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James G. Wakefield;
James G. Wakefield
Carolyn A. Moores
The cover image shows microtubule catastrophe, the moment at which the polymer begins to fall apart by outward peeling of longitudinally connected stretches of tubulin, known as protofilaments. This mode of depolymerisation has long been observed thanks to nanometre-scale electron microscopy, but it lacked mechanistic explanation. Recent work addressed the structural basis of microtubule dynamic instability using cryo-electron microscopy (cryo-EM) at near-atomic resolution. This and other major insights into microtubule biology revealed by cryo-EM are reviewed by Manka and Moores in this issue (see pages 737–751). The atomic surface model was created in ChimeraX using experimentally determined tubulin conformations and was kindly provided by Szymon Manka (Birkbeck, University of London).
MAPs and motors on MT tracks
Tubulin conformational landscape and structural basis of dynamic instability
Emerging mechanisms of MT stabilizing agents
Beyond the lattice: MT end structures
Review Article| October 12 2018
Microtubule structure by cryo-EM: snapshots of dynamic instability
Szymon W. Manka;
Szymon W. Manka
1Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London, London, U.K.
Correspondence: Szymon W. Manka (s.manka@mail.cryst.bbk.ac.uk)
Essays Biochem (2018) 62 (6): 737–751.
https://doi.org/10.1042/EBC20180031
Revision Received:
James G. Wakefield, Carolyn A. Moores, Szymon W. Manka, Carolyn A. Moores; Microtubule structure by cryo-EM: snapshots of dynamic instability. Essays Biochem 7 December 2018; 62 (6): 737–751. doi: https://doi.org/10.1042/EBC20180031
The development of cryo-electron microscopy (cryo-EM) allowed microtubules to be captured in their solution-like state, enabling decades of insight into their dynamic mechanisms and interactions with binding partners. Cryo-EM micrographs provide 2D visualization of microtubules, and these 2D images can also be used to reconstruct the 3D structure of the polymer and any associated binding partners. In this way, the binding sites for numerous components of the microtubule cytoskeleton—including motor domains from many kinesin motors, and the microtubule-binding domains of dynein motors and an expanding collection of microtubule associated proteins—have been determined. The effects of various microtubule-binding drugs have also been studied. High-resolution cryo-EM structures have also been used to probe the molecular basis of microtubule dynamic instability, driven by the GTPase activity of β-tubulin. These studies have shown the conformational changes in lattice-confined tubulin dimers in response to steps in the tubulin GTPase cycle, most notably lattice compaction at the longitudinal inter-dimer interface. Although work is ongoing to define a complete structural model of dynamic instability, attention has focused on the role of gradual destabilization of lateral contacts between tubulin protofilaments, particularly at the microtubule seam. Furthermore, lower resolution cryo-electron tomography 3D structures are shedding light on the heterogeneity of microtubule ends and how their 3D organization contributes to dynamic instability. The snapshots of these polymers captured using cryo-EM will continue to provide critical insights into their dynamics, interactions with cellular components, and the way microtubules contribute to cellular functions in diverse physiological contexts.
cytoskeleton, cryo-electron microscopy, cryo-electron tomography, microtubule, MAPs, molecular motors
Since microtubules (MTs) were first observed in cells [1,2] and tubulin was first purified and proposed to be the building block of MT [3], electron microscopy (EM) has been a crucial technique for investigating MT molecular mechanism and functional context in cells and tissues. Early EM work helped define the organization of individual tubulin αβ-heterodimers, aligned head-to-tail within protofilaments (PFs) and linked via lateral connections between PFs to form the cylindrical, polar MT tube. Initial progress on MT ultrastructure–and the diversity of other oligomers and polymers that tubulin can form—was made using heavy metal staining [4–6]. However, the advent of cryo-electron microscopy (cryo-EM) [7] meant that MTs could be captured in their solution-like state [8], paving the way for decades of insight into their dynamic mechanisms and interactions with binding partners including molecular motors.
Early cryo-EM micrographs provided compelling 2D visualizations of these beautiful polymers (Figure 1). The ultra-fast freezing (vitrification) in cryo-EM sample preparation not only revealed the appearance of the MT lattice, but also enabled the conformational variability of dynamic MT ends to be captured [9]. MT ends are the major sites of dynamic transitions—a behavior termed dynamic instability [10]—and interpretation of tubulin conformations visualized in these regions still challenges the field (see below).
Unstained frozen-hydrated MTs stabilized with DCX and Taxol
Electron cryo-micrograph was obtained with a 120 kV microscope (Tecnai T12, FEI). Straight and curved MTs with a uniform diameter (13-PF architecture) are darker than ice (vitrified buffer). Some polymers are bundled, flattened or otherwise disrupted. In intact MTs, PFs are visible by glancing along their length and their number can be deduced from the striation pattern (so called moiré pattern) [8]. MT edges appear darker due to the presence of several PFs in the projection.
MTs polymerized in vitro from mammalian brain-purified tubulin (still the most practical source of tubulin) are built from a range of PF numbers, typically from 11 to 16. This contrasts with the predominance of 13-PF MTs seen in most eukaryotic cells [4,11]. Although different polymerization conditions can alter this PF distribution [12], how this is determined and its significance is still not understood. Nevertheless, the diversity of polymer architecture has helped define the rules by which the lattice is built [13], highlighting the presence of a lattice discontinuity/discontinuities—known as seams—in which the otherwise helical arrangement of tubulin subunits within the MT lattice is interrupted.
As well as allowing direct visualization of MTs, the 2D images collected in cryo-EM experiments can be used to computationally reconstruct the 3D structure of the polymer. An important early landmark was determination of the 3D structure of the tubulin dimer itself, not from cryo-EM images of MTs in the first instance but from 2D crystals of zinc-induced sheets of anti-parallel PFs [14] (see below). This work defined the PF structure and the positions of the exchangeable (E-site) and non-exchangeable (N-site) GTP/GDP-binding sites within the tubulin dimer, subsequently allowing the PF configuration within MTs to be defined [15].
The combination of cryo-EM imaging and 3D reconstruction has allowed binding site determination for numerous components of the MT cytoskeleton, including motor domains from many members of the kinesin superfamily of molecular motors, and the MT-binding domains (MTBDs) of dynein motors and a host of MT-associated proteins (MAPs) (Figure 2). In these experiments, the cylindrical geometry of a MT is exploited to determine the structure of a MTBD in its MT-bound conformation. Multiple copies of the MTBD of interest attach regularly along the lattice according to their particular binding site—e.g. kinesin motor domains bind every tubulin dimer, centered over the intra-dimer interface. The many views of the MT-bound MTBD present in each MT image are computationally averaged to reveal its 3D shape. In turn, this binding serves as a regular marker of the underlying α- and β-tubulin subunits within the lattice, thereby also revealing the position of the seam in the pseudo-helical MT architecture [16,17]. Recently, the availability of direct electron detectors has caused a resolution revolution in cryo-EM [18] and enabled the calculation of near-atomic reconstructions of a diversity of MT-binding protein complexes [17,19–24]. In tandem with software development [25], these data now enable the relatively subtle differences between α- and β-tubulin to be distinguished computationally, enabling near-atomic reconstructions of naked (undecorated) MTs [26], and of MTs decorated with unstructured or flexible MAPs, interacting with MT lattice via relatively short oligopeptides [22,24,27] (Figure 2).
MAPs and motor proteins studied by cryo-EM
Several structures of MTBDs or MT-binding polypeptide regions of MAPs and motor proteins have been determined in association with MT lattice by single-particle cryo-EM performed on decorated MTs. Atomic models of these structures are placed at relevant sites on the surface of an atomic model of MT lattice, all shown in solvent-excluded surfaces (SES) representation generated with ChimeraX [71]. The MT model was obtained by fitting a 6-dimer atomic model (PDB: 6EVZ) into near-atomic resolution cryo-EM map (EMDB: EMD-3964); the polarity of each MT is indicated by (+) and (−) signs. MAP models include: tau (PDB: 6CVN), WHAMM (PDB: 5X1G), NDC80/NUF2 (PDB: 3IZ0), EB (PDB: 3JAK), PRC1 (PDB: 5KMG), DCX (PDB: 4ATU), CAMSAP (PDB: 5M5C) and TPX2 (PDB: 6BJC). Motor models include two copies of an example typical (+) end directed kinesin motor domain (PDB: 5ND7) and four copies of the (−) end directed dynein MTBD (PDB: 3J1T) reconstructed together with MTs. The cluster of four dyneins assembled on dynactin and the MT was identified by cryo-ET [66], with lower resolution than the single-particle studies, which is indicated with isosurface representation of the experimental density (EMDB: EMD-7001). Structures of all dynein domains have been determined at high-resolution by both X-ray and cryo-EM studies not involving MT templates/tracks, hence not fitted into the density, except the dynactin/BICD part of the complex indicated for clarity. Kinesin motor domains are joined (dimerized) by the cartoon of a kinesin neck region and coiled-coil stalk region extending to cargo-binding elements. Similarly, BICD cargo adaptor is extended with a cartoon of cargo-binding elements.
These technical developments have also enabled visualization of the subtle structural transitions within the MT lattice driven by the tubulin GTPase activity and which drive dynamic instability [17,19,23,26]. These structural findings have provided vital insights into the molecular basis of dynamic instability, which is crucial for many MT functions. This includes the highly dynamic MT-based spindle machinery that drives cell division and which is targeted by widely used cancer chemotherapeutics. Here, we concentrate on in vitro studies of MTs using cryo-EM with a focus on molecular fundamentals of MT dynamics, and MAPs and drugs, whose activities modulate structural and functional characteristics of MTs.
MAPs are essential in mediating the physical and biochemical properties of MTs with a wide range of cellular functions. Some of them modulate these properties in vitro and in vivo, for example, by spatially and temporarily regulating MT nucleation, dynamics, stability or post-translational modification patterns ('tubulin code') [28] at various stages of the cell cycle. Motor proteins, on the other hand, are required for various mechanical tasks, such as intra-cellular transport or flagellar beating, using energy from ATP binding and hydrolysis. Different MAP and motor MTBDs characterized by cryo-EM through co-polymerization or decoration of MTs are presented in Figure 2 and will be briefly discussed.
MAPs with linear MT-binding motifs
Tau and its isoforms [29] were the first identified MAPs and, together with related MAPs such as MAP2, are largely unstructured. This has presented a long-running challenge to structural biologists to identify its MT-binding site. Lower resolution (∼30 Å) reconstructions yielded conflicting conclusions that tau/MAP2 bound on either the outer PF ridge [30] or in the MT lumen [31]. However, with direct detector data and more modern reconstruction algorithms, tau's binding mode on the outer surface of MTs has been precisely confirmed at high resolution [22]. Conserved oligopeptide repeats of tau stabilize MTs by binding in tandem across tubulin dimers on the PF ridge (Figure 2). Another critically important interaction of tau with MTs involves the negatively charged C-terminal tails of tubulin (also called E-hooks) [32], but because they are disordered, they remain obscure to structural analyses by cryo-EM. Hyperphosphorylation of tau favors its dissociation from MTs and aggregation into amyloid fibers found in Alzheimer's disease, the structures of which were also recently solved by cryo-EM [33].
TPX2 and WHAMM are examples of modular, multidomain and multifunctional MAPs. TPX2 is important for chromatin-mediated MT nucleation during the onset of mitosis [34]. It facilitates MT nucleation by binding the MT wall via two elements: the 'ridge' and 'wedge', which are flexibly linked in the middle region of the protein to simultaneously span longitudinal and lateral lattice contacts (Figure 2). Only the 'ridge' and 'wedge' are visible in the MT reconstruction due to flexibility of the rest of the TPX2 molecule. WHAMM is a WASP homolog associated with actin, membranes and MTs. It nucleates actin filaments and links them with MTs and membrane vesicles to remodel them into tubular structures upon transport from the endoplasmic reticulum to the Golgi apparatus [35,36]. Cryo-EM reconstructions revealed binding of an oligopeptide in the central coiled-coil region across α- and β-tubulin bridging two dimers in the MT lattice [27,37] (Figure 2), explaining how WHAMM's activities are structurally coordinated.
MAPs with globular MTBDs
Other MAPs bind to MTs via globular MTBDs. Intriguingly the calponin homology (CH) domain is shared among several different MAPs that bind MTs in quite different ways. End-binding proteins (EBs) autonomously and dynamically bind a region immediately behind growing MT (+) and (−) ends via their CH domain [38,39], an activity that causes the classic comet-like behavior of fluorescently labeled EBs in cells. EB CH domains bind preferentially to MTs grown in the presence of the GTP analogs GTPγS and GDP.BeF [40,41], suggesting that EBs track growing MT ends by recognizing a GDP.Pi-like structural state of tubulin within the MT lattice. The first pseudoatomic model of a CH domain bound to MTs was that of the fission yeast EB Mal3 determined at sub-nanometer resolution [40]. This structure revealed that the CH domain of Mal3 bridges PFs at the corner of four tubulin dimers, adjacent to E-sites of two β-tubulins, thereby suggesting a direct route for sensing the nucleotide state of MT lattice. More recently, a near atomic resolution reconstruction of the CH domain from the mammalian EB3 protein decorating GTPγS-MTs allowed atomic model refinement directly in the EM density, characterizing EB–MT interactions in more detail and shedding more light on GTPase-dependent conformational changes in tubulin (discussed more below) and the sensitivity of EBs to them [19].
Both NDC80 and NUF2—the MT-binding components of the NDC80 hetero-tetrameric kinetochore complex— contain a CH domain that can recognize both intra- and inter-dimer interfaces. Species specific variations have been reported [42], but the human NDC80 complex appears to bind MTs uniformly with a tubulin monomer repeat (Figure 2). The disordered cationic N-terminal tail of NDC80 is critical for high-affinity MT binding [43] and is the target of phospho-regulation by Aurora B kinase [44]. The sub-nanometer cryo-EM reconstructions of NDC80-decorated MTs revealed that this region mediates self-association of NDC80 complexes along PFs, shedding light on how they stay attached to spindle MTs while these MTs depolymerize, driving segregation of chromosomes during cell divisions [45–47].
The calmodulin-regulated spectrin-associated protein (CAMSAP)/Patronin family of proteins participates in organizing non-centrosomal MT networks important for cell division, polarization and differentiation by regulating MT (−) end dynamics [48,49]. The globular C-terminal CKK domain of CAMSAPs autonomously recognizes MT (−) ends and also binds to the MT lattice between two tubulin dimers at the inter-PF interface [50] (Figure 2), a binding site that lies 4 nm away from that of the EBs. Cryo-ET experiments provided insight into the 3D structure of the transition zone between the regular lattice and the more curved, sheet-like PFs at MT ends. The CKK MT (−) end preference was proposed to arise from CKK recognition of the intrinsic polarity by which tubulin curvature develops at MT ends [50].
The neuronal MT-nucleating and stabilizing protein doublecortin (DCX) contains two ubiquitin-like MTBDs separated by a 42-residue unstructured linker. It was shown to strongly promote 13-PF lattice architecture in vitro [51,52]. Via a single DC domain, DCX binds in the vertex of four tubulin dimers in the lattice [53] (Figure 2), the same binding site as EBs [40]. DCX stabilizes both longitudinal and lateral lattice contacts and is sensitive to the nucleotide state of the lattice [23]. The length of the linker between the DC domains would theoretically permit simultaneous binding of both N- and C-DC domains to the MT lattice, but due to their highly similar fold, current sub-nanometer resolution reconstructions have not yet clarified the extent of their individual involvement in this interaction.
The MTBD in PRC1 (protein regulator of cytokinesis 1) is elongated and projects from the MT wall [54]. It cross-links MTs by dimerizing via its N-terminal domains and binding to MTs via the C-terminal spectrin domains. This facilitates generation of anti-parallel MT arrays during mitosis. The spectrin domains bind on the crest of PFs like kinesins, each to a single tubulin dimer (Figure 2), determining the geometry of the resulting PRC1-MT arrays [21,55].
Kinesins and dyneins
Kinesins and dyneins are complex, multidomain and multimeric motors that navigate around the MT cytoskeleton. Globular ∼40 kD kinesin motor domains bind MTs and undergo ATPase-linked conformational changes that drive motor movement (Figure 2). Cryo-EM studies of motor domains from a wide range of kinesins have yielded key insights into their mechanochemistry [56–61]. The dynein motor (ATPase) domain is completely unrelated to kinesin and is a 6 AAA (+) ('ATPases associated with diverse cellular activities') ring separated from the MT track by a long coiled-coil stalk ending with its relatively small (∼10 kD) MTBD (Figure 2). Cryo-EM structures of MT-bound dynein MTBD [62,63] have shown that its MT-binding site is very similar to that of kinesins and is centered over the intra-dimer interface of the tubulin dimer. Larger complexes of cytoplasmic or ciliary dyneins bound to MTs have been studied by tomography (cryo-ET) [64–67]. An emerging theme in the way that MT-based motor transport is regulated is the response of kinesins and dyneins to MAPs present on the motors' MT tracks [68–70]. The reconstitution and structure determination of multicomponent motor, MAP-MTs, is an exciting future direction that offers structural insight into the cross-talk between various components of the MT cytoskeleton. Thus, we will gradually become equipped with more and more sophisticated tools to literally look at MT biology and its associated pathologies.
Over three decades since the discovery of MT dynamic instability [10], a full understanding of its mechanism is still lacking. We know that it is driven by GTP hydrolysis in the β-tubulin subunit within the MT lattice, but it is unclear why the GTP lattice is stable and forms a cap that supports MT growth, while the GDP lattice is unstable, resulting in MT catastrophe when the cap is lost (i.e. when hydrolysis outpaces growth). Two sets of structural observations are likely important aspects of this behavior: (1) X-ray and cryo-EM studies have shown that tubulin adopts a bent conformation outside the context of the MT lattice [9,72,73], suggesting that the lattice-induced straight conformation generates mechanical strain balanced by contacts formed within the lattice [9,74–77] and (2) the MT lattice compacts longitudinally by ∼2 Å per dimer following GTP hydrolysis. This was first shown in mid 1990s [78,79], and recently visualized in detail by high-resolution cryo-EM: MTs bound by the slowly hydrolyzable GTP analog GMPCPP maintain an extended lattice state while MTs in which GTP hydrolysis has occurred adopt a so-called compacted state [17,19,23,26]. These studies show that lattice compaction arises from shortening of the tubulin–tubulin inter-dimer separation as α-tubulin moves closer to the β-tubulin in the adjacent dimer in response to GTP hydrolysis.
Cryo-EM structures of the MT lattice in different nucleotide states provide structural snapshots of these dynamic polymers. However, it is important to bear in mind that, to date, most of the cryo-EM MT structures considered in the context of elucidating dynamic instability mechanisms have been determined in the presence of MAP or kinesin MTBDs [17,19,23]. Furthermore, a number of these binding partners were shown to also elicit specific changes in MT lattice conformation (Figure 3). One exception is DCX, which does not appear to strongly influence MT lattice parameters in any nucleotide state (Figure 3, compare DCX-decorated and undecorated lattices). Thus DCX—with its MT-nucleating and stabilizing properties—is a valuable tool for elucidation of purely nucleotide-dependent lattice transitions underlying dynamic instability [23]. Although such studies do not show how this compaction affects lattice energetics or strain, taken together they can be used to decipher aspects of tubulin mechanochemistry and deduce the structural basis of MT dynamic instability.
Modulation of MT-lattice conformation by MT-binding proteins and drugs
Top, schematic representation of various MT lattices used in studies addressing the mechanism of dynamic instability. Bottom, table summarizing influence of PF-number, nucleotide state (color-coded along the rows) and MT-binding proteins and drugs on major MT lattice parameters: the axial repeat distance of tubulin dimers (lattice spacing) and PF skew. *EB3 induces hydrolysis of GMPCPP leading to compaction of MT lattice—only a small subset of GMPCPP-EB3 MTs has been shown to exhibit the extended lattice conformation. The impact of different drugs on MT structure depends on whether they are present during MT assembly (pre) or are added after the assembly (post). The summary is limited to mammalian tubulin due to the lack of unambiguous characterization of these parameters in yeast [80–82] and other organisms; K, kinesin-1 motor domain.
Looking for the trigger of catastrophe
Considering that tubulin dimers in the lattice are under constant mechanical strain, they can be envisioned as loaded springs that are always poised for release. Therefore, the point at which they eventually do so—driving MT depolymerization—must be a point of imbalance between the lattice binding energy and the lattice strain energy. One can think of three possible scenarios causing such imbalance: the post-hydrolysis lattice compaction may (1) weaken the lattice contacts below the threshold of resisting the existing strain, (2) increase the strain beyond the resistance threshold of the existing contacts or (3) synergize both these effects leading to lattice contacts being overcome by the strain. The first cryo-EM reconstructions of MTs of sufficient resolution (∼4-5 Å) to reveal the structural details of lattice compaction involved kinesin-decorated 14-PF lattices in the GMPCPP (GTP-like) and GDP states. Lattice contacts were still relatively poorly defined at these resolutions, but the source of MT instability was attributed to strain within the MT wall due to changes at the longitudinal inter-dimer contacts on lattice compaction. A subsequent study compared kinesin-decorated 14-PF extended GMPCPP and compacted GDP lattices with the EB-decorated 13-PF compacted GTPγS and GDP lattices at near-atomic resolution. These comparisons identified in GTPγS and GDP states a distortion of a helix (H8) in α-tubulin next to the longitudinal inter-dimer interface, compared with the GMPCPP state [19]. In addition, the compacted post-hydrolysis GTPγS and GDP structures displayed α-tubulin rotation that led the authors to conclude that GTP hydrolysis induces strain. This idea points to scenario (2), listed above, as the underlying explanation for MT instability: the post-hydrolysis lattice compaction increases the strain beyond the resistance threshold of the existing contacts.
At the time of these first high-resolution studies, there were only a few MT structures suitable for accurate cross-comparison. As a result, comparisons between inherently different MT geometries (arising from PF number variation), conflated with binding effects of disparate MT-binding partners, such as kinesins or EBs, were inherent to the approach (Figure 3), making it challenging to attribute conformational changes in the lattice specifically to the nucleotide state. Since structural transitions associated with sequential steps of the GTPase action are subtle (in the range of ∼2 Å), they ideally need to be examined in structures with a single PF architecture and binding partner. We took advantage of DCX's ability to produce uniform 13-PF MTs, as introduced earlier, to obtain a 13-PF extended GMPCPP lattice and 13-PF compacted GDP lattice. We also developed a rapid MT polymerization and plunge freezing protocol exploiting DCX's robust MT nucleation activity that enabled us to capture the bona fide GDP.Pi intermediate state [23]. Comparison of the DCX-stabilized structures at near-atomic resolution unveiled uneven compression of α-tubulin upon lattice transition from GTP-like (GMPCPP) to GDP.Pi state (Figure 4). Such unevenly distributed lattice compaction is dictated by the geometry of the inter-dimer longitudinal contacts, which anchor the intermediate subdomain of α-tubulin (αI), leaving the N-terminal subdomain (αN) more translational and rotational freedom. The resultant αN shift and twist toward the MT (−) end and lumen tightens the longitudinal inter-dimer interaction, while loosening the lateral connection with the more restricted αI domain from the neighboring PF. The subsequent Pi release (GDP.Pi to GDP transition) causes further loosening of homotypic inter-PF contacts—this time between the β-tubulins—and further reinforces the longitudinal lattice contacts (Figure 4). This observation supports scenario (1) above: that dynamic instability arises from the combined two-step lateral weakening and longitudinal strengthening that renders the lattice unable to counteract the strain energy, triggering MT catastrophe via well-documented PF peeling [9,83]. However, it does not exclude the third possibility (scenario (3)), that the lattice strain also increases through those transitions as previously proposed [17,19].
Uneven compression of α-tubulin and the resultant strengthening of longitudinal and weakening of lateral lattice contacts
(A) MT extended lattice fragment (PDB: 6EVW) seen from the lumen and colored by tubulin subdomains: αI, βI, αN, βN–intermediate and N-terminal domains of tubulin α and β subunits, respectively. Tubulin dimer boundaries and lattice polarity are indicated, and longitudinal inter-dimer interface is framed in a perspective rectangle; C domains are not visible from this view as they project from the outside of the MT wall. (B) Top, αN subdomain movements after GTP hydrolysis (transition 1) and after Pi release (transition 2) relative to the αI subdomain; αN in the GDP.Pi state is semi-transparent blue and in the GDP state—gray. Outlines are included for convenient inspection of the movements; αN moves further than αI, as denoted with different sizes of the curved arrows. This causes tensions between the N and I subdomains (zigzag arrow), resulting in their loosening. Bottom, (+) end view of the β subunit's longitudinal interface in different nucleotide states, as color-coded and shaded by subdomains; to aid viewer's orientation, the rectangular frame is related to that in panel (A) by 90° rotation; αN moves further toward the (−) end compared with αI, as it has ample space, whereas αI extensively interacts (white shading) with the βN and βC subdomains of the preceding dimer. The GTPase (bolt symbol)-driven transitions broaden this longitudinal interface—as indicated with outlines and white arrows—through additional interactions with αN. Protein structures are shown with SES generated with ChimeraX [71]. The nucleotides are shown as white and heteroatom-colored sticks, and magnesium ion is shown as a ball.
The study of naked MTs—not decorated with any binding partners—by the Zhang and Nogales labs [26] presents a parallel approach for separating the effects of nucleotide and MT-binding partners on MT structure. The authors focused on reconstructions without imposition of pseudohelical symmetry and highlighted the role of the seam in dynamic instability, showing its deviation from cylindrical symmetry in the GDP state, but not in the GMPCPP or GTPγS states. From the observed ∼1–3 Å relative separation of PFs at the seam, the authors inferred that it is likely to crack open in the absence of the stabilizing GTP cap, triggering MT catastrophe. No other post-hydrolysis changes in any lattice contacts were described.
The GTPγS state—previously widely accepted as a surrogate of the GDP.Pi state—appears to have mixed characteristics relative to the natural nucleotide states (Figure 3). Its tubulin dimer conformation resembles that of the GDP state, but the MT lattice shows tendency for more left-handed PF skew compared with the GDP state [23,26], similar to that of the GDP-Pi state [23] (Figure 3). This may explain preferential binding of EB proteins to GTPγS-MTs and their dynamic MT end-tracking behavior, since EBs themselves induce a left-handed skew (Figure 3) and thus appear to have higher affinity for that particular lattice geometry. On the other hand, similarity of the GTPγS-tubulin to GDP-tubulin explains poor MT nucleation in the presence of this nucleotide analog [84].
The seam-centric structural model of MT catastrophe [26] and the more holistic model [23] are summarized in Figure 5. It is uncontroversial that the seam is the weakest link in the MT after lattice compaction due to the inevitable mismatch between heterotypic lateral contacts. The seam-centric model assumes overall increase in lattice strain after lattice compaction (scenario (2)), potentially corresponding to the greater conformational rigidity of the post-hydrolysis dimer [85] by an unknown allosteric mechanism. But what about truly helical MTs that do not have a seam? We know that in vitro experiments produce a range of MT architectures [8,13,86], including 15- and 16-PF helical MTs, but if dynamic instability relied solely on seam instability, it suggests that a hyperstable subpopulation of MTs would be present in dynamics assays, which has not been reported. This implies that dynamic instability does not strictly require the presence of a seam. On the other hand, a GTPase-dependent hierarchy of strength in all lateral contacts [23] can explain MT growth from GTP-tubulin (stronger lateral contacts) and the complete post-hydrolysis shrinkage (weakening of all lateral contacts) without invoking lattice strain upon compaction. These different scenarios can be computationally modeled [83,87,88], but new approaches to studying the energetic aspects of nucleotide-dependent tubulin transitions and properties in and out of MT lattice are necessary to fully unravel the remaining conundrums.
Proposed models of the structural basis of dynamic instability
Recent cryo-EM studies highlight two potential structural sources of dynamic instability: (i) lateral loosening at the MT seam (white arrow) in the GDP state, but not in the GTP-like (GMPCPP) state or the GTPγS state [26] that shares some characteristics with the GDP.Pi state [23] and (ii) two-step lateral loosening between all PFs in the MT lattice, first on transition to GDP.Pi state and then to GDP state [23]. Both scenarios are depicted at the top part of the figure using schematics of MT cross-sections in transversal plane. Due to PF stagger, the cross-section perpendicular to MT axis runs across both α- (lighter) and β-tubulin (darker), enabling depiction of sequential lateral loosening between the subunits (curved arrows). These observations can be summarized in a single coherent model of MT catastrophe shown in the bottom part of the figure, as the studies agree that the seam appears to be the weakest link of the lattice.
Conservation of MT dynamics mechanisms
It is also worth to mention that MT lattice characteristics, such as those presented in Figure 3, are not universally conserved across species. Because native yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe) tubulin can be purified in relatively large quantities, the dynamics of yeast MTs have been investigated in vitro [89,90,81]. However, they have so far proven more challenging to study by cryo-EM structural analyses with currently available image processing and 3D reconstruction algorithms due to their heterogeneity and unusual architecture compared with mammalian tubulin. This includes more twisted PFs [80] and potentially multiple seams—this latter idea had been previously used to invoke the existence of EB-dependent yeast MTs composed of mostly or completely seam-like heterotypic (α-β, β-α) lateral contacts (so called A-lattice) [91]. Multiple seams would lead to MT reconstructions with mixed α- and β-tubulin registers [82] and thus unreliable estimates of inter- and intra-dimer spacing in the lattice. Significant progress has been made in expressing tubulin recombinantly [92–94] and in isolating sufficient quantities of tubulin from different organisms to undertake structural studies [95]. Future studies will shed light on whether the mechanisms of dynamic instability described for tubulin from mammalian brain are more generally applicable.
Owing to their essential role in cell division, MTs have been targeted by various anti-tumor drugs. Some of them, such as vinblastine or colchicine, perturb MT polymerization and their mode of interaction with curved tubulin dimers was visualized by X-ray crystallography [96,97]. Others, such as Taxol, zampanolide and peluroside, block MT depolymerization. Recent high-resolution cryo-EM studies shed light on the distinct impact of each of these drugs on the MT lattice [98]. Taxol and zampanolide bind to the same pocket at the lumenal side of the β-subunit (Figure 6), and peluroside binds directly to lateral contacts but on the outside of the MT cylinder. MTs stabilized by Taxol or zampanolide are flexibile and characterized by slight deviations from a cylindrical shape along the MT. On the other hand, peloruside stiffens (regularizes) the lattice, eliminates the opening otherwise present at the seam in the GDP state (as discussed above) and overrides the flexibility from stabilization caused by Taxol in doubly bound MTs.
Mechanism of MT stabilization by Taxol
(A and B) Taxol-bound tubulin in the straight conformation associated into PFs forming either the zinc-induced sheet lattice [100] (A) or the MT lattice (B). Each panel shows a single-layer fragment of each lattice and MT lattice (B) is viewed from the lumen. Taxol stabilizes each lattice against cold temperature depolymerization and aging. The PFs in the sheet (A) run in an anti-parallel fashion and in MTs (B) in a parallel fashion, with a stagger determining helical rise of the MT cylinder. The PF polarity is indicated with (+) and (−) signs and with the arrows running along the PF ridges (outside crests in the MT context). Tubulin backbone and nucleotides are shown as wires, Taxol as sticks, and zinc as balls (Chimera [107]); PDB structure identifiers and the color-coding of features are indicated; this depiction uses fading to indicate depth. (C) Close-up view on the Taxol-binding site and its perturbation upon tubulin transition from straight (gray) to bent (magenta) conformation. Multiple straight tubulin structures (with and without Taxol) are aligned on the β-tubulin subunit together with multiple bent tubulin structures, determined by cryo-EM or X-ray crystallography. Tubulins are shown with backbone stick representation and Taxol with molecular surface representation. The PDB identifiers are listed according to tubulin conformation; 6BOC can be found in both columns, since it represents a complex containing two, Taxol-bound (straight) and unbound (bent) tubulin dimers. Straight-to-bent conformational transition moves the S9–S10 loop and the M loop toward Taxol-binding pocket (curved arrows), which severely clashes with Taxol density (asterisks). Only in the straight conformation the M loop is in the position compatible with forming lateral contacts (boxed with the broken line) in both MTs and zinc-induced sheets.
While the MT-stabilization mechanism of peluroside appears straightforward, since it relies on stabilization of lateral contacts, the mechanism of Taxol and its relatives is less obvious, and a long-running topic of debate. Some have suggested that Taxol allosterically prevents or reverses GTPase-driven lattice compaction, thus inducing the stable extended GTP-cap like lattice state [17]. This is in agreement with earlier work showing an increase in the tubulin axial repeat when MTs were formed in the presence of Taxol compared with GDP-MTs without Taxol [99]. Several subsequent cryo-EM studies have shown no or only minimal (∼0.4 Å) lattice expansion in Taxol-bound MTs [23,98] (Figure 3). Crucially, that minimal expansion was captured only when MTs were polymerized in the presence of Taxol (Figure 3, 'pre') and not when Taxol was added after MT polymerization (Figure 3, 'post'). Thus, most studies now agree that Taxol binding neither requires lattice expansion nor causes it in pre-established MTs.
Another important clue about Taxol's mechanism of MT stabilization comes from the earliest high-resolution tubulin structures solved by 2D electron crystallography of Zn2+-induced sheets of anti-parallel PFs of Taxol-bound tubulin, in which α/β-dimers adopt the MT-lattice-like straight conformation [14,100] (Figure 6). Taxol binding stabilizes such sheets: therefore, Taxol may have a role in stabilizing this straight conformation of tubulin, as also reported elsewhere [101], and this could be the basis of its MT-stabilizing mechanism. This idea is consistent with many X-ray structures of bent tubulin, showing profound conformational change in the M-loop (the major contributor to the lateral lattice contact) and the S9–S10 loop, lining the Taxol-binding site. The straight-to-bent transition of tubulin moves the M-loop away from the MT lateral contact and the S9–S10 loop inside the Taxol-binding pocket, occluding it (Figure 6). No Taxol-bound structures of bent tubulin have been reported; this reinforces the idea that bound Taxol sterically prevents the straight-to-bent transition and thus prevents PF peeling, thereby keeping the β-M-loops in place and stabilizing lateral lattice contacts. The enhanced lattice flexibility upon Taxol binding [102] is consistent with post-hydrolysis lateral loosening of the MT lattice [23]—Taxol binding does not tighten the lateral contacts, but rather holds every other M-loop along the lattice in the lattice-like conformation. The lattice heterogeneity and potential trapping of MTs in some distinct intermediate state when Taxol is present during MT polymerization may be important in reducing the affinity of certain MAPs, like DCX, for such MTs [23,103]. Interestingly, another MT-stabilizing drug, epothilone, binds at a similar site to Taxol [104], but it can also bind to bent tubulin [105,106]. Thus, different MT-stabilizing drugs may exploit different mechanisms to prevent MT depolymerization even while binding in the same site on tubulin.
Much of the forgoing work aimed at addressing the GTPase-linked structural transitions of tubulin at as high-resolution as possible, has focused on the MT lattice, the regularity of which makes it highly suitable to be studied using so-called single particle cryo-EM structural averaging approaches [108]. In contrast, the ends of MTs—where key structural transitions that correspond to dynamic events also take place—are very heterogeneous, and it is therefore challenging to access their 3D structure or structures. Early cryo-EM 2D micrographs clearly demonstrated this structural heterogeneity in vitro [9], while revealing a correlation between the overall phase of MT dynamics and the appearance of MT ends: the ends of MTs captured under assembly conditions showed evidence of PFs finishing unevenly in otherwise cylindrical-appearing MTs, while the ends of disassembling MTs demonstrated elaborately curling and apparently well-separated PFs. Later studies also described the presence in growth-promoting conditions of—sometimes very long—sheets of tubulin PFs, gently curving away from the MT axis while retaining lateral connectivity between PFs [109]. These evocative and compelling 2D images have informed decades of discussions and models concerning dynamic instability. Importantly, they emphasize that the MT end does not correspond to a single position or tubulin structure, but is rather a series of zones that correspond to sets of conformational transitions that capture aspects of dynamic instability.
Cryo-electron tomography offers an unbiased route to determining 3D structures—albeit at lower resolutions— without averaging [110]. MT ends are thus an ideal target and several groups have begun to examine MT ends in 3D by cryo-ET [111]. Visualization of MTs in 3D immediately provides a greater appreciation of the complexity of their structures and largely supports previous concepts derived from 2D images: that at least some extended sheets are seen at growing or stable MT ends, while PFs separate and curl away from depolymerizing ends [20,50,112,113]. However, a very recent and comprehensive study comparing MT ends from a variety of sources in vitro and in vivo and using both cryo-EM and freeze-substituted EM samples has reached a very different conclusion: PFs at the ends of all MTs—growing or shrinking—are all curved and not laterally connected [83]. Thus, the molecular structure of MT ends remains a topic of active debate. Even if a consensus is reached concerning their quaternary organization, without the structural resolution that comes from averaging, it is not currently possible to visualize the nucleotide state of the tubulin in each region of the MT end. Thus, a central and ongoing challenge for the future will be connecting the structure of MT ends to phases of dynamic instability and to the biochemical state of tubulin. This in turn has wide implications for the regulation of MTs by their cellular binding partners.
For all aspects of MT biology, the snapshots of these polymers captured using cryo-EM will continue to provide critical insights into the fundamental mechanisms of their dynamics, the way in which they are regulated by cellular components, and the way in which MTs are recruited to many cellular functions in diverse physiological contexts.
Cryo-electron microscopy allows microtubules to be captured and imaged in their solution-like state.
This approach has provided significant insight into their dynamic mechanisms and interactions with cellular binding partners.
High-resolution 3D structure determination of cryo-EM microtubule images using single particle image processing has allowed the binding sites of numerous microtubule-associated proteins and microtubule-stabilizing drugs to be precisely characterized.
Low-resolution 3D structure determination using cryo-electron tomography has begun to reveal conformational heterogeneity of microtubule ends but mechanistic consensus remains lacking.
S.W.M. and C.A.M. are supported by the Medical Research Council, U.K. [grant number MR/R000352/1].
The authors declare that there are no competing interests associated with the manuscript.
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Euler shows that every body has at least one set of three orthogonal principal axes. This paper gives rise to a branch of algebra.
E292 can be viewed or downloaded from Digitalisierte Akademieschriften und Schriften zur Geschichte der Königlich Preußischen Akademie der Wissenschaften, which includes serial publications of the Prussian Academy of Science in the 18th and 19th Centuries.


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Q: PHP - Find extra/missing class variables from stdObject I am fetching data from an API and it is being normalized to a class object. I am working on writing tests that will fetch data from the API and tell me if the API is sending new data for fields my class doesn't have, or extra fields in the class object that the API is no longer sending.
Lets say I have a User/Address Entity:
class User
{
    public $id;
    public $email;
    /**
     * @var Address
     */
    public $address;
    public $name;
}

class Address
{
    public $id;
    public $street;
    public $city;
    public $state;
    public $zip;
}

The data I am returned is JSON data when I fetch a user is:
{"id":1,"address":{"id":44,"street":"123 Main st","street2":null,"city":"Boston","state":"MA","zip":12345},"email":"test@foo.bar","first_name":"bob","last_name":"smith"}
Formatted:
{
   "id":1,
   "address":{
      "id":44,
      "street":"123 Main st",
      "street2":null,
      "city":"Boston",
      "state":"MA",
      "zip":12345
   },
   "email":"test@foo.bar",
   "first_name":"bob",
   "last_name":"smith"
}

Notice that the API is not sending the name field, but is sending first_name and last_name. The address field is sending street2 as well.
What is the best way for me to know that my User and Address class are missing fields, and that my User class has a field that is no longer being sent?

A: Assuming the decoded JSON is in $result as an array, just check the keys against the class vars.  For the Address class:
$new = array_diff_key($result['address'], get_class_vars('Address'));
$missing = array_diff_key(get_class_vars('Address'), $result['address']);

If you decode to objects then you can cast to an array:
$new = array_diff_key((array)$result->address, get_class_vars('Address'));
$missing = array_diff_key(get_class_vars('Address'), (array)$result->address);

Or get the object vars:
$new = array_diff_key(get_object_vars($result->address), get_class_vars('Address'));
$missing = array_diff_key(get_class_vars('Address'), get_object_vars($result->address));

$new here means present in the JSON and not in the PHP class and $missing means present in the PHP class but not in the JSON.
You should get the idea for the User class.  If this is more deeply nested then you can loop, check for an array key or object property named the same as one of your classes and repeat the tests.

A: The get_class_vars and property_exists functions should be useful. You can get an array of the public properties belonging to your class and check whether your object has them:
class User
{
    public $id;
    public $email;
    /**
     * @var Address
     */
    public $address;
    public $name;
}

class Address
{
    public $id;
    public $street;
    public $city;
    public $state;
    public $zip;
}

$obj = json_decode("{\"id\":1,\"address\":{\"id\":44,\"street\":\"123 Main st\",\"street2\":null,\"city\":\"Boston\",\"state\":\"MA\",\"zip\":12345},\"email\":\"test@foo.bar\",\"first_name\":\"bob\",\"last_name\":\"smith\"}");

$vars = array_keys(get_class_vars("User"));

$missing_vars = array();
foreach($vars as $v) {
    if (!property_exists($obj, $v)) {
        $missing_vars[] = $v;
    }
}
if (count($missing_vars) > 0) {
    echo "obj is missing these properties: " . implode(", ", $missing_vars) . "\n";
} else {
    echo "all properties present and accounted for!\n";
}

redpajama_set_name: RedPajamaStackExchange


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- do you want the whole house woken up?
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House! is a 2000 British comedy film written by Eric Styles and Jason Sutton and directed by Julian Kemp. The film stars Kelly MacDonald, known for independent films such as Trainspotting and mainstream releases such as Nanny McPhee.
"House" is the only single by Babes in Toyland that did not appear on an album by the time Babes in Toyland broke up. "House" was recorded for the Subpop Singles Club.
(Housed) (1) Descriptive of a piece or member fitted into another piece or member that has been modified, as by hollowing, gouging, or chiseling. (2) Enclosed.
(Housed) a woodworking term that describes one stairway or balustrade component that is let into a second component.
(Houses) Like the signs of the zodiac, the sky is divided into 12 houses. Each house encompasses a specific area of life such as love, career and health.
(Houses) or Mansions Receptacles, vehicles, stations; the visible planets are called the houses of the planetary regents. In astrology, the signs of the zodiac are called twelve houses: the sun and moon have one house each, and the other five planets have two houses each.
(HOUSES) Twelve areas of a birth chart that represent specific traits and areas of life. An astrologer interprets the planets that lie within houses to forecast events or read a birth chart.
(Houses) the bodymind energies move through the 12 houses on a daily cycle represented in theChinese Clock. The houses represent stages of human development, from birth to self-knowledge and death.
Astrological Houses are created by different methods (called House Systems). Every astrological chart has 12 different houses. Each house is related to a specific area of life or interest (for example the 1st house relates to sense of self, the 2nd house relates to money, and so on).


text:
New visual device could make stethoscope thing of the past
The old stethoscope could soon be replaced by a smarter device developed by researchers from IIT-Kharagpur and an institute here which converts sound into images to make it easier for doctors to diagnose lung diseases.
Developed by researchers of the premier technological institute in collaboration with the Institute of Pulmocare and Research (IPCR), Kolkata, the core technology has been patented after successful field trials.
"The lung sound analyser is non-invasive and very easy to use. It converts signals into images, which makes detection of a disease easier and in-depth. Detection is much more precise," IIT-Kharagpur, Director, Partha Pratim Chakrabarti told reporters here today.
Pulmonologist PS Bhattacharyya, who developed the device along with IIT electronic engineer Goutam Saha, said they have so far identified a digital signature of interstitial lung disease and once they come up with digital signatures of six or seven other diseases, it could be introduced in the market.
The device would have special relevance in rural areas where health infrastructure was inadequate, he said.
"Even a lay person will be able to use it and then transmit the image using a smartphone. The doctor sitting at a remote location will then interpret the disease after seeing the image," the pulmonologist said.
He said that the product was now at a very basic stage and may find application in newer areas gradually.
Saha said the embedded system could be produced at a cheap cost to ensure its mass utilisation.
This entry was posted in Doctor and tagged Doctor, Engineer, General, Professional, The Endoc.
First Electric Plane Flies Over English Channel
The future of travel? A tube called Hyperloop

redpajama_set_name: RedPajamaCommonCrawl


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All County, All the Time Since 2010 MAKE THIS YOUR PRINCE EDWARD COUNTY HOME...PAGE! Sunday, January 17th, 2021
White Pines project deemed complete; application moves to review
Administrator | Mar 11, 2014 | Comments 27
wpd Canada, the renewable energy company developing the 29-turbine White Pines wind project in South Marysburgh, received word from the Ministry of the Environment (MOE) that the Renewable Energy Approval (REA) application for the project has been
deemed complete. The process will now proceed to the technical review stage before it can be determined if a REA will be issued for the project.
The proposal has now been posted for a 60 day public review and comment period ending May 10, 2014. Comments are to be considered as part of the decision-making process by the Ministry of the Environment if they are submitted in writing or electronically using the form provided in the notice and reference EBR Registry number 012-1279.
"Meanwhile, in order to keep to potential project timelines, wpd continues the planning process for possible construction and operation activities," said Ian MacRae, wpd Canada president.
"This is a first but important step in achieving the Renewable Energy Approval for the White Pines project. Our staff and consultants have been working with various ministries and members of the community for nearly four years to ensure that the application meets the vigorous requirements of the permitting process".
MacRae said if he project is permitted to proceed, White Pines will feed an estimated 169,464,000 kWh annually into the electricity grid – equivalent to the average annual power usage of 9,683 homes.
"Electricity generated by wind projects has the ability to provide stability to electricity prices," added MacRae. "The fuel input – wind – is free, and is not subject to speculation, market fluctuations or political instability. Wind is a clean source of energy that will not be depleted, and does not discharge harmful emissions into the atmosphere."
Filed Under: Local News
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Doris Lane says:
Sunday, March 16th, 2014 at 3:06 pm
Susan when White Pines is up and running then South Marysburgh will be a place to leave
When Wynne has all the IWT's in the province up and running none of us will be able to afford to use hydro.
Re John he should not be spraying harmful substance into the air–it is going to kill the bees
BH says:
The White Pines project will place 12 turbines on the same Important Bird Area where Ostrander Point is situated. The current 550 metre set back from homes seems meager, given health concerns for people living near the planned 29 turbines.
By increasing the distance of iwts to 1.5 or 2.0 km away from homes and not placing wind turbines on environmentally sensitive areas, County citizens could work together with government in support of wind energy.
A setback to 1.5 or 2.0 km would reduce or eliminate property value, tourism, and heritage protection issues. It is not the government's job to maximize profit for corporations.
Instead, the Liberal government is causing societal conflict, jeopardizing the health of people, and harming nature.
Sunday, March 16th, 2014 at 11:12 am
Wind energy supporters are not supposed to quote the article commissioned by Environmental Defence because it is one-sided, but opponents have no problem quoting articles by the fossil fuel funded Fraser Institute? Good job at being credible.
Sam and John Thompson, I so appreciate you contributing the facts about renewable wind energy while braving this anti-wind site!
Hopefully when White Pines is up and running, all this negativity will be blown away.
Chris Keen says:
Sunday, March 16th, 2014 at 9:35 am
Another interesting article on Ontario's "Green" Energy Plan by Robert Lyman:
"The Ontario government has to date committed almost $60 billion over the next 20 years to building industrial wind turbines and solar power generators. In the case of wind turbines, the construction of hundreds of plants across the province has given rise to major conflicts between those who stand to benefit from the huge electricity ratepayer subsidies and those whose health and property values are threatened. Ontario's electricity generation capacity is already 40% above the peak requirements, and yet the provincial government continues to contract for more power. When those concerned about the economic, social and health impacts of adding so much "green" energy complain, they are often confronted with an argument expressed with almost religious conviction. Supporters of green energy insist we need it to "save the planet" from the threat of climate change…."
"The Fraser Institute published a report in 2013 entitled, "Environmental and Economic Consequences of Ontario's Green Energy Act". Here is an excerpt from that report:
"Electricity supply is divided into base-load capacity, which comes from sources like hydroelectric and nuclear that deliver a fixed amount of power that cannot easily be adjusted up or down on short notice, and peak capacity, which can be scaled up and down as system demand changes through the day. Ontario power demand currently averages about 18,000 MW and reaches a maximum annual peak of about 26,000 MW. Using figures from the Ontario Power Authority and the Independent Electricity System Operator, the Provincial Auditor General projects average demand to decline to about 16,000 MW and peak demand to fall to about 24,000 MW. Nuclear and hydroelectric facilities alone currently provide 18,000 MW of base-load capacity. In addition, Ontario has 9,500 MW of gas capacity as well as 4,500 MW of the coal-fired power plants much of which is unused. The AGO estimates Ontario will have at least 10,000 MW of surplus generating capacity through 2025."
In other words, Ontario's electrical generating capacity already so far exceeds needs that the coal-burning power plants (the principal sources of GHG emissions) could have been shut down without adding a single new wind or solar plant."
The rest of the article is found here:
http://freewco.blogspot.ca/2014/03/can-ontarios-green-power-program-save.html
@John Thompson – Here's Parker Gallant's take on the Globe article you cite.
" Environmental Defence, one of those environmental charities being investigated by the CRA, released a report March 14, 2014 prepared by Power Advisory. The consulting firm is where Jason Chee-Aloy formerly of the Ontario Power Authority (OPA), now works. Mr. Chee-Aloy's clients at Power Advisory include Samsung, Pattern Energy Group and NextEra; interestingly, his role at the OPA was Director of Generation Procurement, where he was responsible for procuring over 13,000 MW of generation.
With that background in mind, it is easy to assume that Power Advisory's report commissioned by Environmental Defence would weigh in on the side of wind and solar generation, and come to a conclusion that would put those two sources of power generation on the climate change pedestal.
That is exactly what Power Advisory's report did. And it did so by completely ignoring other facts associated with wind and solar power.
The Globe and Mail opened its article on the report with this: "The cost of green energy has a relatively small impact on residential electricity bills in Ontario, a new study to be released Friday suggests, and that will not change significantly even as renewables take up a bigger portion of power generation.That should be a lesson for other provinces considering the shift to renewables, says the study's sponsor, Environmental Defence Canada, because it shows Ontario's pro-green energy plan is not as costly as some critics claim."
The rest of his comments are here:
http://freewco.blogspot.ca/2014/03/environmental-defence-report-on.html
John Thompson says:
http://www.theglobeandmail.com/report-on-business/green-energy-costs-minimal-for-consumers-study-says/article17484685/#dashboard/follows/
Gary Mooney says:
Daily peak demand is easily managed in energy generation, except when it reaches maximum available supply — most likely during the hottest days of summer due to air conditioner load. Ontario's available supply is way above peak annual demand.
On other days, peak demand is handled by intermediate generators which are dispatchable — i.e. output is readily adjustable. The most versatile / quick to adjust are hydro and natural gas.
The least useful power generators are wind turbines, because they produce the most power when least needed (spring and fall, and at night) and can't be relied upon when additional power is needed (e.g. summer).
In this regard, solar is more useful than wind in summer, and it doesn't produce during the night. But the cost of solar is about six times the blended cost of other sources.
With at least 75% of our power produced by sources that don't pollute and don't generate CO2 (nuclear and hydro), Ontario has one of the cleanest supplies of power in the world. If the U.S. and China were to meet our standards, the problem of pollution and excess CO2 production would be solved.
Saturday, March 15th, 2014 at 8:20 pm
Every day has it's peak demands. In the great lakes region, the wind tends to be light first thing in the morning and pick up during the morning and into the afternoon. Those of us who do field spraying live with this reality. This would explain why the system operator notes a downward pressure on peak demands but the armchair quarterbacks have not.
Saturday, March 15th, 2014 at 10:27 am
John Thompson said: "Although a statement of the obvious, the ISEO recently stated that renewable wind and solar power has put downward pressure on peak demands on the electricity system without impacting reliability."
In any given year, Ontario's peak demand is typically during the hottest days of summer (due to air conditioning load), during which time the wind hardly blows.
Throughout the year, wind blows more during the night than during the day, which does nothing to reduce peak loads.
The words "wind turbine" and "reliability" should never be used in the same sentence.
Saturday, March 15th, 2014 at 9:20 am
Here's an absolutely brilliant letter by Christine Hall to the MOE that sums up all the reasons why this madness should stop.
http://ccsage.wordpress.com/2014/03/14/pec-resident-pulls-no-punches-in-letter-to-moe-re-amherst-island-36-turbine-wind-project/
Pamela is correct.
The reason we are in this IWT mess is because of big money, big business and big oil.
The electricity business is controlled by the \ceo of big business and government is afraid to go against them
if you think his is a democracy -think again
Somewhere on face book is some little Spanish towns they want to give away–looks good and anyway PEC oounty used to be called "Little spain"
@John Thompson – If what you say is true, then the "Delivery Charge" line, which on my Hydro One bill always exceeds the charge for electricity used should drop significantly. We both know that isn't going to happen. Except for the landowners, there is absolutely zero benefit for Country residents from these monstrosities.
Mark, I didn't say that there were only slight increases to electricity rates, I said "slight increase in electricity prices due to the integration of renewable energy".
Yesterday's articles in The Star and The Globe And Mail:
http://www.thestar.com/business/2014/03/14/renewable_energy_not_to_blame_for_high_bills_report.html
http://www.theglobeandmail.com/report-on-business/green-energy-costs-minimal-for-consumers-study-says/article17484685/
Pamela Stagg says:
Somehow in this argument, Niagara Falls seems to have been overlooked. There it is, with the hydro infrastructure all built, able to supply electricity at 5 cents per kilowatt hour. But the Ontario government, in its (lack of) wisdom, has decreed that when wind power is being generated at more than double that cost, water may "have" be diverted away from the Niagara power plant in order to give wind power producers first dibs at our grid.
It would be cheaper to buy Quebec's surplus hydro-electricity than it would be to build more wind power. Those who favour renewable energy should be satisfied, since hydro-electricity is the original green energy.
I don't have a degree in economics, but if I'm offered a commodity at two different prices, I'll take the cheaper version. The Ontario government is doing exactly the opposite, choosing super-expensive wind energy over cheap hydro-electricity and driving our industry to Quebec. And those of us who don't have the option of moving to Quebec or the United States are going to be paying through the nose.
Read all the comments with great interest.
Check Gary Mooneys comments seem to be closer to mark.
Friday, March 14th, 2014 at 11:00 pm
I don't know where you are getting your electricity from but if you can claim they are slight increases I would like to know your supplier as I am sure millions other would. Projections of an additional 40 per cent increase the next 4 years to support the payments to foreign companies for our renewables!
Friday, March 14th, 2014 at 9:35 pm
Enbridge has requested a price increase of 40% for natural gas. The price of propane (if you can even get it) has skyrocketed. Fuel oil has increased over 15% between September and January. Municipal water and sewer rates have become difficult to swallow. Municipal taxes increase several percent per year. But all of this is OK because the slight increase in electricity prices due to the integration of renewable energy is the only thing that is making our lives financially difficult.
At least the increasingly more expensive fossil fuels offer the advantage of global warming to help offset our future heating needs.
"Downward pressures on peak demand". So we can look forward to reduction in energy costs right. Hmmmm.
Wpd's power would connect to the high voltage transmission line at the Elmbrook station. As electrons take the shortest route to demand points, the County would get supplied first and any surplus will move into the provincial grid. Ostrander Point if built will connect to the local distribution side of the system so the power will be locked into the County for our use. Local power production has the same effect on the grid as reduced demand which translates into conserving gas at the gas plants.
Although a statement of the obvious, the ISEO recently stated that renewable wind and solar power has put downward pressure on peak demands on the electricity system without impacting reliability.
County Steve says:
It's funny how the pro- and anti-wind people are sold on their own propaganda. Perhaps we should read each others!
Nothing will change Sam's mind, until we're 10 years down the road, and we get to offer a lame "I told you so." After our wallets are empty and our ground is completely covered.
Sam seems to be up on the facts, so how about some info about how natural gas is needed as a backup/startup source for wind generators?
How about some facts on how the province of Ontario spends more than any other province or state in all of North America … which clearly is going to increase to laughable, and painful, amounts.
And if you think wind will replace nuclear in Ontario, or even gas-powered turbines, then you got your engineering degree out of a cereal box!
Dan Wrightman says:
Thursday, March 13th, 2014 at 9:17 am
@Sam Google "Generators Output and Capability Report" Under each wind generator if the actual Output is lower than the Forecast output than curtailment is occurring and Ontario is paying the wind developer to not produce wind energy. The IESO switched reporting to Forecast from Capability which allows indication of curtailment. Today the Greenwich wind plant has been paid to not produce wind energy for most of the morning.
Wednesday, March 12th, 2014 at 9:37 pm
Keep in mind that we are comparing new generation to old generation. Once Pickering Nuclear is mothballed and new generation is needed, do you really think that conventional generation (new nuclear or new gas turbines) will be willing to provide power for the same rate that the old stations are accepting? Also, I see now that the price of power in Ontario is up to $89.88 per mWh. Getting closer to the wind rate. Finally, you stated that "Ontario has been paying some wind developers to not produce wind energy for most hourly periods today." I would like to have a look at that information. Do you have a link to a site that shows when wind producers are paid not to generate? Thanks.
@Sam and the ratepayer loses for every MW of wind energy produced. The hourly Ontario energy price today has ranged from a low of $9.97MW to a high of $44.46/MW. Meanwhile FIT contracted wind energy (like the White Pines project) is paid $135/MW. To add even more salt to the wound, Ontario has been paying some wind developers to not produce wind energy for most hourly periods today.
Wednesday, March 12th, 2014 at 12:00 pm
According to Sygration, right now wind is producing 10% of Ontario's power. 1.86gW out of 18.6 gW total. It is only the 10:00 report, but so far wind is producing more more power every hour today than the total of all the gas fired generators in Ontario. Blown away!
Wednesday, March 12th, 2014 at 11:30 am
@ Gary Mooney – The same New York state that is actually courting businesses in Ontario to relocate there in order to take advantage of their cheap electricity rates! Utter madness.
If built, wpd's 169,464 MWH fed into the grid will be 100% unneeded, due to Ontario's oversupply of electricity.
We consumers' will pay wpd $23 million per year (at $135 per MWH) and Ontario will sell the unneeded power to neighbouring jurisdictions for $5 million per year (at $30 per MWH), for a net loss to us of $18 million per years.
Oh, and the 9,683 homes that wpd mentions? They'll actually be homes located in New York State, which will have gratefully accepted the cheap power offered by Ontario.
Alternatively, wpd's power will supply manufacturers in New York State, including some that will have relocated from Ontario to access affordable power.
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The United Arab Emirates (UAE) receives an arid climate ranging from arid sub-tropical regions on the coast to large areas of desert inland. Summers in the area are invariably uncomfortable with excruciatingly high heat all over the federation and outrageous humidity in coastal areas. Winters are a great relief and certainly the best time to visit the federation: still hot but pleasantly so. Rainfall is low at all times and very erratic from year to year; the whole area is regularly in a state of drought. Violent sandstorms are common throughout the year. The mountains in the southeast receive a slightly cooler climate year round.
Summer, from April till October, is unbearable. Inland regions receive average highs soaring from the mid 30s up to the low 40s and can spike up to 50°C; Al Ain, which is in the interior of the federation sitting in the northeast of the state of Abu Dhabi, sees an average high of 43°C in July. It is actually an oasis city, nicknamed 'Garden City', but is still affected by the dry desert air surrounding it. Coastal regions have this horrendous heat tempered very slightly by the sea; Abu Dhabi, the city on the central Gulf coast, receives an average high of 41°C in July. However, coastal regions are distinctly more unpleasant than desert regions in the summer as humidity soars above ninety percent while it stays very low inland. Coastal summers are pretty sweaty affairs; if you think you can survive without air-conditioning you're probably crackers. If you visit during this season you should pack some light layers in preparation for the extreme cold of the hotels and shopping malls that you are likely to prefer to the intense heat, no matter your views on the importance of cultural exposure. Night times cool off to a much greater extent in the desert due to insolation, though because of the desert's higher starting point, 30°C is around the normal night time low for most areas of the federation in the peak month of July; that's right: 30°C is the average low. Madness.
Any rain falling in this season is usually concentrated on the Al Hajar al Gharbi mountains in the southeast and falls in short but heavy thunderstorms. These mountains act as a barrier to rain-bearing weather systems; blocking them, the mountains force clouds to form and precipitation to fall on them, so depriving the rest of the federation. For the great majority of the summer the northwest shamal wind prevents rain from reaching the federation. The heat of the federation is heightened by the khamsin wind from the south, and the humid south-eastern sharqi wind that blows at the end of the summer and is said to make coastal regions 'particularly unpleasant'; as if things could really get any worse. All of these winds whip up regular sandstorms which are as dangerous as they are inconvenient. These sand blizzards eat away at every available surface and can reduce visibility to mere metres. It goes without saying that getting sand in your eyes stings, especially when travelling at such high speeds.
Dubai, the fastest growing city in the world.
The positive side to a UAE summer is that rain is unthinkable; the sun shines for upwards of ten hours per day; you'll sweat all of your toxins out in a about three seconds and the sea gets up to 33°C. The penultimate plus point is not backed by scientific fact.
Winter, thank God, is cooler. It is a short-lived blessing that soothes the federation from November till May. Across the federation average highs fall to the balmy mid 20s and the humidity relents. Night time temperatures drop to a delightfully cool 14°C in January and February on the coast, while interior regions get positively chilly with temperatures regularly dropping below 10°C. The shamal wind continues to blow in winter and helps to cool the federation. The khamsin also persists in stirring up sandstorms. The sun shines a little less but most would agree that eight hours per day is more than enough when the alternate, sunnier, season is so beastly in other respects. Rainfall is always rare and very erratic to the point that it can be said that 'it rains most years during winter', meaning that sometimes it doesn't. February and March are the wettest months, during which northerly regions see around 50mm of rain. When rain does fall, it falls in torrential downpours. Hard-baked earth, impermeable concrete and loose sand are not great surfaces for dealing with rain. It soaks straight through dunes and sits on top of earth to be evaporated by the sun in moments. In urban areas it causes flash flooding which can lead to mud slides.
Dubai city lights over the marina.
The federation is made up of seven states: Dubai, Abu Dhabi, Fujairah, Ajman, Sharjah, Um al-Quwain and Ras al-Khaimah. It sits on the southeast Persian Gulf coast of the Arabian Peninsula in the southwest of Asia. It also has a short eastern coast on the Gulf of Oman. Saudi Arabia and Oman share the federation's land borders. The country is mostly desert. The Al Hajar al Gharbi mountains stretch along the southeast border with Oman and salt pans, large areas of desert covered in salt and minerals, surround the coast, stretching far inland. The beaches are white and the waters clear turquoise, shallow and full of coral.
If travelling to this part of the world, considerations other than the weather must be made. The ex-pat community is segregated and the local culture is very different. Laws are very conservative, varying from state to state, and in many areas the human rights situation is in a deplorable mess. Luckily for white people, racism in favour of those with lighter skin is widespread and these factors are unlikely to have much affect further than shrugging on a long-sleeved top or slipping a cheap ring onto the ring fingers of unmarried couples.