Tang J, Olson N, Jardine PJ, Grimes S, Anderson DL, Baker TS (2008) DNA poised for release in bacteriophage phi29. Structure 16:935–943 Chattoraj DK, Inman RB (1974) Location of DNA ends in P2, 186, P4 and lambda bacteriophage heads. J Mol Biol 87:11–22 The head-to-tail method is employed as described above and the resultant is determined (drawn in red). Its magnitude and direction is labeled on the diagram. SCALE: 1 cm = 5 m Proximodistal is a term used in anatomy to describe the direction of growth or development from the center of the body or a local structure outwards towards its distal ends. This term is often used to refer to the growth of limbs, for example, where the shoulder joint is located more proximally than the fingertip. It can also be used to describe how certain structures develop and grow throughout the body, such as organs or muscles. In general, it describes an outward-growing pattern from a central point. Understanding Proximodistal Development Liu X, Zhang Q, Murata K, Baker ML, Sullivan MB, Fu C, Dougherty MT, Schmid MF, Osburne MS, Chisholm SW, Chiu W (2010) Structural changes in a marine podovirus associated with release of its genome into Prochlorococcus. Nat Struct Mol Biol 17:830–836

Bhardwaj A, Olia AS, Walker-Kopp N, Cingolani G (2007) Domain organization and polarity of tail needle GP26 in the portal vertex structure of bacteriophage P22. J Mol Biol 371:374–387 Cardarelli L, Lam R, Tuite A, Baker LA, Sadowski PD, Radford DR, Rubinstein JL, Battaile KP, Chirgadze N, Maxwell KL, Davidson AR (2010a) The crystal structure of bacteriophage HK97 gp6: defining a large family of head-tail connector proteins. J Mol Biol 395:754–368 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. Saigo K (1975) Tail-DNA connection and chromosome structure in bacteriophage T5. Virology 68:154–165 Katsura I (1987) Determination of bacteriophage lambda tail length by a protein ruler. Nature 327:73–75

Referenced on Wolfram|Alpha

Arnaud CA, Effantin G, Vivès C, Engilberge S, Bacia M, Boulanger P, Girard E, Schoehn G, Breyton C (2017) Bacteriophage T5 tail tube structure suggests a trigger mechanism for Siphoviridae DNA ejection. Nat Commun 8:1953 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.

Cuervo A, Carrascosa JL (2011) Viral connectors for DNA encapsulation. Curr Opin Biotechnol 23:529–536 The magnitude and direction of the sum of two or more vectors can also be determined by use of an accurately drawn scaled vector diagram. Using a scaled diagram, the head-to-tail method is employed to determine the vector sum or resultant. A common Physics lab involves a vector walk. Either using centimeter-sized displacements upon a map or meter-sized displacements in a large open area, a student makes several consecutive displacements beginning from a designated starting position. Suppose that you were given a map of your local area and a set of 18 directions to follow. Starting at home base, these 18 displacement vectors could be added together in consecutive fashion to determine the result of adding the set of 18 directions. Perhaps the first vector is measured 5 cm, East. Where this measurement ended, the next measurement would begin. The process would be repeated for all 18 directions. Each time one measurement ended, the next measurement would begin. In essence, you would be using the head-to-tail method of vector addition. To see how the method works, consider the following problem: Eric leaves the base camp and hikes 11 km, north and then hikes 11 km east. Determine Eric's resulting displacement. 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 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.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. 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. Guasch A, Pous J, Ibarra B, Gomis-Rüth FX, Valpuesta JM, Sousa N, Carrascosa JL, Coll M (2002) Detailed architecture of a DNA translocating machine: the high-resolution structure of the bacteriophages phi29 connector particle. J Mol Biol 315:663–676 in vivo screens for inhibitors of any target protein of interest. In particular, the Split Intein Circular Ligation of Protein and Peptides (SICLOPPS) system exploits spontaneous protein splicing of inteins to produce intracellular cyclic peptides. A previous SICLOPPS screen against Aurora B kinase, which plays a critical role during chromosome segregation, identified several candidate inhibitors that we sought to recapitulate by chemical synthesis. We describe the syntheses of cyclic peptide hits and analogs via solution-phase macrocyclization of side chain-protected linear peptides obtained from standard solid-phase peptide synthesis. Cyclic peptide targets, including cyclo-[CTWAR], were designed to match both the variable portions and conserved cysteine residue of their genetically-encoded counterparts. Synthetic products were characterized by tandem high-resolution mass spectrometry to analyze a combination of exact mass, isotopic pattern, and collisional dissociation-induced fragmentation pattern. The latter analyses facilitated the distinction between targets and oligomeric side products, and served to confirm peptidic sequences in a manner that can be readily extended to analyses of complex biological samples. This alternative chemical synthesis approach for cyclic peptides allows cost-effective validation and facile chemical elaboration of hit candidates from SICLOPPS screens.

Tavares P, Zinn-Justin S, Orlova EV (2012) Genome gating in tailed bacteriophage capsids. Adv Exp Med Biol 726:585–600 Then it performs the vector addition, which is very simple and where the vector sum can be expressed as follows:The best exmple of cephalocaudal development is seen in the way babies grow and develop. At birth, the baby’s head is much larger than its tail, showing the beginning of growth from head to tail. As a baby grows, the head begins to take on more shape and structure while their body develops length and strength. Over time, the baby will gradually add more weight to their limbs and trunk as they begin to crawl and stand up on their own. By the time they become toddlers, their heads will be proportionally smaller than when they were born while their limbs will be longer and stronger. This gradual development from head to tail reflects the cephalocaudal pattern of growth. Difference Between Cephalocaudal and Proximodistal 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 The middle domains are known to form coiled-coils, with protein helices coiled together like the strands of a rope. However, previous efforts to work out the structure of the ClpB complex did not clearly establish where these coiled-coils were positioned relative to the rest of the ring. Thomas JO (1974) Chemical linkage of the tail to the right-hand end of bacteriophage lambda DNA. J Mol Biol 87:1–9

For cryo EM of ClpB alone, the views were randomly oriented and the initial strategy was to extract clearly identifiable side views by MSA and classification. 7606 side views were used to generate a starting model by angular reconstitution, which was refined to 29 Å resolution ( Figure 1—figure supplement 3) by projection matching. And of course, you can use this calculator to calculate vector difference as well, that is, the result of subtracting one vector from another. This is because the vector difference is a vector sum with the second vector reversed, according to: Trus BL, Cheng N, Newcomb WW, Homa FL, Brown JC, Steven AC (2004) Structure and polymorphism of the UL6 portal protein of herpes simplex virus type 1. J Virol 78:12668–12671 Casjens S, Hendrix R (1988) Control mechanisms in dsDNA bacteriophage assembly. In: Calendar R (ed) The bacteriophages, vol 1. Plenum Press, New York The BAP hexamer has overall outer dimensions of ∼150 × 100 Å, similar to previous structures of ClpB/Hsp104 ( Parsell et al., 1994; Lee et al., 2003; Wendler et al., 2007, 2009; Figure 1C). It encloses a ∼30 Å wide central channel, comparable in size to that in the crystal structure of ClpC ( Wang et al., 2011; Figure 1C,D). In the reconstruction it is possible to identify regions accounting for all the domains, such as L-shaped densities for the AAA+ domains and a rod-like density for the coiled-coil MD.

Vector Addition: Head-to-Tail Method

The reviewers are correct: we do not see a decrease in donor fluorescence while observing acceptor fluorescence when determining FRET upon ClpB oligomerization in absence of ATP. A correlation between loss and gain of donor and acceptor fluorescence is observed in presence of ATP and in all cases for the repressed ClpB-E432A variant, which shows stronger motif 1–motif 2 interactions. We cannot provide a straightforward explanation for this difference, which is now stated in the Results section. Bazinet C, King J (1985) The DNA translocating vertex of dsDNA bacteriophage. Annu Rev Microbiol 39:109–129 Olia AS, Casjens S, Cingolani G (2007b) Structure of phage P22 cell envelope-penetrating needle. Nat Struct Mol Biol 14:1221–1226