Asymmetric reconstructions allowed the authors to reveal heterogeneity in the hexameric ring, with only 3 to 5 subunits in an ATP-binding competent conformation. This is a very exciting finding and consistent with studies on other AAA+ ATPases, showing that only a subset of the 6 subunits has nucleotide bound at any given time. To biochemically confirm their structural data, the authors analyzed nucleotide occupancy by measuring ADP binding in ITC experiments. However, it is unclear to me why they used ADP and not ATPγS, or ATP in combination with a hydrolysis-deficient Walker-B mutant of AAA-2. As the authors well know, ADP is not able to induce an active ring conformation that can bind substrate or the ClpP peptidase. The conformation of an ADP-bound subunit is likely more similar to an empty state than to an ATP-bound state. Therefore, the overall ring-conformation probed in the ITC experiment with ADP probably differs significantly from the hexamer state observed by EM in the presence of ATP, and it is questionable whether an ADP titration experiment indeed provides the right answer regarding the nucleotide occupancy. The authors should consider repeating the ITC experiment for instance with ATP and a Walker-B mutant AAA-2 that is deficient in hydrolysis but can adopt all possible conformations depending on its occupancy. Draw the resultant from the tail of the first vector to the head of the last vector. Label this vector as Resultant or simply R.

Proximodistal is a term used to describe the direction of development or maturation of motor skills. It refers to the tendency for individuals to acquire motor skills from the center of their body outward, progressing towards their extremities. For example, when children are learning to move, they may first learn how to move their head, trunk, and arms before they learn how to move their hands and feet. This progression is known as proximodistal development. Examples of Proximodistal and Cephalocaudal Pattern Using the BAP complex facilitates orientation determination in EM reconstruction, as previously observed for ClpA-ClpP ( Effantin et al., 2010). Side views of the elongated BAP-ClpP complex are easily recognisable ( Figure 1B) whereas in 2D projections of ClpB alone, which has a globular shape, it is hard to distinguish side from tilted views. By restricting the dataset to side views the angle assignment is more reliable, and these views are sufficient to generate the full 3D structure ( Figure 1C). Using H/D exchange experiments, which report on the solvent accessibility and structural flexibility of amide hydrogens, we found that BAP, either alone or bound to ClpP, displays the same protection pattern as ClpB, implying the same MD conformation ( Figure 1—figure supplement 1).

Isidro A, Santos MA, Henriques AO, Tavares P (2004b) The high-resolution functional map of bacteriophage SPP1 portal protein. Mol Microbiol 51:949–962 A step-by-step method for applying the head-to-tail method to determine the sum of two or more vectors is given below. Orlova EV, Gowen B, Dröge A, Stiege A, Weise F, Lurz R, van Heel M, Tavares P (2003) Structure of a viral DNA gatekeeper at 10 Å resolution by cryo-electron microscopy. EMBO J 22:1255–1262 Then it performs the vector addition, which is very simple and where the vector sum can be expressed as follows: Step 6. To get the direction of the resultant, measure the angle it makes with the reference frame using a protractor. (Note that in most calculations, we will use trigonometric relationships to determine this angle.)

T1 - Head-to-tail cyclization of side chain-protected linear peptides to recapitulate genetically-encoded cyclized peptides 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 91:1164–1178A) Basal and substrate-stimulated ATPase activities of ClpB wild type and indicated MD deletions were determined in the absence and presence of casein. Relative ATPase activations by casein were calculated (inset). ( B) Unfolding of Casein-YFP by BAP wild type and indicated MD deletions in the presence of ClpP was monitored by YFP fluorescence. Initial YFP fluorescence was set to 100%. ΔM1: ΔE410-Y455, ΔM2: ΔS456-E520, ΔM1/M2: ΔE410-E520. ( C) E. coli ΔclpB cells expressing the indicated plasmid-encoded clpB alleles under control of an IPTG-regulatable promoter were grown overnight at 30°C. Various dilutions (10 −1–10 −7) were spotted on LB plates containing the indicated IPTG concentration and incubated at 37°C for 24 hr.

Cardarelli L, Maxwell KL, Davidson AR (2011) Assembly mechanism is the key determinant of the dosage sensitivity of a phage structural protein. Proc Natl Acad Sci U S A 108:10168–10173 Grundy FJ, Howe MM (1985) Morphogenetic structures present in lysates of amber mutants of bacteriophage Mu. Virology 143:485–504 Head-to-Tail Addition is a Concept Builder that provides learners with an exercise in recognizing the proper vector addition diagram for a given equation. To be successful with the activity, learners will need to understand the basics of constructing a head-to-tail (or tip-to-tail) vector addition diagram. There are 18 total questions organized into nine Question Groups and spread across three levels of difficulty. Each question provides a vector addition equation involving the addition of three vectors. The magnitude and direction of each vector is shown. Learner must toggle through six diagrams and identify the one that is consistent with the given vectors and the given vector addition diagram. Bazinet C, King J (1985) The DNA translocating vertex of dsDNA bacteriophage. Annu Rev Microbiol 39:109–129It is true that the ADP conformation does not support substrate and ClpP interaction. Both interactions are mediated by flexible loop structures located either at the central channel or at the bottom of the ClpB ring. Nucleotide-driven conformational changes of these mobile elements do not however necessarily reflect substantial conformational changes within the bulk of the AAA+ domains. In fact, soaking of ClpX crystals with either ATPγS, ATP or ADP led to comparable structural changes and ADP, presumably generated by residual hydrolysis, fits best the electron density of an ATPγS-soaked crystal [Glynn, SE, et al, Cell, 2009]. Notably, soaking of ClpX with nucleotides led to asymmetric binding, supporting a model in which ADP also binds asymmetrically to ClpB hexamers. Similarly, binding of either ATP, ATPγS or ADP to ClpX led to the same degree of FRET changes in an experimental setup monitoring the formation of “L” subunits, which show tighter interaction between the large and small subunit of the AAA domain [Stinson, BM, et al, Cell, 2013]. Together these data indicate that nucleotides, irrespective of their identity, cause similar overall structural changes in AAA+ domains. The Pythagorean theorem is a useful method for determining the result of adding two (and only two) vectors that make a right angle to each other. The method is not applicable for adding more than two vectors or for adding vectors that are not at 90-degrees to each other. The Pythagorean theorem is a mathematical equation that relates the length of the sides of a right triangle to the length of the hypotenuse of a right triangle. Veesler D, Cambillau C (2011) A common evolutionary origin for tailed-bacteriophage functional modules and bacterial machineries. Microbiol Mol Biol Rev 75:423–433 Katsura I, Tsugita A (1977) Purification and characterization of the major protein and the terminator protein of the bacteriophage lambda tail. Virology 76:129–145 A variety of mathematical operations can be performed with and upon vectors. One such operation is the addition of vectors. Two vectors can be added together to determine the result (or resultant). This process of adding two or more vectors has already been discussed in an earlier unit. Recall in our discussion of Newton's laws of motion, that the net force experienced by an object was determined by computing the vector sum of all the individual forces acting upon that object. That is the net force was the result (or resultant) of adding up all the force vectors. During that unit, the rules for summing vectors (such as force vectors) were kept relatively simple. Observe the following summations of two force vectors: