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What To Do
The Breeders' Cup has the Bluegrass bursting with excitement, but not all the thrills will be at Keeneland. Explore all Lexington has to offer and the special celebrations happening around town during Breeders' Cup Festival WeekBreeders' Cup Festival VisitLex
Breeders' Cup Spotlight
Maker's Mark Distillery Tour
As the official and preferred bourbon partner of the 2015 Breeders' Cup, Maker's Mark invites you to make the most of your trip by enjoying a complimentary tour of the Maker's Mark Distillery while you're in town for the races. We're only an hour's drive from Keeneland. When you show us your Breeders' Cup ticket, you can see all the sights at our historic distillery, meet some of the friendly folks of the Maker's Mark family, enjoy a personal, up-close look at how we hand-make our bourbon and even hand-dip your own bottle. We hope to see you soon!Maker's Mark Distillery
3350 Burks Spring Road
Loretto, KY 40037
(270) 865-2881
Maker's Mark Distillery Tour
South Hill Gallery
The South Hill Gallery, established in 1971, has evolved from a custom frame shop selling limited edition prints into a gallery that shows unique and original art from all over the U.S. and abroad. Visit the Gallery to view and purchase the official 2015 Breeders' Cup poster by Steve Penley and a custom-designed Breeders' Cup silk scarf.1401 Versailles Road
Lexington, KY 40504
(859) 253-3885
Stuart Mercer Gentleman's Shoppe
Stuart Mercer focuses on custom luxury clothing as well as bringing unique, quality sports and casual wear to the Bluegrass and beyond. Stuart Mercer is proud to carry the official Breeders' Cup tie, custom designed and made from the finest silk for the 2015 World Championships.119 Clay Avenue
Lexington, KY 40502
(859) 309-0357
Exhibits and Tours
Headley-Whitney Museum
The Headley-Whitney Museum was founded in 1968 by jewelry designer George Headley and his wife, Barbara Whitney, and is noted for its collection of jewelry, bibelots, and dollhouses, along with a library and rose garden. The Museum features two special exhibits this fall: "Treasures from the Farm," showcasing the beautiful artwork and trophies housed at some of the area's most famous horse farms, and "Highlighting the Championships: A Breeders' Cup Collection," a collection of memorabilia from the first 31 years of the Breeders' Cup.4435 Old Frankfort Pike
Lexington, KY 40510
(859) 255-6653
Keeneland Breeders' Cup Tours
Celebrate the 2015 Breeders' Cup World Championships with an insider's look at the rich connection between Keeneland and the Breeders' Cup. Learn about the history surrounding two of racing's most iconic names as they come together for the first time! Tours are available through October 23 on Tuesdays, Thursdays, and Fridays at 8:30 a.m. Visitors are encouraged to purchase tickets in advance through the Keeneland website.4201 Versailles Road
Lexington, Kentucky 40510
(859) 288-4142
Horse Farms Tours
Join the Breeders' Cup Festival for a rare opportunity to experience "behind the scenes" tours at Kentucky's iconic stud operations and nurseries, world-renowned veterinary clinics, and more. Hear the stories of Breeders' Cup champions, and meet the sires, dams, sisters and brothers of this year's Breeders' Cup runners — and maybe the champions themselves! Choose from various tours running from Sunday, October 25 to Wednesday, October 28.Various Locations
Old Friends
At Old Friends Thoroughbred Retirement Farm, the horses may not be racing anymore, but they are winners every day. A "living history museum of horse racing," Old Friends allows fans to get up close and personal with such stars of the turf as 1997 Kentucky Derby winner Silver Charm, three-time Santa Anita Handicap victor Game on Dude, Breeders' Cup Champions Gulch and Amazombie, and Hollywood Gold Cup winner Rail Trip.1841 Paynes Depot Road
Georgetown, KY 40324
(502) 863-1775


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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.
Defects in protein folding are associated with a wide variety of human diseases, including cancer and amyloid diseases such as Huntington's, Parkinson's, and Alzheimer's diseases (Gregersen et al, 2000; Dobson, 2004). The eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) folds ~5–10% of newly synthesized cytosolic proteins, including many essential proteins such as actin, tubulin, and cell-cycle regulators (Rommelaere et al, 1993; Chen et al, 1994; Thulasiraman et al, 1999; Spiess et al, 2004; Yam et al, 2008). The TRiC conformation induced by ATP hydrolysis is closely associated with the productive folding of the substrate (Meyer et al, 2003).
Chaperonins have diverged into two structurally and mechanically distinct families: prokaryotic group I chaperonins such as GroEL–GroES from E. coli (Xu et al, 1997; Bukau and Horwich, 1998) and group II chaperonins, which are further divided into archaeal types (including the thermosome, Ditzel et al, 1998; KS-1, Shomura et al, 2004; and Mm-cpn, Kusmierczyk and Martin, 2003a, 2003b; Reissmann et al, 2007), and the eukaryotic chaperonin TRiC (Frydman et al, 1992). Chaperonin subunits share a common architecture, that is, each consists of three domains (apical, intermediate, and equatorial). Unlike GroEL, which employs an additional cofactor (GroES) acting as a detachable lid (Xu et al, 1997; Bukau and Horwich, 1998), group II chaperonins have a built-in lid formed by a protrusion at the tip of the apical domain of each individual subunit (Ditzel et al, 1998). In addition, GroEL and archaeal chaperonins consist of 1–3 types of subunits, whereas each ring of TRiC contains eight different subunits sharing 27–39% sequence identity (Archibald et al, 2001; Cong et al, 2010). The hetero-oligomeric nature of TRiC implies an inherently asymmetric arrangement among the subunits; however, little is known about how such structural organization is related to its functional activity. Notably, the eukaryotic TRiC has unique functions that the archaeal chaperonins cannot perform, despite their similar architecture. For instance, many TRiC substrates cannot be folded by any other chaperonin (Tian et al, 1995; Frydman, 2001; Hartl and Hayer-Hartl, 2002). This suggests that TRiC must have unique specificity and properties required for maintaining protein homeostasis in the eukaryotic system.
There have been several cryo-EM studies of TRiC in the apo and a variety of nucleotide-containing states (Llorca et al, 1999, 2001; Booth et al, 2008; Cong et al, 2010). The 4Å cryo-EM structure of the closed state suggested a specific model for the 8-subunit arrangement (Cong et al, 2010). Recently, two crystal structures of TRiC bound with tubulin or actin and ATP analogue were reported (Dekker et al, 2011; Munoz et al, 2011) and suggested an alternative 8-subunit arrangement different from the cryo-EM model. Therefore, the true ordering of the eight subunits in TRiC complex remains uncertain.
The present investigation is aimed at determining the quaternary structure changes undertaken by TRiC during its ATPase cycle. We report five cryo-EM maps of TRiC in the apo state and the chemically distinct nucleotide-containing states throughout the ATPase cycle. We did not impose symmetry in the 3D reconstruction process, and unlike other group II chaperonins, this study revealed a surprising degree of asymmetry in the conformation of the open, nucleotide-free state. Our composite structures have revealed a distinct interaction pattern of the 16 TRiC subunits in each of these biochemical states, led to a plausible mechanism of lid closure and could illuminate the positive and negative cooperative phenomena occurring in TRiC upon ATP hydrolysis.
To investigate the structural mechanisms of TRiC conformational transitions during its ATPase cycle (Figure 1), we carried out single particle cryo-EM studies of bovine TRiC in well-defined biochemical states: apo (nucleotide-free), ATP-bound (mimicked by non-hydrolysable ATP analogue AMP-PNP), ADP-bound, and two ATP-hydrolysis transition state analogues induced by ADP-AlFx and ATP-AlFx, respectively (Meyer et al, 2003). Each nucleotide state was obtained by incubation of TRiC with the respective nucleotide analogue and buffer (see Materials and methods).
As the first step of the analysis, we carried out reference-free 2D image analysis on the end-on view images to evaluate whether there is any obvious conformational heterogeneity in each state. TRiC–ADP-AlFx showed two drastically different 2D image patterns, suggesting structural heterogeneity. One has the characteristic appearance of a both-ring open conformation (Supplementary Figure S1A) while the other shows a unique twisted pattern of all the subunits (Supplementary Figure S1B). The 2D images of the other states appeared more homogeneous. Therefore, a multireference refinement procedure in EMAN1 (Chen et al, 2006) was adopted for the TRiC–ADP-AlFx data set, while the standard refinement procedure was applied to the other states.
Since the eight subunits of TRiC are all different, no symmetry was imposed in the reconstruction process for all the five states presented here (Figures 2 and and3),3), allowing us to assess structural differences among the eight distinct subunits. The closed ATP-AlFx state was previously determined at 4.7 Å resolution (Cong et al, 2010), while the resolutions of the other four states were found to be 10.5–13.9 Å (Supplementary Figure S2). This resolution limit can be attributed to preferred particle orientation on the cryo-EM grid, continuous conformational variations, and/or subtle structural heterogeneity in TRiC, which have been found in cryo-EM studies of group II chaperonins (Clare et al, 2008; Zhang et al, 2011).
The end-on view of apo-TRiC reveals an obvious asymmetric pattern among its eight distinct subunits (Figure 2A). However, we cannot determine the subunit identity in this map determined at 10.5 Å resolution. Nevertheless, the structural features of the subunits are distinct enough to annotate them from a1 to a8 for one ring and a′1 to a′8 for the other ring. A cylindrical 'unwrapping' was applied to the map, and exhibits a pseudo two-fold symmetry between the two rings (Figure 4A). This symmetry was confirmed quantitatively by the rotational correlation analysis between the two rings (Figure 5A). This pseudo two-fold symmetry is located at the inter-ring interfaces between subunits a1 and a′1, and a5 and a′5 (Figure 2A). Therefore, both of the densities (a1 and a′1) should correspond to the same CCT subunit; and the same applies to a5 and a′5. The presence of the pseudo two-fold axis in this symmetry-free reconstruction reinforces our previous observation in the 4.7Å resolution cryo-EM structure of TRiC–ATP-AlFx (Cong et al, 2010). Therefore, the subunit ordering is identical in both rings but arranged in opposite directions as viewed from the side (Figure 4A). Since the question of subunit ordering of TRiC still remains uncertain (Cong et al, 2010; Dekker et al, 2011), we will only refer to the subunits in the complex as a1–a8. Our conclusions are valid for both proposed models, since both agree on the existence of an inter-ring two-fold symmetry axis.
In the apo-TRiC map, both a1 and a′1 subunits display a significantly weaker density than all the other subunits (Figure 4A). This structural characteristic may be caused by the extremely dynamic nature of this subunit as shown in a movie generated from a superposition of a set of reference-free 2D averages of end-on views (Supplementary Movie 1A). Such subunit flexibility is analogous to a large crystallographic B-factor, making a high-resolution structural determination of the apo state impractical (Zhang et al, 2010). Contrary to a1 and a′1, the densities of both a5 and a′5 are better resolved in both rings, and thus more rigid (Supplementary Movie 1A; Figure 2A).
Another characteristic subunit in this apo-TRiC map is a3, which appears to tilt outward from the central chamber (Figure 5B) and lean towards one of its neighbouring subunits (a4), thus separating it further from the other neighbouring subunit (a2) (Figures 4A and and5B).5B). The unwrapped displays (Figure 4) show the relationship between the subunit pairs across the rings, for instance, a3–a′7, a4–a′6 etc. Figure 4A shows that the line between subunit a3 in one ring and subunit a′7 in the other ring has a larger tilt angle and is less parallel (Figure 4A) to the lines joining other subunit pairs (e.g., a2 and a′8).
Based on the above observations, it is tempting to postulate that the conformations of the very dynamic subunit (a1) and/or the outward tilting one (a3) might make them more likely to encounter the substrate proteins in solution and be critical for substrate recruiting of TRiC.
The distinct open conformational states of TRiC occur at essential steps during the folding cycle and facilitate the binding of substrate proteins (Meyer et al, 2003; Reissmann et al, 2007). Here, our maps in the apo, ATP-, and ADP-bound states reveal that the apical domains of TRiC generally extend towards the central chamber in different angles with a slight upward tilt (Figure 2; Supplementary Figure S3A–C). Importantly, akin to the apo and ADP-bound states (Figure 2A and C), our maps clearly demonstrate that TRiC remains open in the ATP-bound state (TRiC–AMP-PNP; Figure 2B). This is consistent with previous biochemical and biophysical studies of TRiC (Szpikowska et al, 1998; Gutsche et al, 2000; Meyer et al, 2003; Reissmann et al, 2007) and recent structural studies on group II chaperonins (Huo et al, 2010; Douglas et al, 2011; Munoz et al, 2011; Zhang et al, 2011). Thus, our observations unambiguously show that nucleotide binding alone is not sufficient to trigger lid closure.
Upon nucleotide binding (AMP-PNP or ADP state), TRiC remains open in both rings similar to the apo state (Figure 2), and a pseudo two-fold symmetry between two rings persists in these conformational states (Supplementary Figure S4). Strikingly, after nucleotide binding the densities of subunits a1 and a′1 appear to be better defined based on the 2D end-on view image analysis (Supplementary Movie 1B and C), and the entire complex appears slightly more symmetrical in 3D (Supplementary Movie 2). These observations are confirmed by the 3D reconstructions, for instance, the missing density corresponding to subunits a1 and a′1 in apo-TRiC is restored in the AMP-PNP and ADP states (Figure 4B and D). Of note, the TRiC–AMP-PNP map demonstrates that the apical domain of subunit a′1 in the bottom ring is less well resolved than its counterpart a1 in the top ring (Figure 4B). This finding indicates that a1 is possibly better poised than a′1 for the lid closure of the top ring, which might require all the subunits in one ring to be more symmetrically arranged. Such asymmetry between a1 and a′1 may reflect the negative inter-ring cooperativity of TRiC (Kafri et al, 2001; Reissmann et al, 2007).
In total, our data reveal that nucleotide binding can cause the subunit to be more rigid and the arrangement to become more symmetric while still open. However, nucleotide binding is not sufficient to close the folding chamber. This mechanism is in strong contrast to group I chaperonins such as GroEL, which undergo their major conformational rearrangement upon binding, not upon hydrolysis of ATP (Xu et al, 1997). This difference could be related to the fact that GroEL has a detachable lid (GroES) to cover the chamber, while TRiC instead has a built-in lid. Hence, the energy needed for chamber closure might be different.
Interestingly, the outward tilt of the subunits a3 and a′3 in the apo state is preserved upon nucleotide binding, as seen in the AMP-PNP state (Figure 5C and F; Supplementary Movie 2). This outward tilting subunit is also seen in the crystal structure of TRiC–ATP-γ-S in complex with tubulin (2XSM) (Munoz et al, 2011). This ATP-bound state (ATP-γ-S) is equivalent to our AMP-PNP state. These two structures match well (Supplementary Figure S5), providing strong evidence for the existence of this unique conformation of TRiC in the ATPase cycle.
All open states display an extra density in the equatorial domain inside the chamber around subunit a6 in one ring and around subunit a′7 in the other ring (indicated by the black arrow in Figure 2). These densities may correspond to the C and N termini (Supplementary Figure S6A), which have also been observed in the 4.7Å resolution TRiC–ATP-AlFx map (Cong et al, 2010; Supplementary Figure S6B).
The characteristic outward tilt of subunits a3 and a′3, the location of the pseudo two-fold symmetry axis, and the extra density in the equatorial domain allowed us to put the structures of these three states in register. Both the self-correlation function for each map (Supplementary Figure S7) and the 2D end-on view movie (Supplementary Movie 1) reveal that the ADP state (Figure 4D), which is the end state of the ATP-hydrolysis cycle and might be the substrate acceptor state, appears to be slightly more symmetrical than the other two states (Figure 4A and B).
TRiC–ADP-AlFx, the trigonal-bipyramidal ATP hydrolysis transition state (Melki et al, 1997), has been demonstrated to represent the physiological substrate folding state (Kafri et al, 2001; Kafri and Horovitz, 2003; Meyer et al, 2003; Reissmann et al, 2007; Douglas et al, 2011). This biochemical state contains a mixture of two TRiC conformations (Supplementary Figure S1A and B). In all, 35% of the data were classified into a conformation with both rings open (Supplementary Figure S1C). The map generated from the remaining data displays an asymmetric conformation with one ring closed (cis-ring) and the other ring open (trans-ring) (Figures 3A and and5D).5D). Using the correlation between this asymmetric TRiC–ADP-AlFx map and the TRiC–AMP-PNP map (Figure 5E), we can register their subunits in both rings (Figure 5G and H). This registration is also apparent in the match of the characteristic outward tilting subunits a3 and a′3 in the two maps (Figure 5G and H).
In the closed cis-ring, we observe a collective inward tilt motion of all eight subunits (Figure 6A; Supplementary Movie 3) and a more symmetrical arrangement relative to all the open states (Figures 2, ,3A,3A, and and5G).5G). The apical domain protruding densities from each subunit come together and have more contacts to form a lid covering the central chamber of the cis-ring. Although the characteristic subunit a3 tilts inwards slightly, it displays relatively less contact with its neighbouring a2 subunit (Figure 5G) and leaves a small opening in the covering lid at the rendering threshold (indicated by black arrow in Figure 3A, and also shown in Figure 3D). Conversely, the apical protruding density of a3 is closer to subunit a4. It is not known if such an arrangement exists in the presence of substrate. If it does, a productive folding of a substrate inside the chamber may not require a complete closure of the lid.
Careful comparison of the closed cis-ring of TRiC–ADP-AlFx with the closed ring of TRiC–ATP-AlFx reveals that on average the chamber of the cis-ring of TRiC–ADP-AlFx is slightly wider (~8 Å) than that of the other state (Figure 3C and D). This expansion results in a volume of ~180 000 Å3 for the closed folding chamber. It has been suggested that group II chaperonins can assist folding of proteins around 40–70 kDa (Spiess et al, 2004; Yam et al, 2008). Such chamber size in TRiC is comparable to that of the closed cis-ring of GroEL–GroES (~175 000 Å3), which can accommodate substrates up to 70 kDa (Xu et al, 1997).
The subunits in the open trans-ring of TRiC–ADP-AlFx exhibit a visible outward tilt motion of the intermediate and apical domains as compared with the AMP-PNP state (Figure 6A). This motion leads to a noticeable chamber enlargement of the trans-ring (Figure 6A), which would be the physiological acceptor state for the next round of substrate and nucleotide binding.
Our cryo-EM maps of TRiC in multiple nucleotide states depict different intra-ring subunit interaction patterns throughout the ATPase cycle. In the open states, the maps reveal contacts among the apical domain in some but not all of the neighbouring subunits (Figure 5B and C). The eight subunits in each ring appear to group into four subunit pairs (a1–a2, a3–a4, a5–a6, and a7–a8) that create a pseudo four-fold symmetry in TRiC, whereby each ring would be formed by a tetramer of dimers. This can be clearly observed especially in the map of TRiC–AMP-PNP (Figure 5C). This pattern of four subunit pairs within a ring might be related to the differential ATP binding and hydrolysis rate among different TRiC subunits (Reissmann, 2007). Upon ATP hydrolysis, the apical-domain interactions among all subunits become more extensive in the closed cis-ring (Figure 3A) and result in shutting the cis-ring chamber (Figure 3A and D).
As for TRiC–ATP-AlFx, a similar intra-ring contacting pattern to that in the cis-ring of TRiC–ADP-AlFx was detected (Figure 3B and D). Interestingly, our map also reveals additional contacts between the apical/intermediate domain of some subunits and its neighbouring equatorial domain, similar to that in the closed thermosome structure (Ditzel et al, 1998). These extra interactions formed in TRiC–ATP-AlFx might be the potential forces making its chamber more compact and even more symmetrical than that of the cis-ring of TRiC–ADP-AlFx (Figures 3B, D, and ((4E).4E). In summary, the apical-domain interaction is maintained or enhanced throughout the ATPase cycle, which suggests its key role in maintaining the association and communication of the TRiC subunits within one ring.
A number of efforts have been made to describe the mechanism of TRiC chamber closure based on cryo-EM studies (Llorca et al, 1999, 2001; Booth et al, 2008). However, this issue remains uncertain because (a) eight-fold symmetry was imposed in their reconstructions and (b) none of these studies included multiple intermediate states. In the present study, we obtained symmetry-free maps of TRiC for four nucleotide-containing states in addition to the apo state. The asymmetrically closed intermediate (TRiC–ADP-AlFx), especially, representing the key physiological substrate folding state, is structurally determined for the first time.
By not imposing symmetry, we uncovered structural details not previously seen in group II chaperonins (Xu et al, 1997; Llorca et al, 1999, 2001; Booth et al, 2008; Clare et al, 2008; Pereira et al, 2010; Zhang et al, 2010). Our analysis clearly shows that the ATP hydrolysis-induced chamber closure motion for each of the subunits is different (Figure 5G; Supplementary Movie 3). A comparison of the subunit arrangement in Figure 4A–E shows a trend of increasing pseudo symmetry during the ATP hydrolysis cycle. The most characteristic and dramatic change is found in the a3–a′7 subunit pair. They may possibly play a critical role in initiating the formation of pseudo symmetry by increasing the extent of apical-domain interactions among all the subunits of that ring. This symmetry would lead to positive cooperativity among subunits in one ring and result in chamber closure of the cis-ring.
Though the motion of each subunit is unique (Supplementary Movie 3), the overall ATP hydrolysis-induced chamber closure motion is similar for each subunit. To demonstrate the motion (Supplementary Movie 4), we use the subunit a5 and a′5 pair as an example, which appear to have minimum motion among the subunits. The cis-ring subunits move 'upwards' (towards the top of the barrel), and each domain of each subunit tilts inwards with slightly different motions. In the trans-ring subunits, the apical domains move neither downwards nor inwards. Rather, in some of the trans-ring subunits, the apical and intermediate domains tilt outwards (Figure 6A); while the equatorial domains of most trans-ring subunits move radially outward slightly. This outwards motion could prevent the closure of the trans-ring. This opposing motion is a plausible indication of the negative cooperativity of the two rings.
Our characterization of the one-ring closed intermediate TRiC–ADP-AlFx provides some unexpected insights into the structural basis of its negative inter-ring cooperativity. ATP hydrolysis causes a spatial rearrangement at the interface between the two rings, as in the case of GroEL (Xu et al, 1997; Ma et al, 2000). This rearrangement results in an observable increase in separation between the rings in TRiC–ADP-AlFx (Figure 6B; Supplementary Movie 4), while the two major inter-ring contacts are maintained, as in all group II chaperonins (Ditzel et al, 1998). Such a distance increase could reduce the buried surface between the two rings and potentially modify the salt bridges formed between certain opposing subunit pairs (Ditzel et al, 1998), which will vary the interaction network between the rings.
Closely related to the above-discussed subunit a3 and a′7 pair (Figure 4B), our open-state maps reveal that in general if two neighbouring subunits in one ring have formed a pair with contact in the apical domain (e.g., a3 and a4), their connected subunits in the other ring (a′7 and a′6, respectively) tend to be separated in the apical region (Figure 4A and B). This pattern has not been previously observed, and may play a part in both intra- and inter-ring subunit interactions. Such interactions could contribute to the inter-ring negative cooperativity, making TRiC work as a two-stroke molecular machine. This more complex mechanism of inter-ring negative cooperativity of TRiC compared with GroEL is likely related to their different inter-ring interaction patterns, that is, the interactions are registered in TRiC and other group II chaperonins, while it is staggered in GroEL.
Our structures of TRiC reported here show different intra-ring interaction patterns from its archaeal homologue Mm-cpn or prokaryotic homologue GroEL. In the apo state, all these chaperonins share a common neighbouring equatorial-domain interaction within one ring. However, the interactions between the other domains of adjacent subunits vary substantially. In apo-TRiC, we identified apical-domain interactions between pairs of subunits in the same ring (Figures 2A and and4A).4A). Apo-GroEL contains interactions between adjacent apical domains and has contacts between apical and intermediate domains of neighbouring subunits (Supplementary Figure S8A; Braig et al, 1994; Ludtke et al, 2008). In contrast, apo-Mm-cpn has no apical- or intermediate-domain contact in the open state, based on the available symmetric structures (Clare et al, 2008; Pereira et al, 2010; Zhang et al, 2010; Supplementary Figure S8B). This is consistent with the report that the intra-ring cooperativity is relatively weak in archaeal chaperonins, and in the T. acidophilum thermosome it appears to be absent (Bigotti and Clarke, 2005).
These three chaperonins have thus each evolved a different pattern of intra-ring interactions, indicating that the intrinsic inter-subunit coordination strategy is different among them. The enhanced intra-ring subunit contacts seen in TRiC as compared with Mm-cpn may play an important role in stabilizing and specifying the correct assembly of the eight distinct subunits of TRiC; this could also promote a coordinated rotation between the apical and intermediate domains, beneficial for the lid formation from the eight different subunits in TRiC.
The diverse pattern of intra-ring interactions among the open states of TRiC, Mm-cpn, and GroEL (Supplementary Figure S8; Figure 2; Clare et al, 2008; Pereira et al, 2010; Zhang et al, 2010) suggests that different starting points in the chamber closing process lead to differing trajectories of chamber closure. For apo-Mm-cpn, chamber closure appears to involve primarily rigid-body motion of the entire subunit when comparing only two states (Pereira et al, 2010; Zhang et al, 2010) and shows some interdomain movements within a subunit when an additional intermediate state is seen (Zhang et al, 2011). In both TRiC and GroEL, however, the allosteric rearrangement of their lid closure involves relative domain motion within each subunit. However, the direction of the domain motion is different: in TRiC, the apical and intermediate domains move mostly in the same direction (e.g., in subunits a1 and a3, Supplementary Movie 3), while those of GroEL move in the opposite directions (Xu et al, 1997; Booth et al, 2008). In addition, our study depicts a complex inter-ring subunit cooperativity in TRiC (Figure 4), and cross talk between its intra- and inter-ring allosteric networks which may facilitate its chamber closure. Together, the structural mechanism differences in chamber closure among different chaperonins are related to their differences in intra- and inter-ring subunit interaction pattern, with pronounced differences seen in the open conformations.
This study demonstrates how evolution uses a common structural framework but adopts different mechanical switches due to specific gene sequences and consequent allosteric networks. Furthermore, it is possible that the ATPase cycle of chaperonins might be customized to the unique intracellular environments of various organisms or physiological states by interaction with co-chaperones, substrate proteins and perhaps by distinct post-translational modifications (Southworth and Agard, 2008; Tsutsumi et al, 2009).
TRiC was purified from bovine testis essentially as described (Ferreyra and Frydman, 2000; Feldman et al, 2003). In brief, the tubules of bovine testis (500 g) separated from the tunica albuginea by dissection were homogenized in buffer H (20 mM HEPES/KOH (pH 7.4), 5 mM MgCl2, 0.1 mM EDTA, 50 mM NaCl, 1 mM DTT) containing the protease inhibitors leupeptine (2 μg/ml), aprotinin (0.5 μg/ml), pepstatin (0.5 μg/ml) and PMSF (0.2 mM), and the lysate was clarified by centrifugation for 30 min at 20 000 g followed by a 1-h centrifugation step at 100 000 g. The lysate was subjected to a 35% ammonium sulphate cut, and the resulting supernatant was precipitated with a final concentration of 50% ammonium sulphate. The pellet was resuspended in a small volume of MQ-A buffer (20 mM HEPES/KOH (pH 7.4), 5 mM MgCl2, 0.1 mM EDTA, 50 mM NaCl, 10% glycerol, 1 mM DTT) and 30 ml aliquots, respectively, were loaded on sucrose cushions (lower layer: 2 ml 60% sucrose in MQ-A buffer; upper layer 5 ml 20% sucrose in MQ-A buffer). After 20 h ultracentrifugation in a SW-28 rotor at 26 000 r.p.m. and 4°C, the sucrose cushions together with all sedimented materials were pooled, dialysed against MQ-A buffer, and loaded on a Q Sepharose FF column (60 ml, GE Healthcare, USA) equilibrated in MQ-A buffer. Bound proteins were eluted with 0.4 M NaCl in MQ-A buffer. Fractions containing proteins were pooled and diluted 1:1 in MQ-A buffer before they were loaded on a High-Trap Heparin column (20 ml, GE Healthcare, USA) equilibrated in MQ-A buffer containing 0.2 M NaCl. Bound proteins were eluted by a NaCl gradient ranging from 0.2 to 1 M NaCl. Fractions containing TRiC were pooled, concentrated using an Amicon Ultra-15 10K concentrator (Millipore Corporation, USA), and loaded on a Superose 6 10/300 GL column (GE Healthcare). Fractions containing the oligomeric chaperonin were pooled, and aliquots were flash frozen in liquid nitrogen.
TRiC samples were prepared for cryo-EM studies by dilution of purified TRiC to 0.5 mg/ml. The TRiC sample was proven to be folding active under our freezing conditions by an actin refolding assay (Meyer et al, 2003). Nucleotide-free samples were frozen immediately. TRiC–ATP-AlFx/TRiC–ADP-AlFx samples were incubated in the presence of 1 mM ATP/ADP, 5 mM MgCl2, 5 mM Al(NO3)3, and 30 mM NaF for 1 h at 30°C prior to freezing. For TRiC–ADP/TRiC–AMP-PNP, the sample was incubated in the presence of 1 mM ADP/10 mM AMP-PNP and 5 mM/15 mM MgCl2 for 15 min at 30°C prior to freezing. Since commercial AMP-PNP typically contains impurities (Meyer et al, 2003), we performed purification on it by anion exchange chromatography as described (Horst et al, 1996). In all, 3 μl of sample was deposited onto a glow-discharged 400/200 mesh Quantifoil holy carbon grid (1.2 × 1.3 μm hole size, Quantifoil Micro Tools GmbH, Jena Germany). The grid was flash frozen in liquid ethane using a Vitrobot (FEI, Hillsboro, OR, USA). Data were recorded on a JEM 3200FSC microscope (JEOL, Tokyo) equipped with a field-emission gun operated at 300 kV and an in-column energy filter (using 20 eV slit). The spot size was set to 1; the condenser and objective aperture were set to 50 and 120 μm, respectively. Specimen temperature was maintained at 101 K. Images were recorded at × 50 000 microscope magnification on Kodak SO-163 films with the specimen dose of 18 electrons/Å2. The images were digitized on a Nikon Super CoolScan 9000 ED scanner with 6.35 μm/pixel scanning interval.
The particles were boxed out from digitized micrographs by boxer from EMAN 1.8 (Ludtke et al, 1999, 2001) and e2boxer.py from EMAN2 (Tang et al, 2007). CTF parameter estimation was carried out using the EMAN1 program ctfit. Reference-free 2D analysis was carried out using refine2d program in EMAN1 (Ludtke et al, 1999, 2001). For the ADP-AlFx state, there are two conformational states that coexist based on the 2D image analysis (Supplementary Figure S1A), so that multirefine procedure in EMAN1 as described previously (Chen et al, 2006; Booth et al, 2008) was employed. All the 3D reconstructions using EMAN1 were completed with no symmetry imposed. In addition, a recently developed 2D fast rotational matching (FRM2D) algorithm (Cong et al, 2003, 2005; Cong and Ludtke, 2010), available in EMAN 1.8+, was adopted in the final refinement. This method has been used to obtain a number of subnanometer resolution cryo-EM maps (Cong et al, 2009, 2010). Micrographs with a defocus range of ~1.2–3.5 μm were used in the final reconstruction process. For each of the five states, the particle number included in the final 3D reconstruction was listed in Supplementary Figure S2A. Except for the closed ATP-AlFx state (4.7 Å; Cong et al, 2010), the resolutions of the rest of the four states are assessed to be in the 10.5–13.9 Å resolution range based on the 0.5 cutoff for the Fourier Shell Correlation (Saxton and Baumeister, 1982; Harauz and van Heel, 1986).
Map segmentation and visualization were done with CHIMERA (http://www.cgl.ucsf.edu/chimera/; Pettersen et al, 2004).
The existence of the one-ring closed asymmetric conformation induced by ATP hydrolysis is validated by our reference-free 2D image analysis and 3D reconstruction. First, the 2D image analysis of TRiC–ADP-AlFx particles depicts a unique end-on view in which all the subunits appear twisted (Supplementary Figure S1B), not seen in any of the other four states. If the two rings were in the same conformation, there would be no handedness in the end-on projection of TRiC as seen here. The twisted pattern is comparable to computed projections of the true bullet-shaped GroEL–Aacpn10–ADP complex (Chen et al, 2008), as well as a simulated asymmetric map (composed of a closed ring from TRiC–ATP-AlFx and an open ring from apo-TRiC). Moreover, our 3D reconstruction using multirefine in EMAN1 (Chen et al, 2006) indicates that the majority of TRiC particles (~65%) in this data set adopt the one-ring closed conformation (and were used in the reconstruction of the map shown in Figure 3A), while the remainder is in the conformation with both rings open (Supplementary Figure S1A and C), similar to the other open conformations of TRiC. The above analyses clearly confirm the existence of this asymmetric intermediate of TRiC in the ATP hydrolysis transition state (mimicked by ADP-AlFx).
To interpret the structural mechanism in more detail, a pseudo atomic model for each state was generated. We first built a homology model of the TRiC complex in the closed state (TRiC–ATP-AlFx). Since the current reconstructions were completed at relatively low resolution, we began with a homology model corresponding to the CCT2 subunit using thermosome (1A6D) as template and fit it as rigid body to all the subunits in the closed TRiC–ATP-AlFx map. In order to fit the closed-state model of the entire TRiC complex to each of the maps in different biochemical states, we used the real-space refinement/sampling program DireX (Schroder et al, 2007, 2010). DireX uses a geometrical sampling approach to flexibly fit a model into the density map in an iterative manner while ensuring correct stereochemistry and non-overlapping atoms. The model of the closed state was first placed into the density map of apo-TRiC using CHIMERA (Pettersen et al, 2004). Strong elastic restraints were used to avoid overfitting the density. The restraints were chosen between randomly chosen atoms that are within a distance range of 3–15 Å and that are separated by not >100 residues along the protein chain. The number of restraints was chosen as two times the number of atoms. The fitted model of apo-TRiC then served as the starting point for the refinements of other states. For the fitting, 500 optimization steps were performed with DireX, which took on average about 7 h on a single cpu (Intel Nehalem 2.93 GHz). In all refinements, a restrained grouped occupancy refinement was used, which adapted the contribution of the residues to the model density. This was done to account for reduced or missing density. This, however, adds only a small shift to the global position and orientation of the individual subunits and has almost no influence on the local structure. Finally, the models were subjected to minimization with CNS (Brunger, 2007). As shown in Supplementary Figure S3, these models fit to the corresponding density maps well particularly for equatorial and intermediate domains. Our models do not include some regions of the apical domain in several subunits, for example, in apo-TRiC, because the map in those regions was not very well resolved due to the dynamic nature of those subunits. However, such models are useful and simplified to represent the structural complexity and variations of TRiC in the ATPase cycle.
The density maps are deposited to EMDB (accession numbers EMD-1960, EMD-1961, EMD-1962, and EMD-1963) and the associated models are deposited to PDB (PDB IDs: 4A0O, 4A0V, 4A0W, and 4A13). They will be released upon publication.
We thank Dr John F Flanagan IV for the help in composing the map unwrapping tools. This research is supported by grants from NIH through the Common Fund (PN1EY016525), NIGMS (GMR01-074074), and National Center for Research Resources (P41RR002250).
Author contributions: YC, JF, and WC designed research; ASM and SR prepared the specimen and defined the biochemical conditions for attaining different states; YC and JJ collected the cryo-EM data; YC carried out all the image processing and map analysis with the advice of SJL and MFS, and BM involved in the data preprocessing; YC and GFS built the models; YC, MTD, and WC generated the movies; YC, ASM, MFS, JF, and WC interpreted the structures; YC, GFS, ASM, MFS, SR, ML, SJL, JF, and WC wrote the paper.
The authors declare that they have no conflict of interest.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.

subset: commoncrawl


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Donnie Avery, WR at NFL.com
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Wikipedia: Donnie Avery
Donnie Dion Avery (born June 12, 1984) is an American football wide receiver for the Indianapolis Colts of the National Football League. He was drafted by the St. Louis Rams in the second round of the 2008 NFL Draft. He played college football at Houston.
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legacy.com: Donnie Paul Avery
RAVENNA - Graveside services for Donnie Paul Avery, 65, of Ravenna, Texas will be held at 11 a.m., Tuesday February 28,2012 at the Gum Springs Cemetery Pavilion. Serving as
findagrave: Avery, Donnie L.
01/10/1954 - 08/18/1976, Hamilton (Marion County, Alabama)
findagrave: Avery, Donnie Paul
07/5/1946 - 02/25/2012, Carson (Fannin County, Texas)
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Donnie Avery Breaks a Camper's Ankles - …
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Chiefs release WR Donnie Avery; Waive WR A.J. Jenkins
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#17. Donnie Avery. Rec, Yards, TD. 15, 176, 0. Team: Kansas City Chiefs, Ht/Wt: 5-11/200, NFFC ADP: 291.76. Position: WR, Born: 6/12/1984, Birthplace: ...
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The following real estate transactions occurred in Spartanburg County from Jan. 17 to Jan. 25, 2002
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People. A familiar form of Donald, including: Donnie Abraham (born 1973), American football cornerback; Donnie Allison (born 1939), NASCAR driver; Donnie Avery (born ...
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Donnie Avery - JT-SW.com
Played: WR -- 2008-2014: Born: Jun 12, 1984 in Houston, TX: High School: Hastings (Alief, TX) College: Houston: Drafted: 2008 by St. Louis Rams (Round 2 / 33 overall)
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"I think we are still in big trouble."I don't. I have much more confidence in the ADP data which are based on name, rank and serial number. I think ADP payroll encompasses 26 million employees. An increase of 433,000 net employees reported by ADP in december and january is very significant ( up 1.6%).It would not surprise me if the department of labor february job report shows 300 to 400k private payroll increase. The department of labor has a lousy track record and perhaps the country would be better off if many of these statisticians left gov't for productive work. We're only going to be in big trouble if Fed and State gov't do not put the axe to spending. Look, the private sector cut 8 million jobs during the recession. Now that the private sector is recovering, the gov't sector needs to bleed 3 million job loss. At the same time the retirement age for both ss and medicare need to be increased modestly. Folks are living much longer than 50 years ago and it's not a big sacrifice to move retirement out a few years. That's the reality and Obama's new guy, Bill Daley will make it happen.
article today in WSJ re firms are not hiring. Government payroll will not decrease without Reaganesque leadership. Long term job market is still very bad.
Must be careful with the DOL/BLS 'Unemployment Rate'. That is a guess/seasonally adjusted number of folks receiving unemployment insurance benefits. Look to the BLS U-6 for a better idea. But even U-6 understates by leaving out folks who unwillingly leave the labor force or never got in it.I like the ADP numbers because it is an 'indication' of small-midsized companies hiring. Big companies have their own payroll departments and very small companies do their own: generally neither use ADP which might be the source of your discomfort with their numbers. But alas, I think mid-size companies are the engine of growth of US business. Last Summer I noted that mid sized companies on the coasts and the Mississippi valley were having trouble finding qualified welders. They were setting up training programs to teach welding. Generally welding is a very good blue collar job. I think this trend continues. These are the companies that use ADP whose results you see. So the long and short of it is: The gov't is understating employment for political affect. The US economic engine is accelerating but not the BLS way. There will be setbacks to stock performance during 2011. We will end the year with SP500 above 1400.


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Cora the Smart Cub is a cuddly educational toy for babies. Simply connect this soft learning toy to the online VTech Learning Lodge to personalize Cora with your child's name, routine, birthday, and other activities. You can also download songs, stories, and messages. Connecting and downloading information from your computer is easy to do, and a USB cable is included.
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How poverty is a moral issue
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Poverty is a moral issue, according to William Easterly, a professor of economics at New York University, who recently spoke to Christianity Today about caring for the poor. He said in the interview that more consideration should be taken in considering those in poverty.
"I realized our attitude towards the poor is so often condescending and paternalistic," Easterly said to Christianity Today. "We think of them as helpless individuals. We don't respect their dignity as individuals."
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Over the past year a lot of outstanding web tasks have cropped up and I really need to get a lot of them taken care of. I used to handle all this myself but my technical skills are definitely slipping and I just don't have the time to keep up on this stuff anymore. I've talked to people about contracting them to do the projects, but they're just too small and scattered to be worthwhile to most agencies / developers.
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text: UNIVERSITY OF ILLINOIS LIBRARY AT URBANA-CHAMPAIGN BOOKSTACKS Digitized by the Internet Archive in 2011 with funding from University of Illinois Urbana-Champaign http://www.archive.org/details/investigationofc95jens Faculty Working Papers »- — -r< I «i INVESTIGATION OF COST VARIANCES: A CASE OF I-IULTIPLE-STEP INVESTIGATIONS Daniel L. Jensen and James C. McKeown //95 College of Commerce and Business Administration University of Illinois at Urbana-Champaign FACULTY WORKING PAPERS College of Commerce and Business Administration University of Illinois at Urbana-Champaign March 6, 1973 INVESTIGATION OF COST VARIANCES: A CASE OF IWLTIPLE-STEP INVESTIGATIONS Daniel L. Jensen and James C. McKeown if 9 5 INVESTIGATION OF COST VARIANCES: A CASE OF MULTIPLE-STEP INVESTIGATIONS Daniel L. Jensen and James C. McKeown University of Illinois at Urbana-Champaign Introduction The investigation of cost variances has been the subject of several papers in recent years. These papers develop models that lead to a decision as to whether or not to investigate a process after a cost variance is observed. The investigation is usually conceived as a single-stage investigation process with a constant cost; in other words, the decision does not allow for a choice among alternative investigative 2 procedures. Yet such a choice is apt to be necessary in practice. Accounting variances arise from a multiplicity of causes. \'Jhen a variance is ob- served, it is reasonable to suppose that the structure of any insuing investigation will be influenced by available information about the likelihood of the various possible causes of the variance and about the 3 costs and savings associated with discovery and correction of each variance-causing condition. The purpose of this paper is to consider a multiple-stage invest- igation process in which the decision involves a choice among several investigative techniques and a determination of the order of their 4 application. Although the complexity of the decision rule increases with the complexity of the investigative process, the decision rule may be operated by a simple graphical technique. The Single-Step I nvest igation A process is in one of two states; it is either in control or out of control . If the process is in con rol, then corrective action cannot improve its operation. If it is out cf control, then corrective action will secure certain benefits. Unfortunately j we cannot know vrith certainty which state actually exists unless v-je investigate and incur the attendant costs. Clearly we do not wish to investigate a process when the cost of doing so exceeds the benefits obtained thereby. Since the state of the process is uncertain before investigation, we formulate a decision model that leads to a choice between investigating and not investigating so as to maximize the net expected benefits. Prior to the decision, we formulate probabilities about the process being in or out of control and about the probability of observing variances of different sizes given that the process is in control and given that it is out of control. Pq(IC) designates the prior probability that the process is in control and Pq(OC) = 1 - Pq(IC) is the probability that the process is out of control. P(X;IC) and P(X;OC) designate the conditional probabilities of observing a variance X given that the process is in control and given that the process is out of control, respectively. When a variance, X, occurs, we revise our prior probabilities according to Bayes Theorem as follows: p ac) = p (ic- X) = p(x? ic) p"(ic) ^^n''^^'' ^n'^^^* ^^ P(X;IC).Pjj(IC) + P(X;OC).Po(OC) P^(OC) = 1 - P^(IC) where Pj^(IC) is the revised probability that the process is in control given an observed variance X. Notice that three probabilities are required to reach the revised est3.mates — the prior probability that the process is in control, Pq(IC); the conditional probability of a variance X when the process is in control, P(X;IC); and the conditional probability of a variance X vhen the process is out of control, P(X;OC). Having revised the probability that the process is in control, we proceed to calculate the expected value of the actions open to us. In the single-stage investigation problem only two actions are available — investigate or do not investigate. The loss matrix associated with this simple decision is given below: States Action In Control (IC) Out of Control (OC) Investigate (I) Do not Investigate (NI) c C L The table shows the outlay costs associated with each combination of action and state. Investigation incurs a known constant cost, C, and an out-of-control process occasions a constant loss, L, which is avoided if an investigation is undertaken. The decision maker wishes to choose between investigating and not investigating so that he minimizes the expected outlay cost. The expected cost of investigation, E(I), is the sum of two products: (1) the cost of investigation multiplied by the probability that the process is in control and (2) the cost of investigation multiplied by the probability that the process is out of control, that is, E(I) = C-P(IC) + C-P(OC) The expected cost of not investigating, E(NI), is the product of (1) the loss and the probability that the process is out of control, that is, E(NI) = L*P(OC) The decision maker calculates E(X) and E(NI) and chooses the action with the smaller expected cost. Alternatively, the decision rule may be formulated as a test of the revised probability that the process is in control. The expected cost of investigating will be less than the expected cost of not investigating only if the revised probability that the process is in control is greater than the ratio (C - L) / L. This is the familiar result obtained by Bierman, Fouraker and Jaedicke and by Dyckman. Multiple-Step Investigations The single-step investigation model reviewed above contemplates a fixed set of investigative procedures with a constant cost. In practice, however, it is unlikely that the search for causes of an observed deviation of variance exhibits such simplicity. It is probable that different variance-causing conditions call for different investigative procedure investigation and that the decision is not merely whether or not to investigate, but also how to Investigate. The development of a multiple-step investigation model requires the partitioning of investigative procedures into groups or steps containing only inseparable elements — procedures that must be performed together. As in the single-step model, if a step is undertaken the investigative activity is completely specified, and exactly the same procedures are applied regardless of the occasion on which the step is undertaken. It is therefore, reasonable to view the cost of each step as more or less constant. The multiple-step model considered here constrains the relationship between time and conditions that dislodge a process from control. No more than one such condition may be operative in any given period. Such a constraint simplifies the investigation because once the condition is discovered, we need to look no further. On the other hand, if several conditions may combine to effect an out-of-control state, then the in- vestigation must determine the extent of the variance attributable to a particular condition in addition to the presence or absence of the condition* Moreover, the presence of a condition alone may not bring loss of control although its presence in combination vith other conditions will bring ]^09» of control. Cases of this complexity are beyond the scope of thi5 papejr. Each stage of investigation is directed toward the discovery of >a well-defined condition whose presence creates an out-of-control &i-cuaJ:lon and causes a known loss. The outcome of each stage is binary in the sense that the condition is found to be present or absent. Moreover, the in- vestigative techniques are assumed to be error-free, which precludes an erroneous conclusion as to the presence or absence of the condition under Investigation. Finally, the corrective procedures which follow discovery of such conditions are assumed to be effective in avoiding the known loss. The revision of probabilities, after the observation of a variance X, in the presence of several out-of -control states, requires an application of Bayes theorem. In general if the process is in one of J states, S., we revise each of the J prior probabilities, Pq'^S.), according to Bayes Theorem as follows: P(X;S^)>Po (s,) ^n(Si) = Pn(Si;X) = J Z P(X;S ).Po(S.) j=l ^ ^ In the cases examined here, three states are possible: (1) an in-control state, (IC); (2) an out-of-control state owing to the presence of condition A, (OCA); and (3) a second out-of-control state owing to condition B, (OCB) . After observing a variance X, the three prior probabilities are revised as ' y- follows: P(X;IC)'P (IC) o ^n^^^^ P(X;IC)Po(IC) + P(X;OCA)Fo(OCA) + P(X;OCB)Po(OCB) P"(OCA) = P(X; OCA)-P^(OCA) P(X;IC)Pq(IC) + P(X;OCA)P^(OCA) + P(X:OCB)P (OCB) Pj^(OCB) = 1 - P"(IC) - I^ (OCA) The revised probabilities are conditional on the observation of a variance X and require prior knowledge of the conditional density functions of X given each of the three possible states as well as prior estimates of the probability that each state occurs. The Case of Two Investigative Steps Unconstrained as to Combination or Order of Application The allowable combinations of investigative steps and the order in which they are applied niay be constrained in many different ways. For example, Dyckman considers a case in which two investigative procedures — an exporatory investigation and a full investigation — are alternative to one another and may no^ be applied together in any order. The case considered here imposes no constraints as to the allowable combinations of procedures or as to the order of their application. When the two investigative steps may be undertaken in any order, the decision entails a choice among five alternative actions: (1) do not investigate, (NX); (2) investigate for condition A only, (lA) ; (3) investigate for condition B only, (IB); (4) investigate first for condition A, then for condition B, (lAB); and (5) investigate first for condition B, then for condition A, (IBA) . The loss associated with each action under the three possible states of the process is given in Table 1. If the objective is to rainimize the expected loss, then the decision requires a determination of the expected loss of each action (as given in the far right-hand column of Table 1) and a selection of the action with the smallest expected loss. The selection of the minimum- loss action requires at most ten pair- wise comparisons of the expected values of the five alternative actions. Each of the ten comparisons may be represented by an inequality that specifies the choice of one action over another. For example, the action NX (no investigation) is preferred to the action lA (investigate for A only) when E(NI) is less than E(IA), that is, when Vn^A^ + Vn(B) < C^ + LbPn(B) or, equivalently, VPn(A) - C^ < 0. If the inequality is satisfied, then NI is preferred to lA and lA (and the comparisons involving lA) may be eliminated from further consideration. If La'Pjj(A) - Cg = 0, then we are indifferent between lA and NI. On the other hand, if 1^ ^^^^) ~ C^ > 0, then lA is preferred to NI and NI is eliminated from further consideratioas. We then proceed to compare the remaining (non-eliminated) pairs of expected values in a similar way. Eventually a preferred action will be indicated. Tlie ten pairwise com- parisons of expected values are given in Table 2. Although this procedure is somewhat tedious it may be simplified by a simple graphical method. TABLE 1. — Loss Matrix for Case of Three Investigative Steps where the Order of Their Application is Unconstrainted States Actions IC OCA OCB Expected Value NX La h E(NI)=L^P^(A)+L^ P^(B) lA ^a ^a 'a'-h E(IA)= C^ + Lj^ P(B) IB Cb "^^K Cb E(IB)= % + L^ P(A) lAB Ca+Cb Ca Ca+Cb E(IAB)= C^+Cj^[l-P^(A)] IBA ^a+S Ca-^S % E(IBA)= C^+C^[1-P^(B)] Probability p^dc: Pn(A) PJB) TABLE 2. — Inequalities Leading to a Minimum Expected Cost Action Preference Inequality Condition NX over lA ^a Pn(A) - C^ < NI over IB \ Pn<2) - C^ < NX over XAB <La+Cb) Pn^A>+Vn(B)-Ca-Cb < NI over IBA L^Pn(A)+(Lb+Ca)P^(B)-Cb-Ca < lA over IB LbPn(B>-Vn<A)+Ca-Cb < XA over XAB LbPn(B)+CbPn(A)-C^ < lA over XBA (V^a^ Pn(B) -Cb<-0 IB over XAB (La+C^) Pn(A) -C^< IB over IBA LaP(A) + C^PCB) -Ca< lAB over IBA ■ CaP(B) -Cj^P(A)< 10 Illustration of Graphical Procedure Since the costs and avoidable losses associated with investigation are known constants, each pair-wise comparison of expected values represents a partition of the probability space which gives all possible combinations of P (A) and P"(B). If the space is partitioned for each of the ten comparisons, each resulting region of the space identifies pairs of P^CA) and PjiCB) that signal a unique least-cost action. Once the graph is determined, the choice among the five alternative actions requires only the calculation of P^CA) and PqCB) and the location of the region on the graph with the pair of probabilities is associated. A numerical illustration will clarify the application of the model and the graphical procedure. Suppose that condition A leads to a loss of $100 and Investigation for condition A costs $50; condition B leads to a loss of $300 and investigation for B costs $100. The graph in Figure 1 is derived from this data by a simple procedure. First the data is substituted into each of the ten inequalities given in Table 2 and the corresponding boundary lines are drawn on the grap'i. Each inequality in Table 2 gives rise to a line of the graph. For example, the line specifying the preference relation between the actions lA and IB is given by the equation 300P(B) - lOOP(A) - 50 = or, equivalently, 6P(B) - 2P(A) - 1 = 0. Probability pairs above this line indicate a preference for action IB over lA while pairs below indicate a preference for lA over IB. The other lines are obtained from the remaining nine inequalities in a similar way. Next, each cell in the resulting partition of the probability space is identified with the least-cost action. The performance of this second step is straight-forward, but its rationale requires some explanation. 11 i .^A .' 23. NI _ IBA _- ^•?^ -'.r' 1.0 -• P(A) FIGURE 1 12 In general, we will not investigate if ^^(A) and Pjj(B) are sufficiently snail. Clearly, we will not investigate if P^CA) and Pji(B) are both equal to zero which corresponds to the origin. Nor will we investigate in those cells whose boundaries include the origin. As we move to other regions whose boundaries do not adjoin the origin, we. still do not investigate unless a boundary is crossed that signals an investigation of some kind. When such a boundary is crossed, all points within the region entered signal the kind of Investigation designated as we cross the boundary. In general, moving from any region in which the decision is known, to an adjacent region, leaves the decision unchanged unless the shared boundary involves the decision of the first region; if it does, the decision is changed to the other action involved. In other words, the second step in generating the graph begins by locating the cells that adjoin the origin of the probability space and by designating the probability pairs contained therein as signaling no Invest- igation. We proceed from these "no investigation" regions to adjacent regions and, then, to other adjacent regions until every region of the probability space has been identified with a least-cost action according to the following rule: as one moves from any region in which the least- cost action is known, to an adjoining region, determine whether or not the pair of actions associated with the boundary crossed Includes the least- cost action of the region from which one moves. If the pair does not include the action signaled in the first region, then the least-cost action of the second region is the same as that of the first. If the pair does include the action signaled by the first region, then the least-cost action of the second region is the other member of the pair. In this way every cell of the partitioned probability space is identified with a least-cost action. 13 After construction of the graph, the decision rule may be implemented by merely calculating Pn(A) and P^^^^' Plotting the corresponding point on the corresponding point on the graph, and taking the least-cost action identified with the region within which the point falls. For example, if the revised probabilities, P (A) and P (B), are found to be 0.5 and 0.4, respectively, then the action lAB is signaled. Summary The complexity of the investigation process has an important impact on the decision rule in models for the investigation of cost variances. In general, the more complex the representation of the investigation process, the more complicated the decision rule. In some instances, complexity is an impediment to the application of a model despite the fact that it provides a better fit to reality. In the case examined here, however, a simple graphical procedure permits a straight-forward application of the model despite the complication introduced in the decision rule. 14 FOOTNOTES See, for example, H. Biennan, L. E. Fouraker, and R. K. Jaedicke, "A Use of Probability and Statistics in Performance Evaluation," The Accounting Review , XXXVI (July, 1961), 409-417; and T. R. Dyckman, "The Investigation of Cost Variances," Journal of Accounting Research , VII (Autumn, 1969), 215-244. ^Dyckman (1969) considers an exception in the form of a three-action model for the purpose of choosing between an exploratory Investigation, a full investigation and no investigation. The full investigation is assured of correcting an out-of-control state, if one exists, but the exploratory investigation may fail to do so with known probability. This analysis differs from the analysis in this paper in that the two Investigative procedures are strictly alternative to another and cannot be undertaken together. See pp. 228-230. " . . 3 The analysis presented here assumes that the cost of correcting an out-of-control condition, over and above the cost of the investigation leading to its discovery, is negligible. A cost of correction can be in- corporated into the analysis without great difficulty. See Harold Bierman, Jr. and Thomas R. Dyckman, Managerial Cost Accounting . New York: The Macmillan Company, 1971, pp. 33-53. Bierman and Dyckman (1971) suggest this among other possible extensions of the analysis. See p. 52. ^Dyckman (1969), pp. 228-230. eOU'^^sT


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paralegal jobs near Hartford, CT
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Beckett Law, LLC - West Hartford, CT
Real Estate Paralegal:. Small West Hartford law firm seeks real estate paralegal - some estate planning experience would be helpful....
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Barry & Barall, LLC - Manchester, CT
Barry & Barall, LLC is seeking real estate paralegal. Knowledge of real estate closings from beginning to end is necessary....
Foreclosure Paralegal
Witherspoon Law Offices - Farmington, CT
Witherspoon Law Offices, Farmington, CT is seeking a foreclosure paralegal. The ideal candidate must have five years experience and be conversant in LPS....
TR Grace - Hartford, CT
Hartford based boutique law firm seeks smart, organized and detail oriented paralegal who wants to learn IP law....
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A.R. Mazzotta - Middletown, CT
Busy law firm in the Middletown area is looking for a Paralegal – Legal Assistant to join their team! In this Paralegal – Legal Assistant position, you will...
ELLIOTT & STANEK PC - Southington, CT
Paralegal - Southington law firm seeks experienced paralegal who can handle real estate closings independently Must have knowledge of current TRID regulations....
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Kelly Services - East Hartford, CT
Position will entail a 40 hour work week, offers flex hours and will require the chosen candidate to work alongside and provide timely legal and administrative...
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Staffing Now - Hartford, CT
90% secretarial and 10% paralegal. Commercial real estate secretary/paralegal needed with at least 5 years commercial real estate experience with a mid to large...
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Michael Page US - Bristol, CT
Prior experience as paralegal What's on Offer. Teamed with specific attorneys from which virtually all assignments flow....
Bankruptcy Paralegal
Staffing Now - Hartford, CT
My client is seeking an experienced Bankruptcy Paralegal for a small law firm. A paralegal certificate is required....


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This Irish singer/songwriter traded in a degree in law to pursue music and from all accounts it was a move that has paid off. Glover has been compared to Ryan Adams, David Gray and Bruce Springsteen and has been called one "one of the finest writers in Ireland today."
See more info at benglover.co.uk
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