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Ramona Rousseau-Reid and a few of her neighbors had just formed a group called the Eastwick Action Committee (EAC) when a resident spotted engineers doing land survey work on a vast parcel of wooded land between 86th Street and the John Heinz National Wildlife Refuge.
The neighbor asked what they were doing and the surveyors said they were hired by the Korman Company. Turned out the City government gave Korman permission to develop this land decades ago as part of the transformation of Eastwick from lower-density marshland to suburban style housing. Now they were finally developing it. Two EAC members went directly to the Korman office to ask about it, and learned that they planned to build 722 new apartments and 1034 new parking spaces on floodplain land in their community.
"We were horrified when we got the news," Ramona said. EAC found out that Korman needed to change the lot's zoning from single-family homes to multi-family in order to build apartments. Because Eastwick didn't have a Registered Community Organization (RCO) to host a public meeting to vote on whether apartments should be built, Korman could go straight to the District Councilman for approval. Ramona knew she and her neighbors needed to act fast, though she and some EAC members hadn't been part of community groups since the 1990s and "didn't know each other that well yet."
EAC contacted the Friends of Heinz Refuge (FOHR), who were equally concerned. Though both groups were made up of different people, "we saw we had a lot in common in terms of our interest in land use and the Eastwick community." They decided to "form a united front" as the Eastwick Friends & Neighbors Coalition (EFNC) around stopping the development from happening as it was proposed, which they believed would have major negative environmental impacts on the community.
Environmentalists use advocacy frequently, so the Friends were able to connect Ramona and EFNC to other professionals that helped them advocate for their cause, including the Delaware River Keeper Network, Keystone Conservation Trust, Audubon Pennsylvania, Darby Creek Valley Association, and the Pennsylvania chapter of the Sierra Club.
"Tonight's theme is: FLOODING" at a July 2012 EFNC educational meeting
"Vote NO to Rezoning" at a July 2012 EFNC educational meeting
Amy Laura Cahn from the Public Interest Law Center presents about the environmental issues of the proposed development
The Friends and these other partners educated EFNC on the details of the environmental dangers posed by this development, which Ramona recounts took "hours of meetings." During this organizing process, EFNC also wrote a mission statement, elected officers, and retained free legal counsel from the Public Interest Law Center and Dechert LLP.
Korman was scheduled to present at a EFNC community meeting. Ramona knew she needed to pack these meetings with neighbors if there was any hope of changing this development. She and fellow EFNC board members had to meet all their neighbors, tell them the complicated story of Eastwick and its place on environmentally important marshland, and then convince them to take time out of their personal schedules to protest a development that had been in the works and approved by the City for decades.
Ramona and fellow EFNC members table to meet and educate neighbors
EFNC members table to meet and educate neighbors
Ramona & fellow EFNC members table to meet and educate neighbors
Ramona and EFNC set out to reach all 5,500 Eastwick residents and did just that! They personally financed necessary supplies and sometimes borrowed printer paper from their workplaces to make flyers to hand out door-to-door. They also waited outside the Eastwick Regional Rail station and the Penrose shopping mall parking lot in all weather, where they casually started conversations asking if people knew about the historic problems the neighborhood has had with flooding. Ramona even hopped on the SEPTA Regional Rail train with people sometimes to continue talking, and then turned around and came back!
Ramona described:
We knew we needed to educate people before the meeting, so we took as long as it took to have conversations with any residents who had questions. It was so imperative that residents [came to meetings]. We'd tell them 'You must have a voice, we have to stand strong.'
Thanks to four full days of outreach, there was a huge turnout at the community meeting. Korman was surprised by the emotional reaction of neighbors to the proposed development, which was not only because of the environmental risks and the fact that "you can't build like that in the floodplain, that's preposterous," as Ramona put it.
Korman representatives present their proposed development at an EFNC meeting
EFNC meeting attendees vote in opposition to rezoning the parcel for Korman's proposed development
Their reaction was also due to the fact that this proposal was yet another missed opportunity for the economic development neighbors have been promised since the mid-20th century when thousands of residents were displaced by the promise of urban renewal in their neighborhood that never happened.
The next step was outreach to the Councilman's office. EFNC called his office directly to ask for a meeting to educate the relatively new Council member on Eastwick's history and environmental concerns. They invited him to a community meeting and packed the house; by that time, neighbors recognized Ramona and EFNC outreach team members and knew how important this was. "We needed a movement," Ramona said.
The Councilman still introduced the zoning bill anyway, but environmentalists, their free lawyers, and over 100 neighbors attended the public hearing and gave two dozen testimonies. The Councilman tabled the bill that day and eventually pulled it altogether.
Ramona described:
They didn't expect the community to be as forceful as we did. They had to hear us
Since tackling the Korman development, EFNC has become a Registered Community Organization, received various City grants, been awarded the Empowerment Award from The Bread & Roses Funding, and still works actively to make sure neighborhood interests are well represented in any future planning and development efforts. The Councilman now notifies the group when there are any potential projects in the neighborhood.
In December 2015, Ramona and other EFNC members were there when the Philadelphia Redevelopment Authority terminated their agreement with Korman, returning that large piece of marshland back to City ownership. "Some of us were teary eyed...shows that when you know something to be right in your spirit, if you totally commit to it, people will see that passion and come to your aid."
Ramona calls the EAC's leaders "a great unit of friendship and collaboration" and she still lives by her "do what you have to do" approach to outreach, making whatever time is needed to make sure the community has a voice.
If you see something happening in your neighborhood, ask about it! If neighbors hadn't spoke with surveyors on Korman site, residents may not have known about development proposal until it was too late for them to act.
If you need to speak to a large company or City official, pick up the phone or show up at their door.
Work with partners: find out who can help you and has strengths in areas you don't. Friends of Heinz Refuge and other environmental experts educated Eastwick residents and Councilman Johnson about complex environmental concerns surrounding the proposed development.
Work with City Council when large developments are proposed; if you let them know your needs and opinions, they can be an ally.
Learn the City's rules for development and zoning, as well as when you have influence.
A big turnout at public meetings and Council hearings is a great way to make the community voice heard.
Use face-to-face communication to reach out to neighbors. Ramona and EFNC door knocked and went to shopping mall parking lots and train stations to talk to neighbors about her cause.
Get to know your neighbors and earn their trust. Ramona and EFNC did this in a remarkably short period of time by putting in long hours doing face-to-face outreach and hosting meetings, and showing results.


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At the age of digital information and social media, online or virtual jobs are currently in demand. I myself am a freelancer and I work online, but before I got my full time job, I used to work part-time jobs and it was really difficult for me to look for online jobs because it's either I lack the required skills for the job or other candidates have beaten me to it. Yes, if you apply first, there's a big possibility that they will hire you. It's really challenging to look for the perfect one that can work well with their time, experience, and salary expectation—especially professionals living in the Philippines. Good thing there's been a lot of platforms that help assist job seekers online and one platform that has clients looking for particular freelancers can be found at Rakuboss.ph.
At Rakuboss, you'll find a range of clients looking for different sidelines. We accept doctors, teachers, photographers, videographers, consultants, artists, writers, lawyers, therapists, and even fortune tellers. You name it, and we have it. Rakuboss accommodates the oddest and most in-demand workers and services to connect Filipino professionals to hundreds of clients around the globe. So, if you're actively hunting for sidelines to boost your income, here's a comprehensive overview of how things are done at Rakuboss:
Getting StartedRegister a Rakuboss account if you don't have one yet. Make sure to fill out all the important details about yourself, career, and offered services. As much as possible, be transparent. Rakuboss and our prospective clients value honesty more than anything. Besides, there are serious repercussions if you'll advertise false services.
Afterward, verify your email and navigate the website. Our homepage and your timeline can show the activities within the platform, including active clients looking for services. Visit your profile as often as you can to respond to clients and find the latest job offers.
Posting Your Sidelines
With a verified account, you can use Rakuboss services for free. You can post your sidelines and advertise your services to clients around the world without paying a service fee. What's more, there's no limit to what you can post! Rakuboss welcomes Jacks of all trades. However, avoid posting duplicate listings as our team always verify each post.
To start, click the 'Sell Your Sideline' button on the lower right portion of the website. Fill out the details of your service. Provide a straightforward title, clear photos, and a detailed description to attract clients easily. Use a landscape image that is at least 1000 (width) x 600 (height) pixels. When you're done, submit the sideline for review. Wait for the Rakuboss team to verify your information and approve the sideline.
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If you wish to transact outside the platform, you can still use your Rakuboss' account as a medium. Just click on your Rakuboss profile icon on the upper right side of the screen, hover on Selling, click My Sidelines and click the sideline you want to share. Finally, get the Url and paste it on your Social media account.
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Whenever you're ready, you can get all your earnings using our third-party payment providers such as Paypal, Dragonpay, and other local banks.

subset: commoncrawl


text:
The lovely Evergreen pine needles and berries are Petit Point and the field is Needlepoint. Soft shades of greens make up the bough. Lovely delicate shades of pink and reds make up the ribbon and the striped border.
Petit Point and Needlepoint Pillow comes with poly insert. Feathered down Stuffer available as a luxurious alternative.


text:
Preformed, self-adhesive, synthetic elastomer mastics that adhere to a number of surfaces, such as plastic, metal, glass, concrete etc.
Their strong adhesive properties allow them to affix immediately to the surface, and the mastics will stay in place without being held.
They are non-polymerisable and their properties will not change after ageing for long periods in a wide range of environments.
There is no electrolytic corrosion factor associated with these products.


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Cape Town - You could almost call Pieter-Steph du Toit the Springboks' very own version of a "Twenty20" player right now.
But before any cricket-related confusion takes root, that is simply a reminder of the starkly even split between his international caps: 20 as a starter, 20 as a substitute.
Oh yes, he also sports 20 Test points, courtesy of four tries. And, er, stands 2.00 metres according to his Wikipedia profile, if you want to creatively stretch things further. (There's a twenty in his weight, 120kg, just for cherry-on-top purposes.)
Frame those smartly-aligned statistics quickly, if you fancy … for some of them are, almost certainly, about to change dramatically.
Perhaps you shouldn't really use one match as a definitive judgement on a player. But perhaps if it is a rare-as-hen's-teeth victory over the All Blacks in New Zealand, you can.
For the foreseeable future at very least, the humble, spotlight-shirking – though perfectly pleasant when he does have to blink and chat in front of it – 26-year-old from the Swartland wheat and wine region of the Western Cape suddenly seems undroppable.
More than that, he has vaulted to the very top tier of most valued customers in the Bok ranks.
Supersub? No, those days look over for Pieter Stephanus du Toit, unless there are certain matches up the line against relative minnows – like in group play at the 2019 World Cup, for instance – where the Boks opt to restrict their most prized assets to possible impact presences, if necessary.
He was positively immense for the cause in Saturday's 36-34 Wellington upset of the world champions, almost indisputably also one of the finest teams in rugby history.
It was a match where Du Toit didn't just tick the boxes, as they say, against the very best opponents there are … he banged down his mark with the heaviest, most legible of industrial stamps.
If not already, I invite you to watch the game again (some Bok fans delightedly claim they have it on constant repeat), as I have: my only regret on second viewing was limiting my own verdict on Du Toit's team-topping performance, a task done immediately after the Rugby Championship humdinger, to 8.5 out of 10.
Now I'd be more inclined to adjust that figure to 9, or even 9.5.
In short - and it took a more concentrated, Du Toit-specific perusal of the 80 minutes (he somehow lasted the entire duration) to come to this view - just about everything the blindside flank did was quite truly "impactful".
His tackles, and according to some stats sources he made the most of any South African on a day when defence was frequent and stressful, were proper thump-backers, for want of a more orthodox word.
His carries were seldom less than bruising, constantly abrasive, too, and his laybacks agreeably swift when he took contact, often (necessarily) from multiple All Black defenders.
The Newlands-based customer's whole game just screamed of authority; as if he had been wedded to the Bok No 7 jersey for years … which, of course, he decidedly hasn't.
For Du Toit has, more accurately, had to be a fast learner in the role, probably twigging (even if with a hint of trepidation or reluctance initially?) that an extra string to his positional bow might be useful given the country's usual surplus of high-quality locks.
He has also, just as pertinently, graduated with flying colours from the school of hard knocks as far as his "re-education" is concerned, as his earliest exposure to No 7 was anything but blissful sailing.
Indeed, many less determined, perhaps more impatient players might well have quickly abandoned any thoughts of positional remodelling had they suffered certain of the relative indignities Du Toit did as he painstakingly learned the trade.
Bear in the mind the following: his first start as a flank (in his fifth Test match) coincided with one of the most tumultuous results in Springbok history: the defeat to Japan at Brighton in the 2015 World Cup.
It was all of 13 matches later in Du Toit's international career that he was exposed once more to the No 7 role … and you might well say that occasion went even worse for him.
He got a much-publicised, costly run-around - twice - from gleeful England scrumhalf Ben Youngs at Twickenham, prompting many critics to almost scornfully write him off as a utility factor and simply pigeonhole him back as a second-rower following the 37-21 roasting.
Let's give kudos to usually besieged then-coach Allister Coetzee for having the courage, 11 months later, to put a daring toe back in the water, entrusting the blond-mopped competitor with the blindside cares anew in a gallant, thrilling, Championship-ending 25-24 loss to NZ at Newlands in 2017.
Increasingly more adhesive and alert from a positioning point of view in structured defence (his more spontaneous, sometimes last-ditch defending has always been inspiring) Du Toit gave a fine display.
With increasing levels of stealth, he has looked more and more streetwise in the role subsequently, as evidenced by eight of 11 further appearances in the green and gold coming on the side of the scrum rather than within its very belly.
The stirring confirmation of his "arrival" as a blindside strongman in every sense, of course, came on Saturday when his post-match tears of joy for a landmark win became one of the most iconic photographic images from the Cake Tin fixture.
He had produced a robust, imperious showing in the jersey to make a string of illustrious predecessors proud: into that category, I'd earnestly suggest, would fall such figures from the relatively modern annals as Theuns Stofberg, Gert Smal, Andre Venter, Juan Smith, Danie Rossouw and Willem Alberts.
Du Toit is also a superior athlete to several of those - while not giving an inch to many of them in pure physical dimension and desire, either - which only adds to the burgeoning excitement around his potential to prosper still further.
Remember, he has only started 10 Test matches as the dedicated No 7 for South Africa, so continued improvement seems a racing certainty.
One thing is crystal-clear, I think: regardless of specific area of responsibility (yes, he remains a massively attractive option at No 5 lock) Du Toit henceforth starts all properly blue-chip Test matches for his country.
No more of this bench malarkey for him.
He is my early tip as a likely global stand-out at the 2019 World Cup.
Whether he cherishes such status or not, and more probably the latter, a fully-fledged international star was born in Wellington.
*Follow our chief writer on Twitter: @RobHouwing


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Backbone resonance assignment is a critical first step in the investigation of proteins by NMR. This is traditionally achieved with a standard set of experiments, most of which are not optimal for large proteins. Of these, HNCA is the most sensitive experiment that provides sequential correlations. However, this experiment suffers from chemical shift degeneracy problems during the assignment procedure. We present a strategy that increases the effective resolution of HNCA and enables near-complete resonance assignment using this single HNCA experiment. We utilize a combination of 2-13C and 3-13C pyruvate as the carbon source for isotope labeling, which suppresses the one bond (1Jαβ) coupling providing enhanced resolution for the Cα resonance and amino acid-specific peak shapes that arise from the residual coupling. Using this approach, we can obtain near-complete (>85%) backbone resonance assignment of a 42 kDa protein using a single HNCA experiment.
Backbone resonance assignment is a necessary initial process in structural, dynamic and interaction studies of proteins by NMR. This has traditionally been achieved with a standard set of triple-resonance experiments1, supplemented by newer methods including hNcocaNH2,3, HncocaNH4 and the hNCAnH5. For small or homo-multimeric proteins, projection experiments such as APSY6 help resolve ambiguities in the chemical shift space by delineating a backbone spin system into high-dimensional correlations. However, most of these experiments harbor long-coherence transfer steps and are not optimal for large proteins (>40 kDa) due to relaxation-based loss of coherences. Deuteration in combination with TROSY-based backbone triple-resonance experiments is the standard practice to combat fast relaxation in large proteins7,8. However, multidimensional experiments with long-coherence transfer steps and multiple chemical shift labeling for 13C nuclei still suffer from relaxation loss before the detection period9.
Among the triple-resonance experiments, the HNCA10 is the most sensitive experiment that connects spin systems from internal to sequential residues. In the HNCA experiment, including the TROSY variation, the 13Cα coherence is transverse only during the chemical shift-encoding period, thus minimizing relaxation losses. In comparison, the sensitivity of the HNCACB experiment is theoretically and experimentally approximately one-fourth to that of the HNCA experiment, for high molecular weight proteins at high field9. The HNCA has all the necessary and sufficient information to complete sequence specific assignment. In practice, however, unique assignment of all backbone spin systems from this experiment alone is not possible due to insufficient dispersion of the 13Cα chemical shifts, resulting in multiple degeneracies in peak positions. This degeneracy scales with the protein size and concomitant increase in the number of spins within the given spectral space. This degeneracy problem can be alleviated by increasing resolution in the 13Cα dimension. Since the dipole-dipole interaction between the 13Cα and its attached 1Hα is the main source of 13Cα relaxation, deuteration of a protein is the immediate way to improve resolution of 13Cα signals (Supplementary Figure 1)11,12. Theoretical calculations reveal that the transverse relaxation rate of 13Cα in the deuterated case is ~8 times slower than in the non-deuterated case. The estimated line widths of 13Cα that can be achieved for 40 and 80 kDa deuterated proteins at 800 MHz are 4.4 and 8.8 Hz, respectively, at 298 K (Supplementary Figure 1). Narrow 13Cα line widths can be obtained with a relatively long indirect evolution period (>2 times T2), which corresponds to a maximal 13Cα evolution time of 114 ms (684 complex points for 4.4 Hz digital resolution) and 57 ms (342 complex points for 8.8 Hz digital resolution) assuming a 13Cα sweep width of 6000 Hz and one zero fill prior to the Fourier transform. Experimentally, correlation times (τc) of two large NMR model systems, Maltose Binding Protein13 (MBP, 42 kDa) and Malate Synthase G protein14 (MSG, 82 kDa), have been determined using 15N relaxation measurements. MBP and MSG have τc values of 16.2 ns and 36 ns, respectively, at 310 K. These correlation times give theoretical 13Cα line widths of 3.0 Hz and 6.6 Hz at a magnetic field of 800 MHz, assuming the proteins are deuterated. While these resolutions cannot be obtained in a reasonable time with uniform sampling of the Nyquist grid, they can be theoretically achieved using non-uniform sampling (NUS)15,16,17. Although deuteration coupled with NUS permits access to high-resolution (4–8 Hz) in the 13Cα dimension, in practice, this resolution cannot be achieved because of the inherent Cα–Cβ coupling (1Jαβ), which limits the resolution to 35–40 Hz. This can be seen in the 2D plane of an HNCA experiment on the Protein G B1 domain (GB1)18, uniformly labeled using 13C-glucose (U-13C 2H glucose) and deuterated. (Supplementary Figure 2A).
There have been several efforts to combat this resolution limit including band selective 13Cβ decoupling19, post-acquisition virtual decoupling,20 and constant time evolution21. Each of these techniques have their own drawbacks. Selective 13Cβ decoupling often affects the Cα resonances of glycine, serine, threonine, and proline residues due to their proximity to the decoupled frequencies (Supplementary Figure 2B). Virtual decoupling demands a uniform 1Jαβ coupling across all spin systems and even small differences (~3 Hz) from the chosen deconvolution value will result in non-ideal line shapes, especially when long acquisition times are employed (Supplementary Figure 2C-F). While the constant time (CT) approach removes the coupling, it comes at a substantial cost of sensitivity due to the long delays (~60 ms (2xCT) or ~90 ms (3xCT)) required to achieve high resolution. In constant time experiments, the initial point of the free induction decay (FID) experiences relaxation losses corresponding to the entire CT period22. This loss in sensitivity can be seen even for small proteins like GB1 (Supplementary Figure 2G) and is prohibitive for large proteins.
Yet another approach to remove this 1Jαβ coupling is to use the Stereo-Array Isotope Labeling (SAIL) strategy23 or to use 1-13C-labeled or 2-13C-labeled glucose as the carbon source24. The former approach is expensive, the latter suffers from a low maximum labeling rate (<50%). Given these challenges, engineering an inexpensive and efficient way of labeling proteins with 13C at the Cα position and 12C at Cβ would vastly improve the resolution of the sensitive HNCA experiment.
Isotopic labeling using amino acid precursors such as glycerol and pyruvate has been previously proposed to remove 1JCC couplings25,26,27,28. When pyruvate is used as the only carbon source for Escherichia coli growth, it is metabolized via the gluconeogenesis pathway and the TCA cycle to produce amino acids (Fig. 1). Eleven amino acids (Fig. 1; red, LEU and VAL) are either synthesized directly from pyruvate or semi-directly via the gluconeogenesis pathway. These "direct" amino acids largely maintain the carbon backbone structure of pyruvate. Specifically, the C1, C2, and C3 carbons (Fig. 1) of pyruvate are incorporated into the CO, Cα, and Cβ positions respectively, in these amino acids. Therefore, the amino acids synthesized from "direct" pathways will be 13C labeled at the Cα position and 12C labeled at Cβ, if 2-13C labeled pyruvate is used as the carbon source. Leucine and valine are exceptions in the group of "direct" amino acids. In leucine, CO and Cβ are incorporated from the C2 position of pyruvate, whereas its Cα is incorporated from the C3 position of pyruvate. In valine, both Cα and Cβ are incorporated from the C2 position of pyruvate. Thus, these amino acids are treated separately, along with isoleucine as discussed below (Fig. 1; blue). It should be noted that lysine is synthesized via condensation of pyruvate and an aspartate derivative, β-aspartyl-4-semialdehyde. Since aspartate is an amino acid derived from the TCA cycle, the Cα and Cβ positions of lysine retain the labeling pattern of pyruvate. Thus, we treat lysine as a "direct" amino acid.
The remaining nine amino acids (Fig. 1, green) are synthesized from pyruvate via the TCA cycle. Asparagine, aspartate, isoleucine, methionine and threonine are synthesized from oxaloacetate (Fig. 1). Arginine, glutamine, glutamate and proline are from α-ketoglutarate (Fig. 1). For these amino acids, the C3 position of pyruvate would have a higher chance of being incorporated at the Cα position as compared to the C2 position. In addition, the TCA cycle scrambles the 13C-labeled positions for these TCA-derived amino acids, depending on the number of times the amino acid precursors have passed through the TCA cycle before incorporation into amino acids. Furthermore, the TCA cycle reintroduces the 13C–13C coupling by directly condensing oxaloacetate with acetyl-CoA at the equivalent C3 position from pyruvate. After three cycles through the TCA, a certain population of "TCA-derived" amino acids are doubly 13C labeled in both Cα and Cβ positions when 3-13C pyruvate is used.
Like other branched amino acids, isoleucine is an exception. Isoleucine is synthesized via condensation of a threonine derivative, 2-oxobutyric acid, and pyruvate at C2 position. This results in the Cβ position of isoleucine being exclusively incorporated from the C2 carbon of pyruvate. On the other hand, the 13Cα labeling rate would be the same as threonine. Therefore, Cα and Cβ of isoleucine would both be 13C labeled when 2-13C pyruvate is used as a carbon source.
We explored the use of deuterated 13C pyruvate as a carbon source with the intention of obtaining specific labeling patterns where uncoupled 13Cα nuclei could be generated, which will result in narrow lines to aid backbone assignment from a single HNCA experiment. We have acquired high-resolution HNCA spectra on pyruvate-labeled proteins up to 42 kDa using non-uniform sampling in 3–4 days. These spectra reveal very narrow, high-resolution lines in the Cα dimension, for all amino acids and can resolve systems well below the 35–40 Hz 1Jαβ carbon coupling. In addition, we show that the residual coupling, quantified by the "coupled to uncoupled peak height ratio" (C2UR), is an indicator of the metabolic origin of the amino acid and provides information about the matching sequential system. Finally, we demonstrate that the peak shape along the Cα dimension, along with the narrow, high-resolution lines, can be used to make extensive sequence specific assignments from the HNCA experiment alone.
Incorporation of 13C labeling in amino acids from pyruvate
The theoretical 13C-labeling patterns with 2-13C pyruvate and 3-13C pyruvate were experimentally verified by labeling proteins in growth media with D2O as the solvent, ammonium chloride as the 15N source and pyruvate as the 13C source (see Experimental section). The difference in the labeling pattern between "direct" and "TCA derived" amino acids is exemplified by the A22 and E21 resonances, respectively, of the protein GB1 in a high-resolution HNCA experiment with 13CO decoupling (Supplementary Figure 3). As expected, labeling with 2-13C pyruvate strongly incorporates 13C at the Cα position of alanine, an amino acid derived directly from pyruvate. There is no 1Jαβ coupling present indicating that the Cβ is not 13C labeled. On the other hand, glutamate is poorly labeled at the Cα position by 2-13C pyruvate, and there is no apparent labeling at the Cβ position. When 3-13C pyruvate is used as the carbon source, glutamate is strongly labeled and alanine is poorly labeled at the Cα position. Interestingly, the height of coupled peaks of glutamate is just as intense as the central uncoupled peak. This indicates that glutamate is synthesized predominantly in a doubly, 13Cα and 13Cβ labeled, form after at least three cycles through the TCA. The ratio of "doubly 13C" (Cα and Cβ) labeled against singly 13Cα labeled glutamate is about 2:1. It should be noted that the ratio would vary depending on the expression condition and induction period, which influences the number of passes of the precursor through the TCA cycle. Hence a protein derived from individual expression conditions would have unique peak shape for its TCA-derived amino acids, which is dependent upon the expression conditions. The small but significant 13Cα labeling of alanine with 3-13C pyruvate is rather unexpected, but can be explained by a minor amino acid synthetic pathway via glycine that is produced via a condensation of CO2 and the methylene moiety of 5,10-methylene-THF that originates from the C3 position of pyruvate.
In summary, by using 2-13C pyruvate, the strong 1Jαβ coupling can be avoided for most of the "direct" amino acids, which provide access to sensitive and high-resolution resonances without splitting from the 1Jαβ carbon coupling. However, the 13C-labeling rate at Cα is low for "TCA-derived" amino acids, which, depending on the primary amino acid sequence would result in significant gaps in the backbone assignment efforts depending on the primary amino acid sequence. In contrast, 3-13C pyruvate would mainly label the Cα position of the "TCA-derived" amino acid and thus fill the gap left by using 2-13C pyruvate, however a substantial population would be doubly 13C labeled at the Cα and Cβ positions. Thus, in addition to the central uncoupled frequency, which can provide high-resolution matching of the spin systems, pyruvate labeling results in the emergence of the residual 1Jαβ coupling, which provides unique line shapes that are specific for each spin system, in exchange for some sensitivity.
Generating resolution and amino acid-specific peak shapes
To concomitantly achieve sensitivity and the high-resolution enabled by 2-13C pyruvate with the unique labeling pattern and amino acid-specific line shape provided by 3-13C pyruvate, we decided to simultaneously use both 2-13C and 3-13C pyruvate. Along this line of thought, we tested two different approaches, post-mix and pre-mix. In the post-mix strategy, the protein was individually expressed with either 2-13C or 3-13C pyruvate as the carbon source, purified, and mixed in a 1:1 ratio to make the NMR sample. In contrast, in the pre-mix strategy we grew E. coli cultures by using an equal amount of 2-13C and 3-13C pyruvate as the carbon sources for protein expression. A graphical illustration of two strategies is shown in Supplementary Figure 3.
In the post-mix strategy, the labeling pattern would be the simple arithmetic average of the individual 2-13C pyruvate and 3-13C pyruvate labeling. This sample maintains a strong uncoupled central peak for "direct" amino acids (A22), however the signal of the "TCA-derived" amino acid (E21) is still composed of equal height coupled and uncoupled peaks (Fig. 2a, post-mix). Although the coupling gives additional line shape information, the substantial population of coupled systems significantly reduces the sensitivity of the "TCA-derived" amino acids, as described above.
For the pre-mix strategy, the scrambling caused by the TCA cycle gives a distinct pattern, different from the post-mix sample. The labeling maintains a strong uncoupled central peak for "direct" amino acids (A22), while the signal of TCA-derived amino acid (E21) has a stronger uncoupled peak when compared to post-mix (Fig. 2a, pre-mix). This is because the oxaloacetate, which is derived from 3-13C pyruvate, would have a 50% chance of being condensed with the 2-13C labeled pyruvate-derived acetyl-CoA, which prevents the recoupling of the Cα and Cβ positions and dilutes the population of doubly labeled "TCA-derived" amino acids. These effects are clearly seen in the HNCA experiments utilizing pre-mix samples (Fig. 2a, pre-mix), in which pre-mix present a stronger, uncoupled peak when compared to post-mix (Fig. 2a, post-mix).
A fitting algorithm (Supplementary Figure 4) was used to estimate the coupled to uncoupled peak height ratio (C2UR) for all spin systems in GB1 for both post-mix and pre-mix GB1 samples (Fig. 2b). The post-mix strategy is reasonably good for some of the amino acids, especially the "direct" amino acids from the gluconeogenesis pathway. However, in other cases the post-mix condition is quite poor with ratios approaching 100%, indicating all three peaks are approximately the same height. In the worst case, valine, the intensity of the uncoupled peak is vanishingly small, which prevents meaningful extraction of the C2UR. In the pre-mix strategy, the central uncoupled peak is dominant when compare to the coupled peaks, for all amino acids types. Even in the worst cases of isoleucine, leucine, and valine, the coupled peaks are only half the height of the uncoupled peaks. This would allow us to use the central narrow uncoupled resonance for frequency matching. Thus, to take advantage of its more favorable labeling patterns, which result in high sensitivity and resolution, we decided to use the pre-mix strategy to acquire high-resolution HNCA experiments.
Combining resolution and peak shape for backbone assignment
The utility and advantage of using pyruvate-labeled pre-mix samples and obtaining a high-resolution HNCA through Non-Uniform Sampling (NUS), in comparison to traditional HNCA spectra, becomes apparent even for a small protein like GB1. First, we would like to demonstrate the advantage of high resolution in removing degeneracies in Cα resonances. An HNCA experiment was acquired for the protein GB1 using NUS and the resulting spectrum was reconstructed with hmsIST16. 512 complex points were collected in the Cα dimension, spanning a sweep width of 6032 Hz, yielding a final digital resolution of ~5.8 Hz after zerofilling to 1024 points. In a separate processing effort, we truncated the 13Cα dimension of the above spectrum after reconstruction to obtain a digital resolution of ~42 Hz (corresponding to 72 complex points and zero filled to 128 points). This mimics the standard low-resolution practice that deliberately obscures the 1Jαβ coupling (Fig. 3a). The left panel shows a 1HN-13Cα strip for a system in GB1 with the right panel showing the 1D trace through the peak. Using all 512 complex points yields narrower peaks where the coupling can be seen (Fig. 3b). However, it is clear from the 2D strip and the 1D trace that this peak is composed of two sets of split peaks, one with higher intensity at low field, and the other with lower intensity at high field (see Fig. 3b, right panel, inset). No other resonances are present at or near the corresponding 1H/15N spin system in the HN plane of the spectrum, suggesting that this smaller peak is from the sequential Cα resonance and is overlapped with the intra-residue Cα resonance. Although the high resolution partially resolved the problem, the peaks cannot be readily identified if the coupling is present. In contrast, the same spectrum with identical parameters on a GB1 sample labeled with pre-mix 2-13C/3-13C pyruvate (Fig. 3c) shows two completely resolved uncoupled peaks, corresponding to the internal and sequential Cα resonances. Assignment through conventional means using multiple 3D experiments including HNCACB, have indicated that this system is A26/A25 and that these two peak shapes are consistent with the typical alanine peak shape we expect (Fig. 2). Thus, even for these two systems, which are about 34 Hz apart, one would easily resolve and identify the individual peaks by using the pre-mix strategy, but not with conventional uniform 13C labeling. This clearly demonstrates the utility of the ultra-high resolution achievable when the 1Jαβ coupling is removed.
The utility of the peak shape resulting from the residual 1Jαβ coupling is also demonstrated for the internal peak of A26 (Fig. 3d). Coincidentally, two systems provide a sequential match with their individual chemical shifts within the digital resolution of the spectrum (Fig. 3e, f). Therefore, even at this ultra-high resolution, we might not establish the unambiguous sequential connection from the chemical shift information alone. However, it is trivial to obtain the correct assignment by a mere inspection of the peak shape of the two sequential candidate systems. While the same line shape would be expected for the correct assignment, the first candidate fails to match the line shape of the internal A26 signal (Fig. 3e). On the other hand, the correct assignment shows a perfect line shape match between internal and sequential peaks (Fig. 3f).
To further test and validate the utility of labeling using pyruvate and the resolving power of pre-mix 2-13C and 3-13C pyruvate, we applied this strategy to assign the Maltose Binding Protein (MBP). MBP was labeled using the pre-mix strategy as outlined in the experimental methods. A high-resolution HNCA with TROSY selection in the 1H and 15N dimensions, was acquired on the sample with a final digital resolution of ~3.5 Hz for Cα. The sample concentration of the "pre-mix" MBP sample was 600 μM and the HNCA was recorded in ~4 days with 16 scans using NUS to sample ~9% of a 75(15N)×750(13C) grid (5216 total sampled points). This set up corresponded to an evolution time of 103 ms in the carbon dimension.
Every system present in the spectrum had a stronger central uncoupled peak (Supplementary Figure 5), which provided a precise peak position within a digital resolution of about 4.8 Hz, the expected line width for deuterated Cα in MBP at 310 K. The 2-13C and 3-13C pre-mix strategy gives rise to distinct peak shapes for each amino acid. This can be quantified by the C2UR, in the same way we compared the post-mix to pre-mix line shapes of amino acids above. The fitting algorithm extracts the C2UR for each amino acid type as the ratio k2/k1 where k1 and k2 is the height of the uncoupled peak and the coupled peaks, respectively (Supplementary Figures 4 and 6).
The C2UR can be grouped into three categories: low (0–15%, A, F, G, H, K, S, W, and Y), medium (20–40%, D, E, M, N, P, Q, R, and T) and high (~50%, I, L, and V) ratios (Supplementary Figure 6). The group with a low C2UR is comprised of "direct" or gluconeogenesis amino acids and the group with medium C2UR is composed of "TCA-derived" amino acids.
Note that the amino acid cysteine is absent in the primary sequence of both MBP and GB1. However, cysteine is expected to have low C2UR ratio as it is derived from serine. It should also be noted that individual amino acids in the same group also show some variation in C2UR, these variations are represented as error bars in Supplementary Figure 6 (see Supplementary Figure 7 for the complete and individual results for representative amino acid, valine, from the case of MBP). C2UR is a good indicator of which group of amino acids (direct, TCA, or ILV) a spin system originates from. Therefore, we concluded that labeling with the pre-mix strategy not only restores highly sensitive detection of HNCA resonances for every amino acid type, but also guarantees the generation of a stronger uncoupled high-resolution central peak that is devoid of coupling. In addition, the C2UR encodes auxiliary information that can be used to establish which of the 20 amino acids a spin system may be composed of.
Even at this high resolution in the HNCA for MBP, there are several sequential matches for a given system based on chemical shift alone. For example, the spin system for V259 (Fig. 4a) has at least two sequential candidates that closely match in frequency space. Since the backbone assignment of MBP is known we can infer that one is the incorrect match, I212 sequential (S211), and the other is the correct match, G260 sequential. A mere visual inspection of the line shapes can unambiguously identify the correct assignment. To make this visual analysis quantitative, we overlaid intensity-normalized peaks for V259 internal and I212 sequential (Fig. 4b, top left), constructed a correlation plot of the aligned point intensities and calculated a correlational coefficient (Fig. 4b, top right). A zoom-in of this is presented in Supplementary Figure 8. Note that the intensities are normalized only for clarity in overlaying the peak and the intensity data are not normalized for the correlation plots. The same was done for the correct assignment pair, V259 internal and G260 sequential (Fig. 4b, bottom). The correct assignment showed a higher correlation coefficient, indicating that the correlation coefficient can be reliably used as a score to discriminate correct from incorrect assignments (the significance of the difference between the correlation coefficients is p = 0.00017). Even in cases when both the competing candidates are from the same type of amino acid, the correlation coefficient is able to establish the correct assignment (Supplementary Figure 9). In this example, subtle differences in the splitting frequency of two candidate leucines are significant enough to select the correct candidate (the significance of the difference between the correlation coefficients is p = 0.0013). Thus, peak shape correlation or matching proves to have the potential to make correct assignments above and beyond high resolution and C2UR.
To test the power of peak matching, we did a mock assignment of MBP using just the HNCA spectrum. If a traditional "match in frequency alone" approach is used with a resolution window of 42 Hz that corresponds to the typical resolution of HNCA for uniformly labeled sample, we can only establish unique assignment for <5% of the backbone (Fig. 4c, green). In fact, more than 60% of the residues have 8 or more matching candidates at this resolution. No more than two amino acids in a row can be sequentially assigned (Supplementary Figure 10). By increasing the resolution in the Cα dimension to 4.8 Hz, the approximate line width in our MBP spectrum, we can sequentially link about 40% of resonances by the "frequency match alone" approach (Fig. 4c, red). At this resolution, no system has more than five matching candidates for sequential connectivity. Although this is a substantial improvement, there are 200 amino acids that remain unassigned (Supplementary Figure 11). When the power of resolution is combined with the peak shape correlation analysis using the real data, over 85% of amino acids can be correctly assigned from this single HNCA experiment (Fig. 4b, black). Approximately 10% of systems cannot be uniquely assigned (Fig. 4b, black, assigned "2" to indicate degenerate matches with additional candidates). In addition, about 5% of systems cannot be assigned due to being adjacent to proline or systems that are broadened below the detection limit in our HNCA spectrum, presumably due to chemical exchange phenomena (Fig. 4b black, assigned "0" because sequential assignment is not possible). Long stretches of sequential assignment that were obtained as a result of the peak shape and peak position matching are indicated by a red bar under the sequence for MBP (Fig. 5). It should be noted that the matching algorithm employed above performs a simple pairwise matching between probable candidates and does not consider the matching probability in the context of larger stretch of the amino acid sequence. We have analyzed the cases where degeneracy remains even after peak position and peak shape matching, and the small fraction of cases where the pairwise matching apparently provide an incorrect assignment. Factors that contribute to this continued degeneracy can be classified into three categories: (1) low signal-to-noise (an example of which is discussed in Supplementary Figure 12); (2) an overlap between the internal and sequential peaks within a spin system (an example of which is discussed Supplementary Figure 13); and (3) continued degeneracy even after comparing peak shapes and peak positions (an example of which is discussed Supplementary Figure 14). Some of this can be avoided if we are able to record a spectrum with a better signal-to-noise ratio. A recent publication has shown that NUS/hmsIST can be faithfully used to extend the data set up to three times in the indirect dimension29. The time saved by using NUS can be devoted to more scans in the initial part of the evolution where there is more signal to improve the sensitivity. Furthermore, errors stemming from degeneracies in both peak shape and position can be reduced/eliminated if we consider sequential matches in the context of a larger stretch of the amino acid sequence. An example of this is presented in Supplementary Figure 15.
Sensitivity of HNCA with mixed pyruvate labeling
An important aspect to consider is the difference in sensitivity in the HNCA experiment of a "mixed pyruvate" labeled sample as compared to that of a uniformly labeled sample. On average, 50% of the Cα atoms are labeled when the mixed pyruvate labeling strategy is used. In analyzing the relative sensitivity of a "mixed pyruvate" labeled sample in comparison to that of a uniformly labeled sample in the context of an out-and-back-style HNCA experiment there are two key factors that need to considered: (1) the total transfer efficiency for the N to Cα magnetization transfer and the subsequent refocusing of N with respect to Cα and (2) the gain in peak height of the central uncoupled peak in a pyruvate-labeled sample as compared to the split Cα resonance in a uniformly labeled sample. These are two independent factors and can be treated separately. A detailed analysis of this treatment is provided in Supplementary Note 1. Briefly, in the "mixed pyruvate" labeled sample there are four possible labeling combinations, (1) 13C(i-1)-N(i)-13C(i), 2) 12C(i-1)-N(i)-13C(i), 3) 13C(i-1)-N(i)-12C(i), and 4) 12C(i-1)-N(i)-12C(i), each with equal probability (p = 0.25) of occurrence. Note that the case where the labeling is 13C(i-1)-N(i)-13C(i) is identical to the uniform labeled sample and the case where the labeling is 12C(i-1)-N(i)-12C(i) does not yield any signal. The efficiency of transfer and transfer rates depend on the labeling pattern (Supplementary Figure 16). Combining the transfer efficiency (weighted to the probability of occurrence) and the gain in peak heights, the ratio of sensitivities of the HNCA experiment on a pyruvate-labeled sample as compared to a uniformly labeled sample is 0.6104 (ILV), 0.7181 (TCA), and 1.2207 (GNG) for 22 ms of transfer time (the case for the spectra used in this manuscript). We posit that better efficiency for mixed pyruvate samples can be achieved with transfer times closer to 42 ms. The calculations indicate sensitivity gains of 0.8695 (ILV), 1.0229 (TCA) and 1.7389 (GNG) when comparing the ratio of sensitivities of an HNCA experiment at transfer times of 42 ms and 29 ms for mixed pyruvate and uniformly labeled samples, respectively. These calculations and associated sensitivity gains account for relaxation losses during the transfer time for the protein MBP (42 kDa) and are summarized in Supplementary Table 1. To experimentally verify the sensitivity gains of the pyruvate sample described above, we acquired 1HN/13C 2D planes from a TROSY-HNCA experiment with an array of transfer times between 18 and 90 ms on a mixed pyruvate-labeled, perdeuterated GB1 sample. The 2D planes contained sufficient resolution to determine peak heights for many spin systems. The plot of peak intensity for spin system F54 as a function of transfer time both for the internal (red solid line) and sequential (black solid line) is shown in Supplementary Figure 17. We then modeled our calculated sensitivity equations for internal and sequential peaks based on JNC couplings of 11.0 and 8.2 Hz for the internal and sequential peaks, respectively, and a relaxation rate (R2) of 4 Hz. Our calculations for internal (dashed red) and sequential (dashed black) systems predict the data quite well. It should be noted that optimal transfer times for each residue will vary from the calculated value, depending on the individual relaxation time and if the residue experiences exchange broadening.
Since the method described here relies on matching the high-resolution central frequency and the residue specific peak shape information, fidelity in encoding these two components is important. To obtain high-resolution in the Cα dimension the protein should be expressed in its deuterated form, as Cα atoms attached to hydrogen experience faster relaxation. Cα attached to deuterium relaxes ~8 times slower than those attached to hydrogen, which allows longer acquisition times. However, one drawback of deuteration is the incomplete back exchange of the observable amide hydrogens. In some proteins, this incomplete back exchange will result in two populations of amides N–H and N–D. Although there is no magnetization that can start or end with the N–D species, it will create an isotope shift on the Cα resonance30. In a case where a residue i-1 harbors an amide that is incompletely exchanged, the amide of the sequential residue i will encode the weighted sum of two frequencies, each corresponding to the Cα attached to the N–H and N–D, respectively (Supplementary Figure 18). However, the amide of residue i-1 will only encode the frequency corresponding to the Cα attached to N–H. Thus, the match in peak positions will not be ideal This is discussed further in Supplementary Note 2. Effort should be taken to maximize the back exchange of amides to hydrogen. Several established approaches have been used to maximize the back exchange. These include equilibrating the sample at basic pH (~8–9), equilibrating the sample at high temperature, refolding and partial unfolding with refolding. A detailed explanation of the effect of incomplete back exchange is provided in the Supplemental Information.
The use of pyruvate as a carbon source for protein expression has been exploited previously for generating 1H-13C methyl groups31,32 and (1Hα)13Cα-13CO spin-pairs33 in an otherwise deuterated background. The labeling scheme described here is not a mere extension of the 1-13C-labeled or 2-13C-labeled glucose or SAIL strategies. The "mixed pyruvate" labeling strategy opens a new avenue for backbone assignment. This labeling strategy provides two independent sets of information (1) high-resolution chemical shift information and (2) unique peak shape from residual carbon 1Jαβ couplings, which are amino acid specific. The combination provides site-specific information beyond the resolution of amino acid type. The labeling strategy would not only be useful in backbone resonance assignment but could also be expanded to establish unambiguously assigned NOEs, particularly for 15N-dispersed NOESY experiments.
Here we have used deuterated 2-13C-labeled and 3-13C-labeled pyruvate in various combinations to label GB1 and MBP (Supplementary Table 2) with the aim to explore labeling patterns and improve relaxation properties for backbone assignment purposes. The pre-mix labeling strategy allows unique simultaneous access to ultra-high-resolution peaks, amino acid type classification via the C2UR and peak shape correlation tests between candidate systems that enables backbone assignments of large protein systems using the sensitive HNCA experiment alone. The 10% of systems that were incorrectly sequentially aligned when using the intersection of frequency and peak shape can be potentially corrected when attempting to match the C2UR ratios of aligned systems to the amino acid sequence. We anticipate this match will form part of a future automatic assignment procedure. This model could be further expanded to delineate redundant matches that persist. For instance, if there is degeneracy where both the uncoupled center frequency and the correlations are a close match, one could use selective narrow band Cβ decoupling during the Cα evolution period in an additional HNCA experiment. This will collapse the side lobes for the resonances that have a Cβ chemical shift matched with the decoupled frequency. In addition, 13C incorporation at the CO position can be also harnessed to augment peak shapes if the Cα-CO coupling is permitted to partly evolve during Cα encoding. The details of the labeling pattern at the CO position, both in the presence of 13C and 12C carbonate, needs to be explored in a separate study. Furthermore, the 1JCαCβ couplings provided in the spectrum harbor vital information about the local backbone conformation34. Although we have not specifically harnessed this aspect in this study, the dependence of the coupling on the backbone geometry could aid the matching process, especially to differentiate degeneracy involving the same amino acid type.
Based on list prices, the cost of making a mixed pyruvate sample grown with 1.5 grams of 2-13C and 1.5 grams of 3-13C pyruvate is ~13.5% more expensive when compared to the traditional uniformly labeled sample cultured with 3 g of 13C-deuterated glucose. It should be noted that the pyruvate used is purchased as a protonated molecule and is easily exchanged to a deuterated form as described in the Experimental Methods section. In addition, if the demand for protonated 13C pyruvate goes up, the price of labeled pyruvate should decrease, thus making the price of the mixed pyruvate sample comparable or cheaper to the perdeuterated, uniformly labeled sample. Although the effect of induction lengths and varying ratios of 2-13C/3-13C pyruvates in expression media on the subsequent labeling pattern could benefit from further optimization, the pre-mix strategy, as currently implemented, will nevertheless yield ultra-sharp uncoupled resonances and amino acid dependent peak shapes, which extends the resolution and the power of resonance differentiation beyond what has been previously possible. This unique feature will allow us to push the limit of backbone resonance assignment for larger biological systems.
Preparation of growth media with deuterated pyruvate
3 grams of 2-13C and/or 3-13C pyruvate were dissolved in 1 kg of D2O (99.9%) and the pH of the solution was adjusted to ~13.0 by 2.5 mM NaOD to deuterate the methyl protons of pyruvate by proton–deuteron exchange. The exchange was carried out for 30 min at room temperature with occasional shaking. pH was restored to neutral (pH 7.0) by the addition of 4.26 g Na2HPO4, 3.60 g NaH2PO4, and 3.00 g KH2PO4. The media were also supplemented with 1.0 g of 15N or 14N ammonium chloride depending on desired labeling state for nitrogen and 1.0 g of 12C sodium bicarbonate. The media also contained 0.24 g of MgSO4, 0.011 g CaCl2 and necessary antibiotics (50 mg kanamycin, 50 mg carbenicillin or 100 mg ampicillin). The media was sterilized through filtration (0.22 μM) before use.
Culture conditions and sample preparation
A single colony of expression strain (GB1 or MBP) was grown overnight at 37 °C in 10 mL of filter sterilized LB media with 100% D2O substituted for water. The cells were pelleted and resuspended in 10 mL of the deuterated pyruvate media and allowed to grow for 6–8 h at 37 °C. These cells were again pelleted and resuspended in 900 mL of the deuterated pyruvate media and allowed to grow at 37 °C overnight. When the OD at 600 nm reached a value between 0.4 and 0.6, which occurs 16–24 h later, the temperature was decreased to 20 °C and cells where induced by addition of 1 mM IPTG. The proteins were expressed for an additional 24 h. Cells were harvested by centrifugation and protein was purified through standard affinity purification procedures. Briefly, cells expressing GB1 were lysed by sonication in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0). The lysate was purified by a single pass over Ni-NTA resin, which was washed with three column volumes of lysis buffer and eluted with 300 mM imidazole, 50 mM Tris, 150 mM NaCl pH 8.0 buffer. Protein was buffer exchanged into 50 mM sodium phosphate, 50 mM NaCl pH 6.5. SDS–PAGE established the sample was sufficiently pure for NMR analysis. The protein was concentrated to 1 mM for NMR analysis. MBP protein was purified after sonication in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0) by passing the lysate over immobilized amylose beads and eluted with 10 mM maltose in 50 mM Tris-HCl, pH 8.0. The elution was buffer exchanged into 10 mM HEPES, 1 mM EDTA, pH 6.5. The amides of MBP were back exchanged by addition of 1 M urea to this buffer and allowing exchange to take place at 37 °C for 24 h, followed by a buffer exchange back to NMR buffer (10 mM HEPES, 1 mM EDTA, pH 6.5). MBP was concentrated to 600 μM and β-cyclodextrin was added to the sample to a final concentration of 2 mM.
NMR spectra
NMR data collection of GB1 samples was performed on a Bruker 750 MHz instrument equipped with a cryogenically cooled probe. The TROSY-HNCA pulse sequence from the standard Bruker library was used with deuterium decoupling. Sweep widths for the 1H, 15N and 13C dimension were 10,504, 2430, and 6031 Hz, respectively. The indirect dimensions were sampled non-uniformly, selecting 2500 out of a Nyquist grid of 54 × 512 (15N × 13C) complex points (~9% sampling). Sampling schedule was chosen based on the Poisson gap sine weighted protocol16. Data collection on the MBP sample was performed on a Bruker 900 MHz instrument equipped with a cryogenically cooled probe. The standard Bruker TROSY-HNCA pulse sequence (trhncagp2h3d2)35 was used with modification to permit non-uniform sampling with Topspin 2.1. Sweep widths for the 1H, 15N, and 13C dimension were 12,626, 3375, and 7243 Hz, respectively. The indirect dimensions were sampled non-uniformly, selecting 5216 out of a matrix of 75 × 750 (15N × 13C) complex points (~9% sampling). Sampling schedule was selected based on the Poisson gap sine weighted protocol. See Supplementary Methods for more details.
NMR data reconstruction
NUS spectra were reconstructed using the hmsIST software package and NMRPipe36. 400 iterations of iterative soft threshold (IST) reconstruction were used16. Each dimension was zero filled before regular Fourier transformation. For GB1, this results in 1024 real points in the 13Cα dimension for a sweep width of 6031 Hz, or a digital resolution of 5.9 Hz. For MBP, a zero fill to 2048 points was used giving a digital resolution of 3.5 Hz. However, a cosine window function was applied to the 750 complex points before Fourier transformation, which made the effective digital resolution ~4.8 Hz.
NMR data extraction and analysis
Data was analyzed using purpose-built Python software and chemical shifts from the Biological Magnetic Resonance Data Bank (BMRB). Specifically, NMRPipe format spectra were directly read and 1D traces along the 13C dimension of systems were extracted based on chemical shifts reported in the BMRB13 (Entry # 7114). It was necessary to make minor adjustments to the reported BMRB chemical shifts to account for changes that arose from differences in sample preparation and experimental conditions. Coupled to uncoupled peak height ratios (C2UR) were calculated after fitting of peaks to a "three- peak" model. Specifically, the Cα peak intensity at a point in the spectrum (x) was modeled as being composed of three Gaussian peaks; a central uncoupled peak (centered at m) and assumed to be of largest height (k1), and two equally sized (height k2) and spaced coupled peaks (distance d from the central peak) on either side of the central peak. Line width (s) was assumed to be equal for all peaks. Thus, the following equation (1) was derived for the three-peak model:
Data exceeding the width of a peak (~80 Hz, or 33 points for MBP HNCA spectrum) was considered for fitting. A curve fit function written in Python was used. The parameters k1 and k2 were used to estimate the peak heights of the uncoupled central peak and the coupled adjacent peaks. The mock assignment procedure was performed by first declaring an assignment as being unambiguous if, based on the known assignments from the BMRB, there was one and only one match between an internal chemical shift in a spin system and a sequential chemical shift in another spin system within a "resolution" window. Resolution limits of 42 Hz (regular HNCA resolution) and 4.8 Hz (high-resolution HNCA) were then used. Then a check that the correct internal-sequential match was done. Afterwards, all remaining ambiguous matches were examined for peak shape matching by performing a correlation coefficient calculation between an internal peak and all frequency matching sequential peaks within the resolution window. In this case, 23 spectral points were used. The highest correlation was declared the best match and then verified based on known assignments from the BMRB. Correct and incorrect assignments after pattern matching were tallied. In addition, systems that could not be sequentially assigned due to non-existent sequential systems that are missing (i.e., proline or exchange broadened systems) were counted separately.
Additional details on the methods used are provided in the Supplementary Methods section of the Supplementary Material.
Data availability
Other data are available from the corresponding author upon reasonable request.
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This work was supported by NIH grants GM047467 and AI03758 to G.W. H.A. acknowledges funding from Claudia Adams Barr Program for Innovative Cancer Research. Maintenance of the NMR instruments used for this research was supported by NIH grant EB002026. A.B thanks Fonds zur Förderung der wissenschaftlichen Forschung (FWF Project J3872-B21) for support. We would like to thank Drs. Ricard Rodriquez Mias and Kendra E. Leigh for the insightful discussions regarding the work presented here.
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Robson, S.A., Takeuchi, K., Boeszoermenyi, A. et al. Mixed pyruvate labeling enables backbone resonance assignment of large proteins using a single experiment. Nat Commun 9, 356 (2018). https://doi.org/10.1038/s41467-017-02767-8
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Emerging solution NMR methods to illuminate the structural and dynamic properties of proteins
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NMR: an essential structural tool for integrative studies of T cell development, pMHC ligand recognition and TCR mechanobiology
Journal of Biomolecular NMR (2019)
Optimal control theory enables homonuclear decoupling without Bloch–Siegert shifts in NMR spectroscopy
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Late metabolic precursors for selective aromatic residue labeling
Journal of Biomolecular NMR (2018)
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Grafton Group IMS
Grafton Group plc, the builders merchanting and DIY Group with operations in the UK, Ireland and Belgium, has issued an Interim Management Statement for the period from 1 July 2015 to 31 October 2015.
12 November 2015
Insight DIY is the only source of market information that I need and they always have the latest news before anyone else.
Neil Anderton - Sales Director, British Ceramic Tile


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Actor Ajay has become one of the most sought after actors in Tollywood. His current flick, Dikkulu Choodaku Ramayya is all set for a release soon, and we caught with him for a brief chat. Let's see what he has to say.
How did you bag this role ?
I knew director Trikoti from the time of Vikramarkudu. He is a very talented technician, and when he came to me with this role, I had absolutely no doubts in saying yes.
You play a dad of a 20 year old son. How do you feel ?
Right from the title to the story, everything is based on me. I am extremely happy for getting a role like this. It was quite a challenge and fun playing a dad and am eager to see the reaction of the audience in the theaters.
Do you think the audience will connect to a unique concept like this ?
Even though such stories rarely exist outside, they would surely connect to the concept. The way our director has handled the film, and has brought out simple humor is quite interesting in the film.
You have mostly done negative roles. How difficult was it doing this clean film ?
I took a lot of care that the audience should not get any negative vibes from me. I have changed my body language, and did my best to ensure that everyone gets to see a new angle of my acting.
How was it working with Sai Korrapati ?
He is one of the most passionate producer whom I have worked with till date. Any other maker would have thought twice to produce a different film like this.
What are the major highlights of the film ?
Story of the film is quite unique, and the way the director has handled the entire subject will be yet another plus.
Will you still continue to act as a dad in the future ?
If the character and genre of the film are interesting, I would surely do roles like these.
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Ad : Teluguruchi - Learn.. Cook.. Enjoy the Tasty food


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It's completely unsurprising to know that someone who enjoys photography would also enjoy collecting old cameras. I am one of those people, so I guess that makes me a cliché.
I'm not collecting just to collect; I have very specific themes that give me direction on what to seek out. I want to understand the lore and legacy of the camera itself, to know more than just who made it and the approximate date of manufacture. Each camera has a story that enriches the collecting experience. I want to know why it was made, who it was marketed to, and if there's any historic background to it. This type of information is beyond what appears in antique references and books.
At the moment, I own cameras from as early as 1920′ and as recent as the mid-80's. Most are from the 50's, 60's and 70's. The collection comprises mix of Polaroid, 35mm & medium format types, and a handful of rare collectable cameras.
I hunt down the cameras all around the world.
I hope you will find something of interest in my classic camera collection (=


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Hi, this is my very first article, which I am going to post here, I was overwhelmed with the thoughts of selecting the best topic to write on, but I wasn't succeeding in selecting a particular topic. In the past I have written some articles in newspapers, and magazines on varied topics like politics, dowry, the matrimonial issues of our society specially related to girls. But those articles were in my local language and moreover not on regular basis. For me writing articles were time-consuming so I never thought of becoming a professional writer. Moreover, I could not write on any topic suggested by others or by intentional thinking— NO WAY, never I can only write what I can feel deeply. I can write with my heart not with brain. I have been using net for more than a decade now, but just for gathering information specially I am deeply interested in motivational stuff and alternative healing and treatments.
I heard so many times that people are earning a lot of money through Internet, but I never thought of earning through net, not because I didn't like money, but because I wasn't a technical person plus I can't sit so long hours in front of net. Now I often think, if I had found a right person to guide me at that time, it might possible that life would have been turned much easier. Actually a true mentor of course is very important in your life. No amount of experience and time can substitute a true mentor. You see, it was for the last two years that I have been searching Internet earning tips on the net too frequently because of my financial issues, but for a newbie its like a storm of information and the unknown person can easily lost in that storm of information. So I could not go for the option of Internet earning, most of the things were hard for me the only thing which was understand able was to write content, write articles or make blog. But this idea never got me into writing or making a blog. Even in critical financial problems I didn't convince myself for making a blog or writing articles, but now when I have launched a blog for a very big movement and have presented my own case to the entire web community and I am writing my first article I am more than convinced that whatever you say or do, your paths are defined and your destiny will automatically do the rest. So I was also destined to move to a higher place. At first I thought not to mention my blog in the very first post because people will think I am doing any kind of marketing, but then I decided not to disturb the natural flow of this article and decided to be honest.
Now the most interesting part is —– this time it's not for money as I am using a free blog at word press. There was an internal voice to start a blog and for article writing. But when I was writing I was thinking that it might possible that people won't like my articles because people only welcome extra ordinary experts moreover I could not even decide any niche for writing. Then, I decided to go for the most honest approach and decided not to worry about professional writing style, topic, whether people like or not, people read or not because those who are destined to benefit from these articles will definitely read and like. And about topics, niche or extra ordinary headlines are not important, the only thing which matters is whether you are genuine or not. If any word of mine can change someone's life then, it's worth reading and writing both. So I will write on any topic which I can guarantee myself, for example I will write on that positive stuff, the mind-set that can transform the entire life, one special secret which everybody should know (but I will tell this secret to only those few people who can do justice with the miraculous value of this precious secret and to only those who really deserve it) and techniques that literally have changed my life and can change yours as well, I will write on everything about making and launching a free blog because I know what problems a newbie can face, I also motivate people to write articles and share their personal secrets and experiences to help others. And I will share all these good things absolutely free of cost and with everyone except that miraculous valuable secret, this secret is also free, but it will be told only to real deserving people. I can't guarantee how many people will read or like my articles,about professional writing style, error free text, in short I can't guarantee any thing but I can guarantee that I will only share what I can swear by, not about the things that I don't know. I can also guarantee that I will give you 100%real and genuine words not the words that are coming from any article writing software or any other external help I know people are doing this and there's nothing wrong with it because the competition is too tough. But I know if I will talk directly from my heart, then it would be like a "heart to heart talk" and will definitely have a magnetic effect on you. Everything is done by machines these days at least let me give permission to share what I feel and also let me feel you deeply in natural way. I will share "my real stories" of my day to day happenings and also about the things which have dramatically changed my life and in each episode there would we at least something worth your time. I don't want to kill the soft, emotional, sympathetic person inside me just for the sake of competition and money. So ending this long article by a funny slogan of mine which I have created a while before just for the sake of refreshing yourself that "I am here to serve you and if money follows, I will not mind". You also try to write keeping just one rule in mind that give something valuable to people and keep the rest to GOD. Take Care, see you soon.


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WATCH Have you got the perfect home for handsome Harley?
PUBLISHED: 11:53 13 November 2019 | UPDATED: 09:05 14 November 2019
Charlotte Bond
Harley the Yorkie Chihuahua cross is looking for new owners that will provide him with lots of love and plenty of his favourite soft toys.
Described as a "very sweet, affectionate lad", three-year-old Harley is full of personality but struggled to settle in his last home.
The RSPCA Suffolk East and Ipswich Branch said: "Probably due to a lack of socialisation as a pup, Harley can find certain situations hard to cope with and therefore can quickly become reactive.
"His new owners will need to continue with his training plan to help reduce his anxiety around situations he finds stressful.
"Owners who are willing to help Harley through his training and provide him with positive experiences, in a safe manner, would really help him lead a happier, more relaxed life."
Because of his issues with socialising Harley must be rehomed with a family without children or pets.
You can find out more about him on the RSPCA Suffolk East and Ipswich Branch website.
Read more: Hero Bowza set to appear on Channel 5 this Christmas
If you value what this story gives you, please consider supporting the Ipswich Star. Click the link in the orange box above for details.


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Fans of Lebanese pop star Elissa were left in shock after the diva revealed she has spent the last year fighting breast cancer.
The singer shared her journey in the music video for her latest single, Ila Kol Elli Bihebbouni. The seven-minute clip begins by showing Elissa undergoing an MRI scan on December 26, 2017, narrated with poignant voice recordings from the star.
She questions: "You know that moment when you wonder, will I ever see the people I love again? Those who love me back?"
"Is it that moment when it's all over? You won't exist tomorrow?
I do my radiotherapy session, I go to the studio, I finish another session, I rest for two hours. And to the studio again, once a week going live on TV."
The video includes footage from Elissa's Dubai concert in February 2018, where she collapsed during the performance and had to be carried offstage. At the time, the singer told her fans it was "nothing serious".
Thankfully, Elissa reveals at the end of the video "I've recovered, I've beaten the illness, and I won."
"Don't ignore it, face it," she warns, noting that early detection of breast cancer can save your life. "Do it not only for yourself, but for your loved ones."
The video was posted on August 7, and in less than 24 hours has clocked almost three million views on YouTube.
Elissa wrote in an Instagram post: "This music video shouldn't make you cry. It's a reason to smile, be happy, and grateful. We can fight cancer if we know about it in early stages. I love you all


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This week we're having a little fun. We have two Canadian Twinsie Tuesday members and this week we let them call the shots. We split the group up in to two and each of our Canadian sisters was in charge of telling us what kind of mani we were to do for today. I was on Ashley's team and she decided we were all to do a winter themed mani. I'm not much for cold weather myself. Well, really it's the ice I hate. I don't mind the cold temperatures or even the snow, but I really hate the ice. It scares me to drive in slick conditions. My car is all-wheel drive, but I still crawl when there is snow on the roads.
Anyway, I didn't get super creative. I decided to do some snow falling at night. I love watching big, fat, fluffy flakes falling. 🙂 Was that enough "f's" for you? I didn't plan it, it just came out that way.
I did a snowflake mani last year, but they were big snowflakes, so this time I tried to be uber delicate and make tiny ones. I didn't photograph my thumb, but it's sporting some pretty tiny snowflakes. My night sky polish is NYX Jungle with NYX Girls White for my snowflakes.
Be sure the check out all the other manis today:
Amanda of Amandalandish
Anna of Going to the Showing
Anne of Grape Fizz Nails
Ashley of Art Evolve
Brandi of The Collegiate Nail
Chelsea of Nailed Blog
ChiChi of I'm Just Me…ChiChi
Cordia of Seriously Swatched
Elizabeth of Did My Nails
Jenn of All That is Gorgeous
Jenna of I'm Still Thinking
Katherine of Haul of Fame
Maribeth of Obsessive Cosmetic Hoarders Unite!
Marisa of Polish Obsession
Meredith of Polish and Charms
Nory of Fierce Make Up and Nails
Sarah of See Sarah Swatch
Tara of Polishy of Truth


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Dr. Chrishanthy Jayarajah. MBBS, MRCPSPSYCH, DFSRH is a Consultant Perinatal Psychiatrist working at Barnet Liaison Psychiatry service, North London and is to lead a newly commissioned PMH service in NW London. During her core training she worked at the National OCD/BDD Inpatient Unit at Springfield Hospital, SW London, which sparked her ongoing interest and research into OCD. She is particularly … Read More
Speaker Bio: Zainab Yate
Zainab Yate. BSc, MSc, Doctoral Student has a Medical Ethics & Law (Imperial College, MSc) background and her doctorate research is in Bioethics. Her working background is in Public Health and commissioning within the NHS. She is Alt. Vice Chair and named qualitative lead of the North London Research Ethics Committee, with the Health Research Authority in the UK (HRA). … Read More
Speaker Bio: Professor Simon Murch
Professor Murch is Professor of Paediatrics and Child Health at Warwick Medical School. Before taking up this post, he was Senior Lecturer in Paediatric Gastroenterology at the Royal Free and University College School of Medicine, London. His clinical interests include paediatric inflammatory bowel disease (IBD), coeliac disease, food allergy and complex inherited enteropathies. He was the leader of the international … Read More
Speaker Bio: Jennifer Thomas
Dr. Jennifer Thomas MD, MHP, IBCLC, FABM is a American paediatrician and breastfeeding medicine specialist at Aurora Healthcare in Franklin, Wisconsin. She received her MD from MCW in 1993, her MPH in 2011 and became an IBCLC in 2003. She is a Fellow of the Academy of Breastfeeding Medicine (FABM) for her expertise on breastfeeding. She currently serves on the … Read More
Speaker Bio: Nancy Mohrbacher
Nancy Mohrbacher, IBCLC, FILCA, began working with breastfeeding families in 1982 as a peer support counsellor and became an IBCLC in 1991. From 1993 to 2003 she founded and maintained a large private lactation practice around Chicago, USA. In 2008 the International Lactation Consultant Association (ILCA) officially recognised her contributions to the field of breastfeeding by awarding her the designation … Read More


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As the state legislature examines proposals to eventually make Washington's electricity 100 percent clean, they're also taking a good look at emerging technology that could make that happen while also boosting the state's manufacturing industry. A Senate bill would add small modular reactors (SMR) to the state's clean energy strategy and exempt their manufacturing and sales from the state's business and occupation (B&O) tax.
Sponsored by Republican Deputy Leader Sharon Brown (R-8) and cosponsored by three Republicans and one Democrat, SB 5629 is the latest proposal made by Brown addressing SMRs. In 2016, she introduced SB 6224, which managed to clear the Senate Committee on Energy, Environment & Telecommunications, but never cleared Senate Rules. In 2017, she sponsored SB 5475 creating a B&O tax exemption for SMR manufacturers; the bill passed the Senate 27-22, but was sent back from the House.
At a Feb. 6 public hearing of the Senate Environment, Energy & Technology Committee, Brown told colleagues "it's really important that we continue to have this conversation about small modular reactors and microreactors. The wind doesn't always blow, and the sun doesn't always shine, so we really need to have the discussion about what we are going to do to preserve clean baseload sources of power."
SMRs generate 300 megawatts or less, compared to traditional nuclear power plants that generate 1,000 MW or more. As their name implies, they can also be moved by truck or rail. According to the Federal Department of Energy, SMRs are a "key" part of the agency's plan to "to develop safe, clean, and affordable nuclear power options."
Also, Washington is the location for several manufactures such as Oregon Iron Works in Vancouver that produce SMR materials. However, Brown fears that these jobs could shift over to Oregon and Idaho. "This bill is really about keeping the intellectual property and the manufacturing jobs here in this state."
To maintain the B&O tax exemption, qualifying businesses have to develop an apprenticeship or training program with a state college or university. The goal is to increase the number of SMR industry jobs in the state by 10 percent. If that happens, then the legislature will consider extending the exemption beyond its expiration date.
The proposal is backed by entities including the city of Richland, Washington, PUD and Energy Northwest.
"This new technology is something that we've been watching very carefully," Washington Rural Electric Cooperative Association General Manager Kent Lopez told lawmakers. He added that "we need to keep our options open when it comes to the technologies."
Also in support was Isaac Kastama with the Benton and Franklin PUDs. During his testimony, he cited a 2017 study and alternative scenarios that suggested SMRs could potentially reduce the annual incremental cost to achieve 100 percent clean electricity in the Pacific Northwest.
One of the alternative scenarios examined the cost of achieving the 100 percent standard without a reliable energy source such as natural gas or nuclear power ($18 billion), while the other scenario included innovative technology ($6.5 billion).
"While I would not categorize either of those scenarios as low cost, it did dramatically change the cost," he added. "As we look to 100 percent clean and we consider how we're going to get there in the most cost effective manner, it's going to be innovation, and this is one of those technologies that offers that prospect, not just locally but also around the world."
The bill is also backed by the Association of Washington Business. Government Affairs Director for Environmental Policy Peter Godlewski told lawmakers "as our state moves to a cleaner grid and continues to grow population-wise, our need for reliable baseload power will also continue to grow. We believe all options should be available to help meet this need."
No further action is scheduled for SB 5629 at this time.


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The best sounds ever relax us, entice us, evoke fond memories, and force smiles on our faces. They are the opposite of noise. Noise — according to the Centers for Disease Control and Prevention (CDC) — can cause bad things, like stress or anxiety or depression or high blood pressure. Especially if you are regularly exposed to it without hearing...
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Christopher M. MacMillan
Email: firstname.lastname@example.com
Phone: (503) 314-9176
Chris MacMillan joined the firm in 2015 and is a member of the Oregon and Washington State Bar Associations. He is leading Demco Law Firm's expansion into the Portland area.
Mr. MacMillan's practice focuses on real estate transactions and a wide range of litigation matters, including property and contract disputes. He has represented and advised, among others, property owners, buyers, sellers, real estate brokers, and homeowners associations. He is approved as a continuing education instructor for real estate licensees in Oregon and Washington, and regularly teaches courses to real estate professionals in all aspects of real estate law.
Chris is a graduate of the University of Oregon School of Law where he was a member and executive editor of the Journal of Environmental Law and Litigation, a legal research and writing tutor, and a conference co-director of the world-renowned Public Interest Environmental Law Conference.