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Isidro A, Santos MA, Henriques AO, Tavares P (2004b) The high-resolution functional map of bacteriophage SPP1 portal protein. Mol Microbiol 51:949–962 AAA+ chaperones of the Clp/Hsp100 family play a major role in removing aggregated proteins in prokaryotic as well as eukaryotic cells, but their detailed mechanisms remain still largely unknown. The M-domain of ClpB has been revealed to have a crucial regulatory function through direct interactions with DnaK/Hsp70, but its location has never been clearly defined in an oligomeric assembly of ClpB or the related Hsp104. The AAA+ rings have a central opening of ∼30 Å and therefore are not as compact as in one of the previous models ( Lee et al., 2003, 2007, 2010), but not as expanded as in the other ( Wendler et al., 2007). The Arg-fingers are at the interface between subunits, available to catalyse hydrolysis as expected from mutational studies ( Mogk et al., 2003; Yamasaki et al., 2011; Biter et al., 2012). It has become clear that AAA+ proteins are highly dynamic molecular motors unlikely to exist in a homogeneous structural state. Therefore we generated asymmetric reconstructions of ClpB. Although the resolution of the asymmetric structures is not sufficient to support a detailed mechanistic model, these reconstructions provide the first visualization of the MD conformational flexibility that was inferred from biochemical analysis ( Lee et al., 2003; Haslberger et al., 2007; Oguchi et al., 2012). The asymmetric structures show that the MD orientation varies around the ring occupying the repressed, wild type-like and hyperactive positions described by the symmetrised averages. The variable tilts of MDs observed around the ring suggest that 2 to 4 adjacent subunits are available for DnaK binding in the wild-type vs only 1 in the repressed mutant ( Figure 6B,C). This is consistent with the estimated stoichiometry of 2–5 molecules of DnaK per ClpB hexamer required for activation ( Seyffer et al., 2012; Desantis et al., 2014). It also suggests that at least four subunits must have detached MDs to allow activity, perhaps through movements of the AAA+ domains, in agreement with the number of ClpX subunits that hydrolyze ATP in a coordinated manner to unfold GFP in single molecule experiments ( Sen et al., 2013). Moreover, we calculated an ADP binding stoichiometry of 4 for both wild type and mutants ( Figure 6—figure supplement 3), which indicates that although ATP hydrolysis is strongly affected, detachment of the MD does not change the nucleotide binding.
Auzat I, Petitpas I, Lurz R, Weise F, Tavares P (2014) A touch of glue to complete bacteriophage assembly: the tail-to-head joining protein (THJP) family. Mol Microbiol 91:1164–1178 Leiman PG, Chipman PR, Kostyuchenko VA, Mesyanzhinov VV, Rossmann MG (2004) Three-dimensional rearrangement of proteins in the tail of bacteriophage T4 on infection of its host. Cell 118:419–429 The AAA-2 ring density could be almost entirely interpreted using this approach and the fit suggests that 3 to 5 subunits are sufficiently closed to bind ATP ( Figure 6—figure supplement 2). We analyzed nucleotide (ADP) binding to wild type ClpB by isothermal calorimetry (ITC) revealing a binding stoichiometry of 7.5 ± 0.1 ADP per hexamer ( Figure 6—figure supplement 3). ITC experiments using ClpB-K212A, which is deficient in nucleotide binding in AAA-1, allowed us to determine a binding stoichiometry of 3.7 ± 0.3 ADP in AAA-2 of the mutant hexamer ( Figure 6—figure supplement 3). The same ADP binding stoichiometry was also found for the repressed and hyperactive variants ( Figure 6—figure supplement 3). The deduced stoichiometries are in good agreement with the distinct AAA-2 conformations observed in the asymmetric EM reconstructions. Similar calculations have been reported for the AAA+ proteins MCM, ClpX and HslU ( Moreau et al., 2007; Yakamavich et al., 2008). The AAA-1 ring is more asymmetric than AAA-2 and it is not easily interpretable by fitting crystallographic dimer models. There is sufficient density to guide rigid body fitting of all AAA-1 domains, but this fitting does not allow deductions of nucleotide occupancy ( Figure 6—figure supplement 2). Tavares P, Zinn-Justin S, Orlova EV (2012) Genome gating in tailed bacteriophage capsids. Adv Exp Med Biol 726:585–600 In Figure 4C , the authors present the measurements of FRET between an engineered Trp in motif 1 and a Cys-linked AEDANS label in motif 2 for wild-type and mutant ClpB. Overall, the data convincingly show that there is a difference in the spatial separation of motifs 1 and 2 for the repressed versus active conformation of MD. The only surprising observation for ClpB-WT is that the 50% increase in acceptor fluorescence upon oligomerization is not accompanied by a corresponding decrease in donor fluorescence. According to fig. 4 supp. 2 , there is no change (increase) in the Trp donor fluorescence upon oligomerization, so it remains unclear why the acceptor fluorescence can double while the donor fluorescence stays unchanged. The authors should briefly discuss this point to strengthen the presented FRET data further.There are a variety of methods for determining the magnitude and direction of the result of adding two or more vectors. The two methods that will be discussed in this lesson and used throughout the entire unit are: Nováček J, Šiborová M, Benešík M, Pantůček R, Doškař J, Plevka P (2016) Structure and genome release of Twort-like Myoviridae phage with a double-layered baseplate. Proc Natl Acad Sci U S A 113:9351–9356 Wellcome Principal Research Fellowship, Grant/Award Numbers: 089396, 221044; Canadian Institutes of Health Research, Grant/Award Numbers: FDN‐167277, PJT‐152962, MOP‐126129 Funding information People are often faced with difficult decisions between two choices. Flipping a coin can be very useful in these situations. Sometimes, however, you may find that you’re disappointed with the result. In this scenario, instead of letting the coin decide, you may want to go with the choice that you now realize you really wanted.
Step 5. To get the magnitude of the resultant, measure its length with a ruler. (Note that in most calculations, we will use the Pythagorean theorem to determine this length.)Leiman PG, Shneider MM (2012) Contractile tail machines of bacteriophages. Adv Exp Med Biol 726:93–114 Bhardwaj A, Walker-Kopp N, Casjens SR, Cingolani G (2009) An evolutionarily conserved family of virion tail needles related to bacteriophage P22 gp26: correlation between structural stability and length of the alpha-helical trimeric coiled coil. J Mol Biol 391:227–245 The cephalocaudal principle is the idea that development proceeds from the head down to the tail, or top to bottom. This means that the earliest changes during development will occur in the head and facial areas, and then progress downwards towards the rest of the body. For example, a newborn baby might develop their eyes before their legs. Maxwell KL, Yee AA, Arrowsmith CH, Gold M, Davidson AR (2002) The solution structure of the bacteriophage lambda head-tail joining protein, gpFII. J Mol Biol 318:1395–1404 Head-to-Tail Method: The head-to-tail method of graphically adding vectors is illustrated for the two displacements of the person walking in a city considered in the previous lesson. (a) Draw a vector representing the displacement to the east. (b) Draw a vector representing the displacement to the north. The tail of this vector should originate from the head of the first, east-pointing vector. (c) Draw a line from the tail of the east-pointing vector to the head of the north-pointing vector to form the sum or resultant vector \(\mathbf{D}\). The length of the arrow \(\mathbf{D}\) is proportional to the vector’s magnitude and is measured to be 10.3 units . Its direction, described as the angle with respect to the east (or horizontal axis) \(\theta \) is measured with a protractor to be \(\text{29}\text{.}1º\).
Users are encouraged to open the Concept Builder and begin. There is no need for an activity sheet for this Concept Builder. However, the nine different groups of questions can be printed. View Questions (Teachers Only). Lebedev AA, Krause MH, Isidro AL, Vagin A, Orlova EV, Turner J, Dodson EJ, Tavares P, Antson AA (2007) Structural framework for DNA translocation via the viral portal protein. EMBO J 26:1984–1994 The result of adding 11 km, north plus 11 km, east is a vector with a magnitude of 15.6 km. Later, the method of determining the direction of the vector will be discussed. The proximodistal principle states that physical and cognitive development proceeds from the center of the body outward, towards the extremities. This concept suggests that development begins with trunk control and progresses to more distal parts, such as arms and legs. Additionally, this principle applies to the development of fine motor skills, such as grasping an object or using a tool. It is thought that this pattern of development allows for greater control and coordination of body movements which in turn aid in learning new skills and tasks. What is the Meaning of Proximodistal?Farley MM, Tu J, Kearns DB, Molineux IJ, Liu J (2017) Ultrastructural analysis of bacteriophage Φ29 during infection of Bacillus subtilis. J Struct Biol 197:163–171 If you live in Maple Ridge or the surrounding area and need a trusted veterinarian to care for your pets – look no further. Dr. Martin Rebele is a licensed B.C. veterinarian, treating all types of pets. Your pets’ health and well being are very important to us, and we take every possible measure to give your animals the care they deserve. The proximodistal trend is a pattern of motor skill development that begins with the core of the body and progresses outward. This refers to the development of skills such as head control, trunk control, shoulder control, and then arm and finger movement. It can be seen in infants as they learn to control teir heads before their shoulders or arms, and then their fingers. In older children and adults, it is seen in sports activities or other physical activities in which skills develop from the core outward. For example, a tennis player may start by learning how to move their legs correctly before learning how to move their arms and hands for a serve. Importance of Cephalocaudal Development Using a ruler, measure the length of the resultant and determine its magnitude by converting to real units using the scale (4.4 cm x 20 m/1 cm = 88 m). Tang J, Olson N, Jardine PJ, Grimes S, Anderson DL, Baker TS (2008) DNA poised for release in bacteriophage phi29. Structure 16:935–943
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