This article needs additional citations for. Unsourced material may be challenged and removed. (April 2009) () Arrow pushing or electron pushing is a technique used to describe the progression of mechanisms. It was first developed. In using arrow pushing, 'curved arrows' or 'curly arrows' are superimposed over the of reactants in a to show the.

The arrows illustrate the movement of as between are broken and formed. Arrow pushing is also used to describe how positive and negative are distributed around through. It is important to always remember, however, that arrow pushing is a formalism and electrons (or rather, electron density) do not move around so neatly and discretely in reality. Recently, arrow pushing has been extended to, especially to the chemistry of s- and p- elements. It has been shown to work well for compounds. Trajectory of single electron When a bond is broken, electrons leave where the bond was and this is represented by a curved arrow pointing away from the bond and ending the arrow pointing towards the next unoccupied molecular orbital. Similarly, organic chemists represent the formation of a bond by a curved arrow pointing between two species.

For clarity, when pushing arrows, it is best to draw the arrows starting from a lone pair of electrons or filled bonds (sigma, pi) and ending in an unfilled molecular orbital, allowing the reader to know exactly which electrons are moving and where they are ending. Breaking of bonds [ ] A joining atoms in an organic molecule consists of a group of two electrons.

Such a group is referred to as an electron pair. Reactions in organic chemistry proceed through the sequential breaking and formation of such bonds. Organic chemists recognize two processes for the breaking of a chemical bond. These processes are known as homolytic cleavage and heterolytic cleavage. Homolytic bond cleavage [ ] Homolytic is a process where the electron pair comprising a bond is split, causing the bond to break. This is denoted by two single barbed curved arrows pointing away from the bond. The consequence of this process is the retention of a single unpaired electron on each of the atoms that were formerly joined by a bond.

Pushing electrons: a guide for students of organic. By Daniel P Weeks Pushing electrons: a guide for students of organic chemistry. By Daniel P Weeks; Arthur Winter. Fourth edition. Belmont, CA: Brooks/Cole. Pushing electrons, 3. Pushing electrons by Daniel P Weeks. Pushing electrons.

These single electron species are known as. For example, light causes the -chlorine bond to break homolytically. This is the initiation stage of. Heterolytic bond cleavage [ ] Heterolytic bond cleavage is a process where the electron pair that comprised a bond moves to one of the atoms that was formerly joined by a bond. The bond breaks, forming a negatively charged (an ) and a positively charged species (a ).

The anion is the species that retains the electrons from the bond while the cation is stripped of the electrons from the bond. The anion usually forms on the most atom, in this example atom A.

Heterolytic reaction mechanisms [ ] All heterolytic organic chemistry reactions can be described by a sequence of fundamental mechanistic subtypes. The elementary mechanistic subtypes taught in introductory organic chemistry are S N1, S N2, E1, E2, addition and addition-elimination. Using arrow pushing, each of these mechanistic subtypes can be described. S N1 reactions [ ] An occurs when a molecule separates into a positively charged component and a negatively charged component. This generally occurs in highly polar through a process called. Manual For Bearcat 210 Scanner.

The positively charged component then reacts with a forming a new compound. Step 2, substitution.

In the first stage of this reaction (solvolysis), the C-L bond breaks and both electrons from that bond join L (the ) to form L − and R 3C + ions. This is represented by the curved arrow pointing away from the C-L bond and towards L.

The nucleophile Nu −, being attracted to the R 3C +, then donates a pair of electrons forming a new C-Nu bond. Because an S N1 reaction proceeds with the Substitution of a leaving group with a Nucleophile, the S N designation is used. Because the initial solvolysis step in this reaction involves a single molecule dissociating from its leaving group, the initial stage of this process is considered a uni-molecular reaction. The involvement of only 1 species in the initial phase of the reaction enhances the mechanistic designation to S N1. S N2 reactions [ ] An occurs when a nucleophile displaces a leaving group residing on a molecule.

This displacement or substitution results in the formation of a new compound. E1 eliminations proceed with the Elimination of a leaving group leading to the E designation. Because this mechanism proceeds with the initial dissociation of a single starting material forming a carbocation, this process is considered a uni-molecular reaction. The involvement of only 1 species in the initial phase of the reaction enhances the mechanistic designation to E1. E2 eliminations [ ] An E2 elimination occurs when a proton adjacent to a leaving group is extracted by a with simultaneous elimination of a leaving group and generation of a double bond. E2 eliminations proceed through initial extraction of a proton by a base or nucleophile leading to Elimination of a leaving group justifying the E designation. Because this mechanism proceeds through the interaction of two species (substrate and base/nucleophile), E2 reactions are recognized as bi-molecular. Harry Potter E La Pietra Filosofale Libro Pdf Download.

Thus, the involvement of 2 species in the initial phase of the reaction enhances the mechanistic designation to E2. Addition reactions [ ] occur when nucleophiles react with. When a nucleophile adds to a simple or, the result is a 1,2-addition.

When a nucleophile adds to a conjugated carbonyl system, the result is a 1,4-addition. The designations 1,2 and 1,4 are derived from numbering the atoms of the starting compound where the oxygen is labeled “1” and each atom adjacent to the oxygen are sequentially numbered out to the site of nucleophilic addition. A 1,2-addition occurs with nucleophilic addition to position 2 while a 1,4-addition occurs with nucleophilic addition to position 4.

The initial assembly product of bacteriophage φ6, the procapsid, undergoes major structural transformation during the sequential packaging of its three segments of single-stranded RNA. The procapsid, a compact icosahedrally symmetric particle with deeply recessed vertices, expands to the spherical mature capsid, increasing the volume available to accommodate the genome by 2.5-fold. It has been proposed that expansion and packaging are linked, with each stage in expansion presenting a binding site for a particular RNA segment.

To investigate procapsid transformation, we induced expansion by acidification, heating, and elevated salt concentration. Cryo-EM reconstructions for all three treatments produced the same partially expanded particle. Analysis by cryo-electron tomography showed that all vertices of a given capsid were either compact or expanded, indicating a highly cooperative transition. To benchmark the mature capsid, we analyzed filled ( in vivo-packaged) capsids.

When these particles were induced to release their RNA, they reverted to the same intermediate state as expanded procapsids (intermediate 1) or to a second, further expanded state (intermediate 2). This partial reversibility of expansion suggests that the mature spherical capsid conformation is obtained only when sufficient outwards pressure is exerted by packaged RNA.

The observation of two intermediates is consistent with the proposed three-step packaging process. The model is further supported by the observation that a mutant capable of packaging the second RNA segment without previously packaging the first segment has enhanced susceptibility for switching spontaneously from the procapsid to the first intermediate state. Introduction Bacteriophage φ6 offers an attractive system for studying encapsidation of the segmented genomes of double-stranded RNA viruses, due to its relative simplicity. The genome is packaged into a pre-assembled procapsid, as opposed to the co-assembly of the genome and capsid proposed for reoviruses.

The selection of segments and the order in which they are packaged is also well understood, which is not the case for other segmented genome viruses in general., φ6 replication starts with an initial assembly product, the procapsid, consisting of four proteins, P1 (capsid shell ), P2 (RNA-dependent RNA polymerase ), P4 (packaging motor, ), and P7 (packaging facilitator ). The procapsid shell is an icosahedral assembly of 120 P1 subunits, with P4 hexamers bound over the recessed vertices, and P2 monomers nestled between the tips of the vertices on the inside. Then the three segments of single-stranded RNA - s, m and l - are packaged, in that order. Once all three segments are packaged, minus-strand synthesis takes place within the capsid, followed by transcription. According to a model that has been proposed for regulating the order of packaging, the pac sequence of the s segment binds to the outer surface of the procapsid, which is then packaged by the P4 motor, leading to a structural transition that exposes the binding site for the m segment. The m segment is then packaged, leading to further expansion that allows the l segment to bind and be packaged. This model suggests the existence of at least two intermediate states between the compact procapsid and the spherical morphology of the mature fully packaged capsid.

A candidate intermediate structure was reported by Butcher et al. As present in populations of isolated P14 procapsids (i.e. Containing only P1 and P4). They also observed a similar partially expanded particle in preparations obtained by extracting complete virions with salt and EDTA.

The goal of the present study was to investigate structural transformations of the φ6 procapsid by cryo-electron microscopy in the context of the three-step packaging hypothesis. However, to date, in vitro packaging of φ6 procapsids has been hampered by relatively low efficiency (2–5%)., Noting that in some other viral systems, it has been possible to induce procapsid maturation by subjecting them to appropriate physical or chemical perturbations,,, we decided to follow this approach. Thus we investigated the effects of varying several environmental parameters (pH, ionic strength, and temperature) on φ6 procapsids. We also examined procapsids with a point mutation in P1 that is capable of packaging the m segment directly, in order to ascertain whether this property has some structural correlate. To compare the conformations observed with the mature surface lattice, we studied in vivo-packaged mature capsids, both intact and after causing them to release their RNA at elevated salt concentration. Conditions for the expansion of procapsids In an earlier study in which P1’247 mutant procapsids were imaged, we observed a subpopulation of what appeared to be spontaneously expanded particles. This mutant, which harbors the E390A substitution in P1, is able to bind and package the m segment without previously packaging the s segment.

The proportion of expanded particles was somewhat variable, ranging from 10–20%, but substantially greater than for wild-type (P1247) procapsids with a similar preparatory history (. Cryo-electron micrographs of the P1’247 mutant procapsids at pH 5.0 (A) and of the P1247 wildtype procapsids heated to 60°C in 0.3 M KCl (B). Almost all particles appear expanded, however at the lower pH, some exhibit signs of disassembly. We next tested the effects of elevated temperature and ionic strength on procapsid expansion. To facilitate sampling multiple conditions, negative-staining electron microscopy was used.

Preparations of both P1247 and P1’247 procapsids showed increased levels of expanded particles after incubation for 10 minutes at temperatures in the range, 20–70°C (micrographs from selected temperature experiments are presented in and ). In general, the percentage of expanded particles increased with rising temperature, although the data had considerable scatter, probably as a result of aggregation removing ill-defined fractions of capsids from the suspension. Without any added salt, only about 10% of wild type procapsids expanded at 50 C, increasing to ~30% above 60 C (); no particles of any kind were observed at 80 C.

At 0.3 M KCl, the expanded particles increased to ~50% at 50 C, and up to 90% above 60°C (), again, none were observed at 80 C. For the P1’247 mutant in 0.3 M KCl, most particles (>90%) were already expanded at 40 C (). We did not see any evidence of reversibility of procapsid expansion in a sample observed after cooling it down to 20 C, following incubation at 65 (data not shown). Differential scanning calorimetry In order to probe procapsid stability and to ascertain whether an energetic transition that could be correlated with expansion might be observed, we performed differential scanning calorimetry (DSC) ().

With wild-type procapsids at concentrations above 0.2 mg/mL protein, the thermograms were compromised by large exothermic excursions, indicative of severe aggregation effects (not shown). This problem was solved by limiting runs to lower concentrations (0.1 mg/mL) but at the cost of lower signal-to-noise ratios. Procapsids in low-salt conditions, which poorly promote expansion in a heated sample (), exhibited an endothermic event at 70 C that we take to represent thermal denaturation of the procapsid. In 0.3 M salt, this event shifted to 74 C and we interpret it as denaturation of the expanded particle. We observed no event at lower temperature that could potentially represent expansion. However, as transitions other than denaturation tend to involve enthalpy exchanges of less than 10% of that of capsid denaturation (~5% in the case of P22, 2–10% in the case of HK97 ), the prospects to observe such an event were low on account of both the weak signal at low protein concentration and expansion being spread over a wide temperature interval.

Reconstructions of expanded procapsids We performed 3D reconstructions of spontaneously expanded P1’247 procapsids (), P1’247 procapsids subjected to acidic pH (), P1247 procapsids heated in high salt (), and several other conditions (see for details). The damage to and aggregation of P1’247 procapsids subjected to low pH treatment limited the numbers of particles in micrographs that were suitable for reconstruction and hence the resolution of the reconstructions (, ). All the reconstructions of expanded particles are indistinguishable ( and ), suggesting that the same conformation of the P1 shell is reached through each of the expansion-inducing treatments. Comparisons of the expansion intermediate reconstructions by Fourier shell correlation (0.3 cutoff): 1–6: first expansion intermediate; 7–8: second expansion intermediate. (The gray cells present the most similar comparisons) Despite the relatively low resolution of the reconstruction of P1’247 procapsids at low pH, the densities associated with P2 and P4 are present.

This indicates that at least some of these proteins remain attached during acidification. In contrast, all reconstructions from particles expanded spontaneously or by heat and salt (such as those in and C and others not shown) exhibit no P2-related densities on the inside, and very little P4-related density above the vertices. Prior experience has been that retention levels of P4 are variable,,, implying that P4 is dislodged relatively easily (a property used to prepare P4 from nucleocapsids ). Although we observed detachment of P4 molecules from P1247 procapsids after storage at 4°C for two weeks (), there is no reason to believe that expansion per se causes detachment of P4, as the protein is well represented on mature filled particles (see below). This point is considered further in the Discussion section.

The dearth of P2 densities in these expanded particles may be attributed to their detachment from the fixed sites that they occupy in the procapsid. In tomograms, internal densities of appropriate size for P2 molecules are seen (), but not arranged in consistent positions and consequently smeared to invisibility in icosahedrally symmetrized reconstructions. Discussion Packaging of the φ6 genome follows a specific program, whereby the three segments are packaged in the order: s → m → l. During packaging and minus-strand synthesis, the procapsid expands by 150% in internal volume, enabling it to accommodate the complete genome (). A current model holds that the pac site of each segment recognizes a particular conformational state of the P1 shell, and switching between these states specifies the order of packaging. This model predicts that there should be at least four conformational states of the capsid: the procapsid; and maturing particles respectively packaged with the s, s+m, and s+m+l segments.

Throughout the packaging process, the P4 NTPase remains bound to the outer surface at the fivefold vertices, while P2, the internalized polymerase, replicates the genome during or after the last packaging step. Here we endeavored to shed light on the structural transitions of which the procapsid is capable. P2 detaches from the inner surface during expansion P2 binds to the inner surface of the procapsid near the three-fold axes of symmetry., In most reconstructions of expanded procapsids and all reconstructions of RNA-filled and emptied capsids, we could find no trace of P2-related density (, ). However, tomography revealed numerous randomly distributed P2-sized densities inside expanded procapsids ( and ).

It follows that most if not all P2 molecules detach from their initial binding sites when the procapsid expands. Although the distribution of P2 in expanded particles is unclear, there is evidence that the binding of host proteins to P1 alters the activity of P2, indicating that P1 and P2 continue to interact. Sequential steps of procapsid expansion. Here we tentatively correlate the structures produced by perturbing the procapsid or the RNA-filled capsid in vitro by environmental factors (acid, heat and/or salt) with packaging intermediates that it has not. The observed reversion of emptied capsids to the two expansion intermediate conformations implies that pressure from internalized RNA is needed to push the procapsid into its fully mature conformation, and possibly also the second intermediate. In this property, the φ6 capsid differs from the capsids of dsDNA viruses such as HK97 or HSV in which only slight differences are observed between filled capsids and empty but nominally mature capsids.

The volumetric increase in the first intermediate of 112% leaves only a small further increase to the 150% of the fully packaged capsid over the procapsid (). Within this range lies the second intermediate we propose corresponds to the capsid packaged with the s and m segments, as well as the fully packaged capsid before replication (i.e., with ssRNA as opposed to the dsRNA in the particle shown in ) that we have yet to observe. In conclusion we propose that the first expansion intermediate may be equated with the conformation putatively achieved after packaging the s segment, in which the particle is competent to package the m segment. This is strongly supported by the propensity of the P1’247 mutant to expand and its ability to bind and package the m segment in the absence of the s segment. In terms of this scenario, we envisage a mixed population of procapsids with some still unexpanded and packaging the s segment, and others already expanded and packaging the m segment. Preparation of φ6 procapsids and packaged mature capsids Procapsids were produced in Escherichia coli strain JM109, using either the plasmid pLM687 to coexpress wildtype P1, P2, P4 and P7 subunits (P1247) or the plasmid pLM2541 to coexpress the E390A mutant of P1 and wildtype P2, P4, and P7 subunits (P1’247). The RNA-packaged mature capsids were prepared from the strain LM4383 and contained only segments L (6.4 kb) and S (4.0 kb, modified to contain a kanamycin resistance gene)., The capsids were purified as described previously, and stored at 4°C in buffer P (10 mM potassium phosphate, 1 mM MgCl 2, 50% sucrose, pH 7.5).

Prior to experiments, the samples were buffer exchanged into buffer T (20 mM Tris, 1 mM MgCl 2, pH 6.5) with additional salts as indicated, using Zeba-midi buffer exchange columns with a 7-kDa cutoff (Thermo Scientific, Rockford, IL). Differential Scanning Calorimetry DSC measurements were performed using a VP-DSC calorimeter from MicroCal, LLC (Northampton, MA). The instrument was run without feedback with at least 60 min equilibration times prior to, and between, the 60°C/h scans.

Samples, at concentrations over the range 0.1–1.0 mg/mL, were scanned three times from 15 to 90 °C with rapid cooling between scans. Samples at concentrations above 0.2 mg/mL exhibited deep and broad exothermic signal above ~50 °C due to precipitation of procapsids in the sample. DSC data were corrected for instrument baselines (determined by running the dialysis buffer in both reference and sample cells just prior to placing protein in the sample cell) and normalized for scan rate and protein concentration.

Data conversion and analysis were performed with Origin software (OriginLab Corporation, Northampton, MA). The baseline under denaturation peaks was approximated with a polynomial function and the peaks fitted with a non-two-state model with no ΔC p. This model considered cooperativity in procapsid denaturation (ΔH cal ≠ ΔH vHoff) that had to be included to fit the denaturation peaks reliably. Negative-stain electron microscopy For negative staining, the sample was diluted to ~0.1 mg/mL and stabilized at a chosen temperature for ~10 min. A 5 μl aliquot was deposited on a 300-mesh copper grid with a continuous carbon layer. The sample was blotted, grid washed with Mili-Q ultrapure water and stained with methylamine tungstate (Nanoprobes, Yaphank, NY). Micrographs were collected on CM120 electron microscope using a CCD camera at a magnification of 22,000x.

The fraction of expanded intermediates at each temperature was determined for 100–500 procapsids picked from several micrographs that were collected from different parts of the grid. Multi-reference classification The classification was done as described in Heymann et al. Six starting reference maps were chosen to represent the known range of P1 shell conformations: The wild type P147 procapsid, the expanded mutant P1’247 at pH 5.5, 5.0, and 7.5, and initial single reconstructions of the packaged particle at 100 and 200 mM NaCl (obtained using as reference the nucleocapsid map from the EMDB: EMD-1207. After alignment and competitive classification, only the classes derived from the P1’247 references at pH 5.5 and 7.5 and the higher salt reference map had significant numbers of particles assigned to them, and the other three were discarded.

This procedure was repeated 8 more times, each time discarding classes with very low numbers of particles, combining classes where the reconstructions were indistinguishable at the measured resolution, and generating new intermediate references by combining pairs of previous maps. The final classes of particles remained stable through the last three rounds of alignment and assignment. The final result yielded four classes, with one being the fully packaged particle and containing only 3% of the particle images. The other three were kept as representative of the data set of particles ranging from empty to some still containing RNA.