium Prochlorococcus MED4 and its associated cyanophage P-SSP7 have revealed complex gene expression dynamics once infection has begun, but the initial cyanophage-host interactions remain poorly understood. Here, we used single particle cryo-electron tomography (cryo-ET) to investigate cyanophage-host interactions in this model system, based on 170 cyanophage-to-host adsorption events. Subtomogram classification and averaging revealed three main conformations characterized by different angles between the phage tail and the cell surface. Namely, phage tails were (i) parallel to, (ii) ~45 degrees to, or (iii) perpendicular to the cell surface. Furthermore, different conformations of phage tail fibers correlated with the aforementioned orientations of the tails. We also observed density beyond the tail tip in vertically-oriented phages that had penetrated the cell wall, capturing the final stage of adsorption. Together, our data provide a quantitative characterization of the orientation of phages as they adsorb onto cells, and suggest that cyanophages that abut their cellular targets are only transiently in the "perpendicular" orientation required for successful infection.
View details for DOI 10.1038/srep44176
View details for Web of Science ID 000395891400001
View details for PubMedID 28281671
View details for PubMedCentralID PMC5345008
Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM
Proc Natl Acad Sci U S A
2017; 114 (31): 8259-8264
View details for DOI 10.1073/pnas.1704725114
Control of the structural landscape and neuronal proteotoxicity of mutant Huntingtin by domains flanking the polyQ tract.
2016; 5
Many neurodegenerative diseases are linked to amyloid aggregation. In Huntington's disease (HD), neurotoxicity correlates with an increased aggregation propensity of a polyglutamine (polyQ) expansion in exon 1 of mutant huntingtin protein (mHtt). Here we establish how the domains flanking the polyQ tract shape the mHtt conformational landscape in vitro and in neurons. In vitro, the flanking domains have opposing effects on the conformation and stabilities of oligomers and amyloid fibrils. The N-terminal N17 promotes amyloid fibril formation, while the C-terminal Proline Rich Domain destabilizes fibrils and enhances oligomer formation. However, in neurons both domains act synergistically to engage protective chaperone and degradation pathways promoting mHtt proteostasis. Surprisingly, when proteotoxicity was assessed in rat corticostriatal brain slices, either flanking region alone sufficed to generate a neurotoxic conformation, while the polyQ tract alone exhibited minimal toxicity. Linking mHtt structural properties to its neuronal proteostasis should inform new strategies for neuroprotection in polyQ-expansion diseases.
View details for DOI 10.7554/eLife.18065
View details for PubMedID 27751235
View details for PubMedCentralID PMC5135392
Resolution and Probabilistic Models of Components in CryoEM Maps of Mature P22 Bacteriophage
2016; 110 (4): 827-839
CryoEM continues to produce density maps of larger and more complex assemblies with multiple protein components of mixed symmetries. Resolution is not always uniform throughout a cryoEM map, and it can be useful to estimate the resolution in specific molecular components of a large assembly. In this study, we present procedures to 1) estimate the resolution in subcomponents by gold-standard Fourier shell correlation (FSC); 2) validate modeling procedures, particularly at medium resolutions, which can include loop modeling and flexible fitting; and 3) build probabilistic models that combine high-accuracy priors (such as crystallographic structures) with medium-resolution cryoEM densities. As an example, we apply these methods to new cryoEM maps of the mature bacteriophage P22, reconstructed without imposing icosahedral symmetry. Resolution estimates based on gold-standard FSC show the highest resolution in the coat region (7.6 Å), whereas other components are at slightly lower resolutions: portal (9.2 Å), hub (8.5 Å), tailspike (10.9 Å), and needle (10.5 Å). These differences are indicative of inherent structural heterogeneity and/or reconstruction accuracy in different subcomponents of the map. Probabilistic models for these subcomponents provide new insights, to our knowledge, and structural information when taking into account uncertainty given the limitations of the observed density.
View details for DOI 10.1016/j.bpj.2015.11.3522
View details for PubMedID 26743049
Improved Peak Detection and Deconvolution of Native Electrospray Mass Spectra from Large Protein Complexes
2015; 26 (12): 2141-2151
Native electrospray-ionization mass spectrometry (native MS) measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact protein assemblies. However, native spectra derived from these assemblies are often partially obscured by low signal-to-noise as well as broad peak shapes because of residual solvation and adduction after the electrospray process. The wide peak widths together with the fact that sequential charge state series from highly charged ions are closely spaced means that native spectra containing multiple species often suffer from high degrees of peak overlap or else contain highly interleaved charge envelopes. This situation presents a challenge for peak detection, correct charge state and charge envelope assignment, and ultimately extraction of the relevant underlying mass values of the noncovalent assemblages being investigated. In this report, we describe a comprehensive algorithm developed for addressing peak detection, peak overlap, and charge state assignment in native mass spectra, called PeakSeeker. Overlapped peaks are detected by examination of the second derivative of the raw mass spectrum. Charge state distributions of the molecular species are determined by fitting linear combinations of charge envelopes to the overall experimental mass spectrum. This software is capable of deconvoluting heterogeneous, complex, and noisy native mass spectra of large protein assemblies as demonstrated by analysis of (1) synthetic mononucleosomes containing severely overlapping peaks, (2) an RNA polymerase II/α-amanitin complex with many closely interleaved ion signals, and (3) human TriC complex containing high levels of background noise. Graphical Abstract .
View details for DOI 10.1007/s13361-015-1235-6
View details for Web of Science ID 000365116500020
View details for PubMedID 26323614
Gating machinery of InsP(3)R channels revealed by electron cryomicroscopy
2015; 527 (7578): 336-?
Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are ubiquitous ion channels responsible for cytosolic Ca(2+) signalling and essential for a broad array of cellular processes ranging from contraction to secretion, and from proliferation to cell death. Despite decades of research on InsP3Rs, a mechanistic understanding of their structure-function relationship is lacking. Here we present the first, to our knowledge, near-atomic (4.7 Å) resolution electron cryomicroscopy structure of the tetrameric mammalian type 1 InsP3R channel in its apo-state. At this resolution, we are able to trace unambiguously ∼85% of the protein backbone, allowing us to identify the structural elements involved in gating and modulation of this 1.3-megadalton channel. Although the central Ca(2+)-conduction pathway is similar to other ion channels, including the closely related ryanodine receptor, the cytosolic carboxy termini are uniquely arranged in a left-handed α-helical bundle, directly interacting with the amino-terminal domains of adjacent subunits. This configuration suggests a molecular mechanism for allosteric regulation of channel gating by intracellular signals.
View details for DOI 10.1038/nature15249
View details for PubMedID 26458101
Outcome of the First wwPDB Hybrid/Integrative Methods Task Force Workshop
2015; 23 (7): 1156-1167
Structures of biomolecular systems are increasingly computed by integrative modeling that relies on varied types of experimental data and theoretical information. We describe here the proceedings and conclusions from the first wwPDB Hybrid/Integrative Methods Task Force Workshop held at the European Bioinformatics Institute in Hinxton, UK, on October 6 and 7, 2014. At the workshop, experts in various experimental fields of structural biology, experts in integrative modeling and visualization, and experts in data archiving addressed a series of questions central to the future of structural biology. How should integrative models be represented? How should the data and integrative models be validated? What data should be archived? How should the data and models be archived? What information should accompany the publication of integrative models?
View details for DOI 10.1016/j.str.2015.05.013
View details for Web of Science ID 000360312200004
View details for PubMedID 26095030
View details for PubMedCentralID PMC4933300
CTF Challenge: Result summary
2015; 190 (3): 348-359
Image formation in bright field electron microscopy can be described with the help of the contrast transfer function (CTF). In this work the authors describe the "CTF Estimation Challenge", called by the Madrid Instruct Image Processing Center (I2PC) in collaboration with the National Center for Macromolecular Imaging (NCMI) at Houston. Correcting for the effects of the CTF requires accurate knowledge of the CTF parameters, but these have often been difficult to determine. In this challenge, researchers have had the opportunity to test their ability in estimating some of the key parameters of the electron microscope CTF on a large micrograph data set produced by well-known laboratories on a wide set of experimental conditions. This work presents the first analysis of the results of the CTF Estimation Challenge, including an assessment of the performance of the different software packages under different conditions, so as to identify those areas of research where further developments would be desirable in order to achieve high-resolution structural information.
View details for DOI 10.1016/j.jsb.2015.04.003
View details for Web of Science ID 000356115600009
View details for PubMedID 25913484
An atomic model of brome mosaic virus using direct electron detection and real-space optimization
2014; 5
Advances in electron cryo-microscopy have enabled structure determination of macromolecules at near-atomic resolution. However, structure determination, even using de novo methods, remains susceptible to model bias and overfitting. Here we describe a complete workflow for data acquisition, image processing, all-atom modelling and validation of brome mosaic virus, an RNA virus. Data were collected with a direct electron detector in integrating mode and an exposure beyond the traditional radiation damage limit. The final density map has a resolution of 3.8 Å as assessed by two independent data sets and maps. We used the map to derive an all-atom model with a newly implemented real-space optimization protocol. The validity of the model was verified by its match with the density map and a previous model from X-ray crystallography, as well as the internal consistency of models from independent maps. This study demonstrates a practical approach to obtain a rigorously validated atomic resolution electron cryo-microscopy structure.
View details for DOI 10.1038/ncomms5808
View details for Web of Science ID 000342928700012
View details for PubMedID 25185801
Modulation of STAT3 folding and function by TRiC/CCT chaperonin.
PLoS biology
2014; 12 (4)
Signal transducer and activator of transcription 3 (Stat3) transduces signals of many peptide hormones from the cell surface to the nucleus and functions as an oncoprotein in many types of cancers, yet little is known about how it achieves its native folded state within the cell. Here we show that Stat3 is a novel substrate of the ring-shaped hetero-oligomeric eukaryotic chaperonin, TRiC/CCT, which contributes to its biosynthesis and activity in vitro and in vivo. TRiC binding to Stat3 was mediated, at least in part, by TRiC subunit CCT3. Stat3 binding to TRiC mapped predominantly to the β-strand rich, DNA-binding domain of Stat3. Notably, enhancing Stat3 binding to TRiC by engineering an additional TRiC-binding domain from the von Hippel-Lindau protein (vTBD), at the N-terminus of Stat3, further increased its affinity for TRiC as well as its function, as determined by Stat3's ability to bind to its phosphotyrosyl-peptide ligand, an interaction critical for Stat3 activation. Thus, Stat3 levels and function are regulated by TRiC and can be modulated by manipulating its interaction with TRiC.
View details for DOI 10.1371/journal.pbio.1001844
View details for PubMedID 24756126
View details for PubMedCentralID PMC3995649
Validated near-atomic resolution structure of bacteriophage epsilon15 derived from cryo-EM and modeling
2013; 110 (30): 12301-12306
High-resolution structures of viruses have made important contributions to modern structural biology. Bacteriophages, the most diverse and abundant organisms on earth, replicate and infect all bacteria and archaea, making them excellent potential alternatives to antibiotics and therapies for multidrug-resistant bacteria. Here, we improved upon our previous electron cryomicroscopy structure of Salmonella bacteriophage epsilon15, achieving a resolution sufficient to determine the tertiary structures of both gp7 and gp10 protein subunits that form the T = 7 icosahedral lattice. This study utilizes recently established best practice for near-atomic to high-resolution (3-5 Å) electron cryomicroscopy data evaluation. The resolution and reliability of the density map were cross-validated by multiple reconstructions from truly independent data sets, whereas the models of the individual protein subunits were validated adopting the best practices from X-ray crystallography. Some sidechain densities are clearly resolved and show the subunit-subunit interactions within and across the capsomeres that are required to stabilize the virus. The presence of the canonical phage and jellyroll viral protein folds, gp7 and gp10, respectively, in the same virus suggests that epsilon15 may have emerged more recently relative to other bacteriophages.
View details for DOI 10.1073/pnas.1309947110
View details for Web of Science ID 000322112300044
View details for PubMedID 23840063
TRiC's tricks inhibit huntingtin aggregation
2013; 2
In Huntington's disease, a mutated version of the huntingtin protein leads to cell death. Mutant huntingtin is known to aggregate, a process that can be inhibited by the eukaryotic chaperonin TRiC (TCP1-ring complex) in vitro and in vivo. A structural understanding of the genesis of aggregates and their modulation by cellular chaperones could facilitate the development of therapies but has been hindered by the heterogeneity of amyloid aggregates. Using cryo-electron microscopy (cryoEM) and single particle cryo-electron tomography (SPT) we characterize the growth of fibrillar aggregates of mutant huntingtin exon 1 containing an expanded polyglutamine tract with 51 residues (mhttQ51), and resolve 3-D structures of the chaperonin TRiC interacting with mhttQ51. We find that TRiC caps mhttQ51 fibril tips via the apical domains of its subunits, and also encapsulates smaller mhtt oligomers within its chamber. These two complementary mechanisms provide a structural description for TRiC's inhibition of mhttQ51 aggregation in vitro. DOI:http://dx.doi.org/10.7554/eLife.00710.001.
View details for DOI 10.7554/eLife.00710
View details for Web of Science ID 000328620500004
View details for PubMedID 23853712
View details for PubMedCentralID PMC3707056
Cryo-EM model validation using independent map reconstructions.
Protein science
2013; 22 (6): 865-868
An increasing number of cryo-electron microscopy (cryo-EM) density maps are being generated with suitable resolution to trace the protein backbone and guide sidechain placement. Generating and evaluating atomic models based on such maps would be greatly facilitated by independent validation metrics for assessing the fit of the models to the data. We describe such a metric based on the fit of atomic models with independent test maps from single particle reconstructions not used in model refinement. The metric provides a means to determine the proper balance between the fit to the density and model energy and stereochemistry during refinement, and is likely to be useful in determining values of model building and refinement metaparameters quite generally.
View details for DOI 10.1002/pro.2267
View details for PubMedID 23592445
The Molecular Architecture of the Eukaryotic Chaperonin TRiC/CCT
2012; 20 (5): 814-825
TRiC/CCT is a highly conserved and essential chaperonin that uses ATP cycling to facilitate folding of approximately 10% of the eukaryotic proteome. This 1 MDa hetero-oligomeric complex consists of two stacked rings of eight paralogous subunits each. Previously proposed TRiC models differ substantially in their subunit arrangements and ring register. Here, we integrate chemical crosslinking, mass spectrometry, and combinatorial modeling to reveal the definitive subunit arrangement of TRiC. In vivo disulfide mapping provided additional validation for the crosslinking-derived arrangement as the definitive TRiC topology. This subunit arrangement allowed the refinement of a structural model using existing X-ray diffraction data. The structure described here explains all available crosslink experiments, provides a rationale for previously unexplained structural features, and reveals a surprising asymmetry of charges within the chaperonin folding chamber.
View details for DOI 10.1016/j.str.2012.03.007
View details for Web of Science ID 000304214400008
View details for PubMedID 22503819
View details for PubMedCentralID PMC3350567
Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle
2012; 31 (3): 720-730
The eukaryotic group II chaperonin TRiC/CCT is a 16-subunit complex with eight distinct but similar subunits arranged in two stacked rings. Substrate folding inside the central chamber is triggered by ATP hydrolysis. We present five cryo-EM structures of TRiC in apo and nucleotide-induced states without imposing symmetry during the 3D reconstruction. These structures reveal the intra- and inter-ring subunit interaction pattern changes during the ATPase cycle. In the apo state, the subunit arrangement in each ring is highly asymmetric, whereas all nucleotide-containing states tend to be more symmetrical. We identify and structurally characterize an one-ring closed intermediate induced by ATP hydrolysis wherein the closed TRiC ring exhibits an observable chamber expansion. This likely represents the physiological substrate folding state. Our structural results suggest mechanisms for inter-ring-negative cooperativity, intra-ring-positive cooperativity, and protein-folding chamber closure of TRiC. Intriguingly, these mechanisms are different from other group I and II chaperonins despite their similar architecture.
View details for DOI 10.1038/emboj.2011.366
View details for Web of Science ID 000300871700019
View details for PubMedID 22045336
View details for PubMedCentralID PMC3273382
Cryo-EM Structure of a Group II Chaperonin in the Prehydrolysis ATP-Bound State Leading to Lid Closure
2011; 19 (5): 633-639
Chaperonins are large ATP-driven molecular machines that mediate cellular protein folding. Group II chaperonins use their "built-in lid" to close their central folding chamber. Here we report the structure of an archaeal group II chaperonin in its prehydrolysis ATP-bound state at subnanometer resolution using single particle cryo-electron microscopy (cryo-EM). Structural comparison of Mm-cpn in ATP-free, ATP-bound, and ATP-hydrolysis states reveals that ATP binding alone causes the chaperonin to close slightly with a ∼45° counterclockwise rotation of the apical domain. The subsequent ATP hydrolysis drives each subunit to rock toward the folding chamber and to close the lid completely. These motions are attributable to the local interactions of specific active site residues with the nucleotide, the tight couplings between the apical and intermediate domains within the subunit, and the aligned interactions between two subunits across the rings. This mechanism of structural changes in response to ATP is entirely different from those found in group I chaperonins.
View details for DOI 10.1016/j.str.2011.03.005
View details for Web of Science ID 000290815500006
View details for PubMedID 21565698
Dual Action of ATP Hydrolysis Couples Lid Closure to Substrate Release into the Group II Chaperonin Chamber
2011; 144 (2): 240-252
Group II chaperonins are ATP-dependent ring-shaped complexes that bind nonnative polypeptides and facilitate protein folding in archaea and eukaryotes. A built-in lid encapsulates substrate proteins within the central chaperonin chamber. Here, we describe the fate of the substrate during the nucleotide cycle of group II chaperonins. The chaperonin substrate-binding sites are exposed, and the lid is open in both the ATP-free and ATP-bound prehydrolysis states. ATP hydrolysis has a dual function in the folding cycle, triggering both lid closure and substrate release into the central chamber. Notably, substrate release can occur in the absence of a lid, and lid closure can occur without substrate release. However, productive folding requires both events, so that the polypeptide is released into the confined space of the closed chamber where it folds. Our results show that ATP hydrolysis coordinates the structural and functional determinants that trigger productive folding.
View details for DOI 10.1016/j.cell.2010.12.017
View details for Web of Science ID 000286459900009
View details for PubMedID 21241893
View details for PubMedCentralID PMC3055171
MOTIF-EM: an automated computational tool for identifying conserved regions in CryoEM structures
2010; 26 (12): i301-i309
We present a new, first-of-its-kind, fully automated computational tool MOTIF-EM for identifying regions or domains or motifs in cryoEM maps of large macromolecular assemblies (such as chaperonins, viruses, etc.) that remain conformationally conserved. As a by-product, regions in structures that are not conserved are revealed: this can indicate local molecular flexibility related to biological activity. MOTIF-EM takes cryoEM volumetric maps as inputs. The technique used by MOTIF-EM to detect conserved sub-structures is inspired by a recent breakthrough in 2D object recognition. The technique works by constructing rotationally invariant, low-dimensional representations of local regions in the input cryoEM maps. Correspondences are established between the reduced representations (by comparing them using a simple metric) across the input maps. The correspondences are clustered using hash tables and graph theory is used to retrieve conserved structural domains or motifs. MOTIF-EM has been used to extract conserved domains occurring in large macromolecular assembly maps, including as those of viruses P22 and epsilon 15, Ribosome 70S, GroEL, that remain structurally conserved in different functional states. Our method can also been used to build atomic models for some maps. We also used MOTIF-EM to identify the conserved folds shared among dsDNA bacteriophages HK97, Epsilon 15, and ô29, though they have low-sequence similarity. Supplementary information: Supplementary data are available at Bioinformatics online.
View details for DOI 10.1093/bioinformatics/btq195
View details for Web of Science ID 000278689000037
View details for PubMedID 20529921
View details for PubMedCentralID PMC2881380
4.0-angstrom resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement
2010; 107 (11): 4967-4972
The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of approximately 5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-A resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-A resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Calpha backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed approximately 95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.
View details for DOI 10.1073/pnas.0913774107
View details for Web of Science ID 000275714300032
View details for PubMedID 20194787
View details for PubMedCentralID PMC2841888
Mechanism of folding chamber closure in a group II chaperonin
Group II chaperonins are essential mediators of cellular protein folding in eukaryotes and archaea. These oligomeric protein machines, approximately 1 megadalton, consist of two back-to-back rings encompassing a central cavity that accommodates polypeptide substrates. Chaperonin-mediated protein folding is critically dependent on the closure of a built-in lid, which is triggered by ATP hydrolysis. The structural rearrangements and molecular events leading to lid closure are still unknown. Here we report four single particle cryo-electron microscopy (cryo-EM) structures of Mm-cpn, an archaeal group II chaperonin, in the nucleotide-free (open) and nucleotide-induced (closed) states. The 4.3 A resolution of the closed conformation allowed building of the first ever atomic model directly from the single particle cryo-EM density map, in which we were able to visualize the nucleotide and more than 70% of the side chains. The model of the open conformation was obtained by using the deformable elastic network modelling with the 8 A resolution open-state cryo-EM density restraints. Together, the open and closed structures show how local conformational changes triggered by ATP hydrolysis lead to an alteration of intersubunit contacts within and across the rings, ultimately causing a rocking motion that closes the ring. Our analyses show that there is an intricate and unforeseen set of interactions controlling allosteric communication and inter-ring signalling, driving the conformational cycle of group II chaperonins. Beyond this, we anticipate that our methodology of combining single particle cryo-EM and computational modelling will become a powerful tool in the determination of atomic details involved in the dynamic processes of macromolecular machines in solution.
View details for DOI 10.1038/nature08701
View details for Web of Science ID 000273748100049
View details for PubMedID 20090755
View details for PubMedCentralID PMC2834796
Estimating contrast transfer function and associated parameters by constrained non-linear optimization
2009; 233 (3): 391-403
The three-dimensional reconstruction of macromolecules from two-dimensional single-particle electron images requires determination and correction of the contrast transfer function (CTF) and envelope function. A computational algorithm based on constrained non-linear optimization is developed to estimate the essential parameters in the CTF and envelope function model simultaneously and automatically. The application of this estimation method is demonstrated with focal series images of amorphous carbon film as well as images of ice-embedded icosahedral virus particles suspended across holes.
View details for DOI 10.1111/j.1365-2818.2009.03137.x
View details for Web of Science ID 000263758000005
View details for PubMedID 19250460
Mechanism of lid closure in the eukaryotic chaperonin TRiC/CCT
2008; 15 (7): 746-753
All chaperonins mediate ATP-dependent polypeptide folding by confining substrates within a central chamber. Intriguingly, the eukaryotic chaperonin TRiC (also called CCT) uses a built-in lid to close the chamber, whereas prokaryotic chaperonins use a detachable lid. Here we determine the mechanism of lid closure in TRiC using single-particle cryo-EM and comparative protein modeling. Comparison of TRiC in its open, nucleotide-free, and closed, nucleotide-induced states reveals that the interdomain motions leading to lid closure in TRiC are radically different from those of prokaryotic chaperonins, despite their overall structural similarity. We propose that domain movements in TRiC are coordinated through unique interdomain contacts within each subunit and, further, these contacts are absent in prokaryotic chaperonins. Our findings show how different mechanical switches can evolve from a common structural framework through modification of allosteric networks.
View details for DOI 10.1038/nsmb.1436
View details for Web of Science ID 000257412500018
View details for PubMedID 18536725
View details for PubMedCentralID PMC2546500
Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins
2007; 14 (5): 432-440
Chaperonins are allosteric double-ring ATPases that mediate cellular protein folding. ATP binding and hydrolysis control opening and closing of the central chaperonin chamber, which transiently provides a protected environment for protein folding. During evolution, two strategies to close the chaperonin chamber have emerged. Archaeal and eukaryotic group II chaperonins contain a built-in lid, whereas bacterial chaperonins use a ring-shaped cofactor as a detachable lid. Here we show that the built-in lid is an allosteric regulator of group II chaperonins, which helps synchronize the subunits within one ring and, to our surprise, also influences inter-ring communication. The lid is dispensable for substrate binding and ATP hydrolysis, but is required for productive substrate folding. These regulatory functions of the lid may serve to allow the symmetrical chaperonins to function as 'two-stroke' motors and may also provide a timer for substrate encapsulation within the closed chamber.
View details for DOI 10.1038/nsmb1236
View details for Web of Science ID 000246187400017
View details for PubMedID 17460696
View details for PubMedCentralID PMC3339572
Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion
2004; 324 (2): 373-386
Alphaviruses have the ability to induce cell-cell fusion after exposure to acid pH. This observation has served as an article of proof that these membrane-containing viruses infect cells by fusion of the virus membrane with a host cell membrane upon exposure to acid pH after incorporation into a cell endosome. We have investigated the requirements for the induction of virus-mediated, low pH-induced cell-cell fusion and cell-virus fusion. We have correlated the pH requirements for this process to structural changes they produce in the virus by electron cryo-microscopy. We found that exposure to acid pH was required to establish conditions for membrane fusion but that membrane fusion did not occur until return to neutral pH. Electron cryo-microscopy revealed dramatic changes in the structure of the virion as it was moved to acid pH and then returned to neutral pH. None of these treatments resulted in the disassembly of the virus protein icosahedral shell that is a requisite for the process of virus membrane-cell membrane fusion. The appearance of a prominent protruding structure upon exposure to acid pH and its disappearance upon return to neutral pH suggested that the production of a "pore"-like structure at the fivefold axis may facilitate cell penetration as has been proposed for polio (J. Virol. 74 (2000) 1342) and human rhino virus (Mol. Cell 10 (2002) 317). This transient structural change also provided an explanation for how membrane fusion occurs after return to neutral pH. Examination of virus-cell complexes at neutral pH supported the contention that infection occurs at the cell surface at neutral pH by the production of a virus structure that breaches the plasma membrane bilayer. These data suggest an alternative route of infection for Sindbis virus that occurs by a process that does not involve membrane fusion and does not require disassembly of the virus protein shell.
View details for DOI 10.1016/j.virus.2004.03.046
View details for Web of Science ID 000222376800012
View details for PubMedID 15207623
Electron cryo-microscopy of VAT, the archaeal p97/CDC48 homologue from Thermoplasma acidophilum
2002; 317 (5): 673-681
VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member of the AAA-family (ATPases associated with a variety of cellular activities) that possesses foldase as well as unfoldase-activity, forms homo-hexameric rings like its eukaryotic homologues p97 and CDC48. The VAT-monomer exhibits the tripartite domain architecture typical for type II AAA-ATPases: N-D1-D2, whereby N is the substrate binding N-terminal domain preceding domains D1 and D2, both containing AAA-modules. Recent 3-D reconstructions of VAT and p97 as obtained by electron microscopy suffer from weakly represented N-domains, probably a consequence of their flexible linkage to the hexameric core. Here we used electron cryo-microscopy and 3-D reconstruction of single particles in order to generate a 3-D model of VAT at 2.3 nm resolution. The hexameric core of the VAT-complex (diameter 13.2 nm, height 8.4 nm) encloses a central cavity and the substrate-binding N-domains are clearly arranged in the upper periphery. Comparison with the p97 3-D reconstruction and the recently determined crystal structure of p97-N-D1 suggests a tail-to-tail arrangement of D1 and D2 in VAT.
View details for DOI 10.1006/jmbi.2002.5448
View details for Web of Science ID 000175202300003
View details for PubMedID 11955016
Fourier amplitude decay of electron cryomicroscopic images of single particles and effects on structure determination
2001; 133 (1): 32-42
Several factors, including spatial and temporal coherence of the electron microscope, specimen movement, recording medium, and scanner optics, contribute to the decay of the measured Fourier amplitude in electron image intensities. We approximate the combination of these factors as a single Gaussian envelope function, the width of which is described by a single experimental B-factor. We present an improved method for estimating this B-factor from individual micrographs by combining the use of X-ray solution scattering and numerical fitting to the average power spectrum of particle images. A statistical estimation from over 200 micrographs of herpes simplex virus type-1 capsids was used to estimate the spread in the experimental B-factor of the data set. The B-factor is experimentally shown to be dependent on the objective lens defocus setting of the microscope. The average B-factor, the X-ray scattering intensity of the specimen, and the number of particles required to determine the structure at a lower resolution can be used to estimate the minimum fold increase in the number of particles that would be required to extend a single particle reconstruction to a specified higher resolution. We conclude that microscope and imaging improvements to reduce the experimental B-factor will be critical for obtaining an atomic resolution structure.
View details for DOI 10.1006/jsbi.2001.4330
View details for Web of Science ID 000169093800004
View details for PubMedID 11356062
Scaling structure factor amplitudes in electron cryomicroscopy using X-ray solution scattering
1999; 128 (1): 51-57
The structure factors derived from electron cryomicroscopic images are modified by the contrast transfer function of the microscope's objective lens and other influences. The phases of the structure factors can be corrected in a straightforward way when the positions of the contrast transfer function rings are determined. However, corrected amplitudes are also essential to yield an accurate distribution of mass in the reconstruction. The correct scale factors for the amplitudes are difficult to evaluate for data that are merged from many different micrographs. We opt to use X-ray solution scattering intensity from a concentrated suspension of the specimen to correct the amplitudes of the spherically averaged structure factors. When this approach is applied to the three-dimensional image data of ice-embedded acrosomal bundles, the core of a filament in a three-dimensional reconstruction of the acrosomal bundle becomes denser and matches more closely the outer density ascribed to scruin.
View details for Web of Science ID 000084451600008
View details for PubMedID 10600558
Solution X-ray scattering-based estimation of electron cryomicroscopy imaging parameters for reconstruction of virus particles
1999; 76 (4): 2249-2261
Structure factor amplitudes and phases can be computed directly from electron cryomicroscopy images. Inherent aberrations of the electromagnetic lenses and other instrumental factors affect the structure factors, however, resulting in decreased accuracy in the determined three-dimensional reconstruction. In contrast, solution x-ray scattering provides absolute and accurate measurement of spherically averaged structure factor amplitudes of particles in solution but does not provide information on the phases. In the present study, we explore the merits of using solution x-ray scattering data to estimate the imaging parameters necessary to make corrections to the structure factor amplitudes derived from electron cryomicroscopic images of icosahedral virus particles. Using 400-kV spot-scan images of the bacteriophage P22 procapsid, we have calculated an amplitude contrast of 8.0 +/- 5.2%. The amplitude decay parameter has been estimated to be 523 +/- 188 A2 with image noise compensation and 44 +/- 66 A2 without it. These results can also be used to estimate the minimum number of virus particles needed for reconstruction at different resolutions.
View details for Web of Science ID 000079643400049
View details for PubMedID 10096920
View details for PubMedCentralID PMC1300198
1990; 3 (2): 213-226
Both the number and range of applications of cryotechniques in transmission electron microscopy are increasing rapidly. In some cases, most notably the determination of protein structure by electron crystallography, progress has been limited by the performance of commercially available cryo stages. We review the design and performance criteria for stages which will be necessary for wide applicability in high resolution studies of biological specimens. The important criteria include an operating temperature below -140 degrees C with a low rate of contamination of the specimen, ability to tilt to 60 degrees, and perhaps most important, good resolution as judged by an effective modulation transfer function of 0.8 at 0.35 nm. Most applications also require an effective cryotransfer system. Up until now, most work in high resolution electron crystallography has been accomplished with laboratory-built stages which meet some, but not all, of these criteria. The availability of cold stages which fully meet criteria will allow the rapid expansion of high resolution studies by electron microscopy in structural biology.
View details for Web of Science ID A1990EG78800002
View details for PubMedID 2103342
1986; 49 (1): 251-258
Low dose cryoelectron microscopy has been used to record images and electron diffraction patterns of frozen hydrated crystals of the single-stranded DNA binding protein gp32*I. Fourier transforms from 13 image areas, corresponding to approximately 40,000 unit cells, were aligned by a minimal phase residual search and merged by vector addition in reciprocal space. Phases from the resulting composite transform were combined with amplitudes from electron diffraction patterns to reconstruct the projected mass density of the gp32*I crystal at 8.4 A resolution.
View details for Web of Science ID A1986AYX5300072
View details for PubMedID 3513856
1983; 169 (1): 235-248
View details for Web of Science ID A1983RG01200011
1979; 66 (3): 235-242
View details for Web of Science ID A1979GU49700003
View details for PubMedID 439191
1977; 2 (2-3): 207-217
An experimental and theoretical characterization of a fixed-beam transmission electron microscope with a field emission gun has been made with regard to the factors of electron beam brightness, spatial and temporal coherence of the incident electrons, objective lens current fluctuation, mechanical stability, and specimen contamination. It has been found that mechanical stability and temporal coherence are the primary factors that prevent the contrast transfer function from extending to 2.0 A in our microscope. Different amorphous thin films have also been used in order to compare their suitability for testing the imaging capability of the microscope at atomic resolution.
View details for Web of Science ID A1977DT14100011
View details for PubMedID 888240
View details for Web of Science ID A1975AB51500003


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July 29, 2009
This is what I've been working on today. I've had the Maple sitting in the shop for awhile with such a unique grain pattern, just waiting for inspiration to hit.
I made a comment on Twitter this past Sunday that I needed to create something this week, and asked people that follow my tweets to keep me accountable about it. Well, I didn't get back into the shop till today, and saw that piece of Maple sitting there, just begging me to use it. So I sat down to think what to make.
Now, I have a couple different ways that I get inspired. The big one is to just be out in nature, whether walking in the woods, enjoying a mountain trail (yes, I realize that is not possible where I live at the moment, don't remind me), fishing on a quaint lake, or sitting on my back porch in the morning and hearing all the birds sing to name a few ways. Considering that my shop is in town, and even though the empty lot across the road gets its share of deer and a red tailed hawk that lives somewhere near by, the Dairy Queen and the auto-body shop next door kind of ruin that inspiration.
I also can get inspired listening to great music. Maybe its the idea of art begetting art, I'm not sure. I just know that some days, hearing a great country or Americana song, by, say, Willie Nelson, Uncle Tupelo, or Neko Case, can get the creative juices flowing. Other days, it may be the latest indie album from Sufjan Stevens, We Were Promised Jet Packs, or DM Stith that does it. Of course, jazz is always an inspiring choice, but I really prefer my jazz live, and we don't have a decent club anywhere near us. Other times, I need to jam to the likes of My Morning Jacket, Arcade Fire, or Yeasayer to peak my imagination. This morning though, I just didn't turn any music on, not sure why really.
Another excellent source of inspiration for me is just seeing other people's woodworking creations. The works of Sam Maloof are a big one for me. I have ideas rattling around my head for a couple furniture pieces directly inspired by his chairs that I deeply long to make happen. Someday, hopefully soon. I am also intrigued by Thos. Moser. I know he isn't as revered among the studio furniture and craft world, but I really love the simple beauty in his furniture. I also admire his accentuating his joinery. The works of Greene and Greene inspire me every time I see them! If I ever get the chance to go view the Gamble House, I could probably spend a couple days there. I have honestly just begun my journey into getting know the great furniture designers and craftsmen, so I know there are so many more great works to inspire me out there. Honestly though, sometimes it is as simple as flipping through one of stacks and stacks of woodworking magazines when something will catch my eye. Usually, its one aspect of a project that jumps out at and causes me to think in a different direction. Where my imagination takes it could be 180º from what the project was that inspired it, but it took seeing a dovetailed box of two contrasting woods, or a vase carved and sculpted in mahogany, or the choice of a unique grain in a panel of a door on a cupboard to open my mind to endless possibilities.
Now, I know that this little project isn't as detailed as an 18th Century Highboy, as stunning as a natural edge table by George Nakashima, or as graceful as the elegant curves of a Maloof rocker. But as I flipped through one of my woodworking magazines, a cutting board caught my eye with contrasting woods. I immediately thought that a cutting board would perfectly accentuate the beautiful grain of that piece of Maple sitting by my desk. The ends are Cherry, which I think will set off the beauty in the Maple grain quite nicely. I haven't decided what to make the plugs out of yet. Hopefully I can get up to Woodcraft in the next couple days and find something that speaks to me.
Which I guess would be the best inspiration of all, the wood. This simple piece of Maple with an extremely unique grain pattern has caused my imagination to really run rampant. It has given me a couple ideas that I want to pursue further, so stay tuned! Hopefully what comes of my inspiration will inspire creativity of your own, in whatever area you are passionate about.
(By the way, sorry for the green tint to the picture. The fluorescent lights in the shop tend to do that to pictures I take with my phone. Hopefully yet this week, I will have the cutting board completed, and I will be sure to post some better pictures of it at that time.)
Building an Oak replacement door.
July 9, 2009
I got to build an Oak interior door the last couple days to replace this one, which was burned in a fire.
This has been a fun little project. Just waiting for the glass, and for the painter to stain and lacquer the door, then I will install it.


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For the first time, Kushwaha and other senior executives such as chief operating officer Arvind Singhal and chief marketing officer Pushkar Jain at the Ratan Tata-backed jewellery portal will be interacting with customers live through phone and chat in an effort to build a more personal connection with buyers.
"It's a conscious decision we made to be closer to consumers, not only to help them with their purchases and queries, but also to get real feedback on what works and what doesn't," Singhal told ET.
"On festival days like Akshaya Tritiya and Dhanteras, our sales spike six-seven times our usual volume of business and we get at least four times the usual number of calls and chat interactions. This is a great opportunity to have our ears on the ground and understand the customer experience," he said.
Singhal said the company's senior team regularly goes through call and chat transcripts to understand the pulse of consumers, but this is the first time it will be operating together with customer service executives and interacting with buyers in a live environment.
"We've received special training for this. We're being given a crash course in a basic understanding of metals and stones, our policies for purchase and return, website navigation, special offers and the process of raising order complaints," Singhal said.
Telephone etiquette, too, will be a focus of the course, he said. With much fewer opportunities for personalised customer interaction in the e-commerce model, business leaders are trying new and innovative ways to build a rapport with their customer base. Earlier this year, Flipkart cofounder Sachin Bansal had turned delivery executive and dropped off packages to customers in Bengaluru.


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Backup Blu ray/DVD movies with original structure
Description: With Pavtube ByteCopy you can easily backup your favorite Blu-ray and DVD movies from physical disks to computer hard drive with original structure.
Many movies have released their Blu-ray and DVD versions on Amazon.com, such as Pacific Rim, Despicable Me 2, The Heat, Monsters University, etc, which adds more fun for the coming Halloween so you can enjoy these hot movies. But as we all know, Blu-rays and DVDs are usually hard to be backed up due to region code and disc encryption protection, you'd better to use Pavtube BD/DVD copying program to remove the disc protection and copy source disc to computer hard drive with same structure preserved.
Full guide: Make 1:1 Copy Despicable Me DVD and Pacific Rim Blu-ray with Pavtube ByteCopy
Step 1. Install Pavtube ByteCopy on your Windows/Mac.
Pavtube ByteCopy for Windows/Mac is an excellent BD/DVD backing up tool that can make 1:1 copy of your source BD/DVD with original structure including extras and menus. Meanwhile, this app is regarded as a great BD/DVD ripping program to help users convert BD/DVD collections for new how tablets and smartphones including iPad mini, iPad Air, iPhone 5S, Samsung Galaxy Tab, Kindle Fire HDX 7, Google Nexus 7/10 and more.
Top Blu-ray/DVD Copying & Ripping Review - Pavtube ByteCopy
Step 2. Launch the best BD/DVD backup tool, and load your Blu-ray or DVD movie from disc drive to the app.
Step 3. Click the "Browse" button to set output folder where you want to save the copied Blu-ray and DVD movies.
Step 4. Hit the Copy the entire disc icon on ByteCopy main interface and start Blu-ray/DVD backing up process.
1. If you'd like to copy only movie content and leave out the extras and commentary, the Directly Copy is exactly what you need, which copies intact .M2TS/VOB main movie from original BD/DVD disc without extras.
2. Pavtube ByteCopy allows users lossless backup Blu-ray/DVD to MKV with multiple audio tracks and subtitles as well as original chapter markers preserved.
More topics:
Lossless Convert Blu-ray to MP4/MOV for iPad
Rip/Backup DVD to lossless MKV for WDTV player with 5.1 channel
Put Blu-ray/DVD collections to external hard disc for storage
Convert Blu-ray movies for NAS device streaming
Encoding BD to MKV format for streaming to Smart TV
Convert Blu-ray ISO to MKV 1080p for streaming
How to put DVD ISO/IFO movies on Apple new iPhone 5S/5C?
Convert DVD ISO/IFO file to MP4 for Galaxy Tab 3 10.1/8.0
By the way Hurry up and never miss the chance to join the 2013 Halloween Deal-Free and Up to 30% off to Get free DVD Ripper, free Video DVD Converter Ultimate, 30 % off Blu-ray Ripper, 30 % off DVD Ripper, 20% off Video Converter Ultimate and 20% off Video Converter now!
Source: http://video-tips.jimdo.com/2013/10/25/bakup-blu-ray-dvd-with-original-structure/


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The potion of blood and potion of vampire blood are SLASH'EM specific items. They are primarily of interest to Vampire players. Dipping a unicorn horn into a potion of (vampire) blood will turn it into water, which is typically the only use for non-vampires. Despite being potions, drinking either potion will break vegetarian conduct[1]. A thrown potion of (vampire) blood that lands on a non-chaotic altar will convert the altar to chaotic and abuse your wisdom, even if you are chaotic. This will anger an attendant priest, but has no other effects[2].
Potion of Blood Edit
|50 zm
A vampire can drink a potion of blood to gain 30 nutrition if blessed or 20 if uncursed, halved if the potion is diluted. Cursed blood has congealed and will give 0 nutrition. Non vampires that drink the potion will vomit for 10d8 (more) turns.
Potion of Vampire Blood Edit
|vampire blood
|350 zm
A vampire player can drink a noncursed potion of vampire blood to gain 400 nutrition points. If the potion is blessed, your HP and max HP will be increased by the same amount as from a level gain. Vampire monsters can make use of this potion to heal themselves for d8 + d2 hit points if not cursed ("Monster looks full of life.")[3]. If the potion is cursed there is no effect ("Monster discards the congealed blood in disgust."). This will identify the potion if it is seen. If a non-vampire player drinks the potion, they will polymorph into a vampire and abuse their constitution. Note that the resulting polymorph is semi-permanent -- it has no timeout, but you will revert back to your normal form if your HP are drained to zero. Neutral characters will suffer a -3 alignment penalty, while lawful characters and monks will suffer a -15 alignment penalty and anger their god.
Generation Edit
Neither potion is randomly generated. Vampire players playing as a role that normally starts with food will receive potions of vampire blood instead. Potions of vampire blood can be created by a player vampire using the draw blood technique if the player also has a medical kit with phials. Finally, vampires, vampire lords, and vampire mages have a 50% chance of being generated with a potion of either blood or vampire blood; there is a <level> in 30 chance that the potion will be vampire blood, otherwise it is regular blood[4]. Because enemy vampires will drink potions of vampire blood to heal themselves, it is important for player vampires to kill them quickly to avoid losing the potion. Fire vampires and star vampires do not start with potions of blood, but can make use of any they find on the ground.


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Consumer Behaviour and Psychology
Duration 2 weeks
Weekly study 3 hours
Understand how consumer psychology affects the lives of customers and companies
Have you ever wondered why you buy one brand of cereal over another? Or why you might react to different types of advertising? Your decisions are motivated by a number of factors. The study of these factors is called consumer psychology.
On this course you will explore what consumer psychology means and how it's related to the rise of a consumer society. You will be introduced to basic marketing concepts and you will understand how these concepts affect the relationship between customers and the companies. And you will reflect on how consumer psychology impacts the decisions you make everyday.
What topics will you cover?
- What is consumer psychology?
- Consumer psychology and the consumer society
- Consumer-oriented marketing
- Consumer choice and decision making
When would you like to start?
What will you achieve?
By the end of the course, you'll be able to...
Who is the course for?
This course is for people seeking a better understanding of consumer psychology and how it affects the decision-making process of both individuals and companies.
Who will you learn with?
Andre Soares
Andre Soares PhD, MSc, BSc, FHEA, CPsychol – assistant professor in business and occupational psychology at Coventry University
Advance your career with a degree
What's different about this course?
Since 2017 you have been able to take a degree on FutureLearn. This free course is a bit like a starter course for a degree — it should give you a flavour of the degree subject but can also be your first step towards a degree qualification.
If you enjoy this course you can apply to join the program it's part of to start working towards a degree on FutureLearn.
Don't worry, you can still take the course without doing a full degree!
How do degrees work on FutureLearn?
Which program and degree is this course part of?
This course is part of the program Consumer Psychology, which is one of the building blocks that leads to a degree in MSc Business and Organisational Psychology.


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Daughter of Theron, Librarian of Alexandria
When the Christians destroyed the Library of Alexandria under Theodosius in 391 and 396CE, the librarian was Theon (or variously Theron). We can imagine his and his daughter Hypatia's feelings as some 500 000 scrolls representing hundreds of years of collection and research were destroyed. This was a forerunner of the end of "The Golden Age". Many have argued that the death of Hypatia, only 25 years later introduced the real "dark ages" as the last light of reason was extinguished with her death.
Certainly, due to the deliberate destruction of all things perceived as anti- or even, "not sufficiently pro-" Christian, we don't know very much about her, and of that, much of what we do know was written by her enemies. Yet she was such a brilliant beacon that even they could not entirely hide the fact that Hypatia was something quite exceptional. Nicephorus, Philostorgius and Socrates Scholasticus (who wrote an ecclesiastical history in the 5th Century infra) mention her, and even these church apologists and manufacturers of pseudographical praised her characteristics and scholarship even as they damned her values. A few surviving letters by one of her former students who she taught neoplatonic ideas, Synesius of Cyrene (infra) and later Bishop of Ptolemais (who helped create the doctrine of the Trinity) provide some background and a few quotations, such as those used here.
And then there was silence
For a long time. Many centuries later (1510-ish) Raphael would allegedly submit a draft of his work, "The School of Athens", to the church fathers.
"Who is this woman in the middle?" asked the Bishops."
"Hypatia, the most famous student of the School of Athens," answered Raphael.
"Remove her. Knowledge of her runs counter to the belief of the faithful," admonished the priest. "Otherwise, the work is acceptable."
"As you command," replied the artist, given no choice. However, he still "snuck" her back into the picture, using Francesco Maria della Rovere as his model.
18-th Century and Beyond
However, Hypatia's story was memetically compelling and during the eighteenth century enlightenment many authors resurrected and retold it. The first, but by no means the most significant (although the foundation for many of the following works) was that by John Toland, a zealous Protestant in 1720. His, "Hypatia, or the History of a Most Beautiful, Most Virtuous, Most Learned and in Every Way Accomplished Lady; Who was Torn to Pieces by the Clergy of Alexandria, to Gratify the Pride, Emulation and Cruelty of the Archbishop, Commonly but Undeservedly Titled St. Cyril" . Voltaire, Fielding, and Gibbon wrote about her, reawakening the ire of the Roman Catholic Church, which responded with an official rebuttal, "The History of Hypatia, a Most Impudent School-Mistress. In Defense of Saint Cyril and the Alexandrian Clergy from the Aspersions of Mr. Toland."
Throughout the nineteenth Century, she inspired the romantics of England and Europe, the most famous of which was undoubtedly "Hypatia or the New Foes with an Old Face]", written in 1853 by Charles Kingsley (later to publish, amongst others, Westward Ho and Water Babies, probably his most famous books).


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How Does a Swab Drug Test Work?
Oral fluid drug tests are a popular choice with employers and with parents who want to test their teens for drugs 3. Among oral fluid drug tests, the swab type is the most common. The highly absorbent mucous membranes of the mouth and the nature of the glands that produce oral fluids allow swab drug tests to be surprisingly accurate.
Sample Types
The term "oral fluids" refers to more than merely saliva, but saliva is the most abundant of the oral fluids. For this reason, oral fluid drug tests are often called "saliva tests" or "spit tests." Oral fluid samples may be retrieved in two general ways: spitting and swabbing. While swab tests do not allow multiple tests to be performed on the same sample, they make it easy to collect samples without contamination and provide easy, fast, accurate results.
Clinical Setting
When a swab drug test is used in a clinical setting, a multi-purpose medical swab may be used to collect the sample. A medical professional places the swab between the subject's cheek and lower gum or under his tongue and holds it there for several minutes. The clinic then sends the swab to a lab, where it is tested for the presence of target drugs.
Swab Drug Kits
When a swab drug test is performed by an employer or parent, they generally use a commercially-available drug testing kit. The collection device, which is similar to a pregnancy test, contains an absorbent strip housed by a thin, plastic casing. The swab device is placed in the same manner as a clinical swab in order to collect the sample. The "lab" is built into the device. As the sample is absorbed, a colored control line "floats" higher or gradually appears in the device window.
Reading Test Results
With a testing kit, one or more lines indicates whether a certain type of drug is present. In the case of a multiple-drug array, each drug is represented by a different color. If the line appears, the drug is not detected. If a line is missing from the array, it is considered a positive result for the presence of that drug. Electronic versions don't require the reading of lines. Instead, the presence or absence of the target drugs is indicated on a clear, electronic readout.
Drug Detection
Most swab drug test kits test for a standard array of drugs, which may include:
- the following "parent drugs": amphetamines
- methamphetamines
- cocaine
- opiates
- THC (marijuana-type drugs)
- phencyclidine
- benzodiazepines
- methadone
The test does not indicate the presence or absence of a specific drug in the system (e.g., heroin); it only indicates the parent drug (in this case, opium).
Swab drug tests are inexpensive and provide quick results on a wide range of drugs. They are easy to use and generally easy to read. Moreover, they sidestep some of the common problems involved with other methods. Samples can be collected "on the spot," without the modesty issues inherent in urine sample collection. This allows observation throughout the testing procedure and virtually eliminates switching, diluting or tampering with the sample.
While other samples provide a longer history of drug use prior to test administration, oral fluid tests will only indicate drug use in terms of very recent use (typically 24 to 48 hours). Also, while swab tests check for a wide range of drug types, they do not test for some of the most commonly abused drugs, such as alcohol or inhalants.
- Children's Hospital Boston
- The Clinical Biochemist Reviews; Drug Testing in Oral Fluid; Olaf H. Drummer; August 2006
- U.S. News & World Report; 5 Ways Teens Might Cheat on Drug Tests—and How to Catch Them; Lindsay Lion; August 2008
- Mark Dadswell/Getty Images News/Getty Images


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Pizza does not need any upselling of any kind. Why? So many people LOVE pizza! It is no surprise that more and more places are being created not for the purpose of selling pizza, but rather worshipping it, in a sense. Many museums have been found across the U.S. devoted to pizza lovers, but despite that, a buzz worthy big pizza event is happening towards the end of this year where a new museum dedicated to Pizza will open in the big city, New York City to be exact. The Museum of Pizza will open its doors from October 13th to the 28th and heading this opening is Youth media company Nameless Network as mentioned in an online article for Ny.Eater.
So what is the big deal? This does not come at much surprise since sex has been the best tool in selling a product or service in the past decades, but to use sensual images in the promos for this new pizza museum? This place may be the best place for the #selfienation crew or #pizzalovers crew, but biggest question, will the artwork produced in the museum also share a sexual experience with pizza as well? Is it more than just pizza? Should children be able to attend the event? See for yourself in this photo of the Museum of Pizza banner(PHOTO VIA: NAMELESS NETWORK) .
For all you pizza lovers out there, this may interest you. If you go, please share those photos with me in the comments! Would love to see them! Tag @pizzaovensdotcom as well on Instagram and possibly your photo can be featured!


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The History of Stained Glass Windows
Stained glass is made by adding colored metallic salts during its manufacture. The process of creating these windows involves arranging pieces of the colored glass inside a frame to form a design, which is fastened together with strips of lead.
Stained glass windows are a global fascination and have adorned churches and other establishments for centuries.
Early History
Stained glass window use began with church building. In the 10th century, French and German churches featured biblical windows and English churches featured decorative windows.
Around 1100 AD, a monk named Theophilus wrote a "how to" that described stained glass window construction and techniques.
The Gothic Age
The Gothic age ushered in fantastic stained glass window techniques that graced the great cathedrals of Europe with works of art.
The Renaissance
During the Renaissance, stained glass suffered a 300 year period in which the form became less artistic and more garish with the use of thick, heavy paint.
The 1800s
In the mid-1800s, interest in Gothic architecture resurrected in England when art historians rediscovered stained glass techniques of medieval times.
Contemporary Age
After the Second World War the flowering abstract and expressionist movements led to stained glass church windows that are reminiscent of the artistically beautiful Gothic period.


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Addressing the Health Needs of LGBT Missourians
Related Story
ST. LOUIS - The Missouri Foundation of Health issued a report finding LGBT Missourians health below the general population average.
The report found that LGBT Missourians are less likely to report to be in good health and face conditions such as heart disease, asthma, obesity, smoking related illness and substance abuse more then the general public.
These conditions are linked to health behaviors dealing with social and economic environments, clinical care, insurance coverage, and access to preventive care.
Ryan Barker, MFH Health Policy Director, said " the LGBT community turns to things like smoking, higher rates of obesity, eating unhealthy, and those are coping mechanisms, they are a way for people to deal with those daily stresses in their lives like discrimination and oppression".
According to the report sexual and gender minorities are 1.5 times more likely to uninsured. LGBT families are more likely to be poor and underemployed than non LGBT families. Also LGBT are twice as likely not to receive medical care or surgery.
"A lot of this community is fearful of being discriminated against in the doctor's office and so often times the population is reluctant to go to the doctor or access the health care system", Barker said.
The report also found that cultural competent providers are hard to come by in Missouri with less than 15 of 5,704 primary care physicians have registered as LGBT-affirming in the Gay and Lesbian Medical Association on-line database.
For more information click here.