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Claims on multi car policies are usually dealt with similarly to single car policies. All multi car policies are different so it’s important to check with your insurer, but the best multi car insurance policies will not penalise one driver if another makes a claim. If someone else on the policy makes a claim, for example if your partner makes a claim on their car, your no claims bonus will not be affected.
To initiate the process, insured is required to submit a detailed estimate of loss to the insurance company. Independent automobile surveyors with engineering background are given the task of assessing the reason and extent of loss. They carefully inspect the damaged vehicle and submit their survey report with the insurance company who will review and examine it in accordance with the recommendations mentioned therein The usual practice is to authorize repairs with the repairer to whom letter is issued in this regard.
If a LPG or CNG kit is installed in your automobile, you must inform the Road Transport Authority’s office, from where the automobile was registered so that they can modify the necessary changes in the registration certificate of the automobile. The insurance provider should be informed as well so that it can provide the coverage to the kit on the payment of additional premium as per the kit’s value under Own Damage Section.
 Availability varies. Enrollment discount applies during data collection; final discount is calculated on driving behavior and could be zero. Discounts do not apply to all coverage elements; actual savings vary by state, coverage selections, rating factors and policy changes. Final discount applies at the next policy renewal and remains until drivers or vehicles on the policy change.
Comprehensive insurance covers all of the above and damage to the vehicle caused by the driver themselves, as well as vandalism and other risks. This is usually the most expensive type of insurance. It is custom in the UK for insurance customers to refer to their Comprehensive Insurance as "Fully Comprehensive" or popularly, "Fully Comp". This is a tautology as the word 'Comprehensive' means full.
Vehicles kept in the UK must now be continuously insured unless a Statutory Off Road Notification (SORN) has been formally submitted. This requirement arose following a change in the law in June 2011 when a regulation known as Continuous Insurance Enforcement (CIE) came into force. The effect of this was that in the UK a vehicle that is not declared SORN, must have a valid insurance policy in force whether or not it is kept on public roads and whether or not it is driven.
Cheapest Auto Insurance can access the policy offerings from over 30 of the top auto insurance companies in America. We’ll therefore be able to help you pinpoint the correct coverage options for your own needs. In addition to standard auto coverage, we offer non-owners, motorcycle, boat insurance, SR-22 options, high-risk coverage and more. Regardless of your driving history, we will get you coverage.
Auto insurance in India deals with the insurance covers for the loss or damage caused to the automobile or its parts due to natural and man-made calamities. It provides accident cover for individual owners of the vehicle while driving and also for passengers and third party legal liability. There are certain general insurance companies who also offer online insurance service for the vehicle.
We wrote the book on low price car insurance, but the story isn’t just about discounts. We’ll go a step further by giving you the power to tailor the quote to your budget and protection needs. We’ll also make sure you’ve maximized your savings opportunities with helpful hints below. That’s why you’re at the best place to buy cheap auto insurance online.
Several jurisdictions have experimented with a "pay-as-you-drive" insurance plan which utilizes either a tracking device in the vehicle or vehicle diagnostics. This would address issues of uninsured motorists by providing additional options and also charge based on the miles (kilometers) driven, which could theoretically increase the efficiency of the insurance, through streamlined collection.
The insurance certificate or cover note issued by the insurance company constitutes the only legal evidence that the policy to which the certificate relates satisfies the requirements of the relevant law applicable in Great Britain, Northern Ireland, the Isle of Man, the Island of Guernsey, the Island of Jersey and the Island of Alderney. The Act states that an authorised person, such as a police officer, may require a driver to produce an insurance certificate for inspection. If the driver cannot show the document immediately on request, and evidence of insurance cannot be found by other means such as the MID, then the Police are empowered to seize the vehicle instantly.
Gocompare.com Limited is authorised and regulated by the Financial Conduct Authority (FCA) for insurance mediation activity under firm reference number 465053. You may check this on the Financial Services Register by visiting the FCA website. Gocompare.com Limited is registered in England and Wales (Company No. 5799376). Registered office: Imperial House, Imperial Way, Newport, Gwent, NP10 8UH, United Kingdom. Gocompare.com Ltd. All Rights Reserved.
The 12-month policy will generally start on the date that the last car insurance is up for renewal, with short-term cover used to protect the other cars in the interim. For example, if you have one car that needs to be insured from February and another in June, the February car will be insured first but the policy will actually renew in June when both cars are covered. After the last car has been insured for a full 12 months, the policy will renew each year until you choose another policy.
The Road Traffic Act, 1933 requires all drivers of mechanically propelled vehicles in public places to have at least third-party insurance, or to have obtained exemption – generally by depositing a (large) sum of money to the High Court as a guarantee against claims. In 1933, this figure was set at £15,000. The Road Traffic Act, 1961 (which is currently in force) repealed the 1933 act but replaced these sections with functionally identical sections.
In 1930, the UK Government introduced a law that required every person who used a vehicle on the road to have at least third-party personal injury insurance. Today, this law is defined by the Road Traffic Act 1988, (generally referred to as the RTA 1988 as amended) which was last modified in 1991. The Act requires that motorists either be insured, or have made a specified deposit (£500,000 in 1991) and keeps the sum deposited with the Accountant General of the Supreme Court, against liability for injuries to others (including passengers) and for damage to other persons' property, resulting from use of a vehicle on a public road or in other public places.
Products underwritten by Nationwide Mutual Insurance Company and Affiliated Companies. Not all Nationwide affiliated companies are mutual companies, and not all Nationwide members are insured by a mutual company. Subject to underwriting guidelines, review and approval. Products and discounts not available to all persons in all states. Nationwide Investment Services Corporation, member FINRA. Home Office: One Nationwide Plaza, Columbus, OH. Nationwide, the Nationwide N and Eagle and other marks displayed on this page are service marks of Nationwide Mutual Insurance Company, unless otherwise disclosed. ©2019. Nationwide Mutual Insurance Company.
As with any credit check, a record of this search will remain on your file – and your credit rating will affect the amount of APR that you’re charged. If you have a poor credit rating, you may be charged up to 20% APR. And if you’ve struggled to pay credit in the past, or if you have a CCJ to your name, then you may be denied the option to pay monthly. If you think you’ll have issues with your credit rating, you can read our guide on improving your credit score.
Third-party vehicle insurance is a mandatory requirement in Indonesia and each individual car and motorcycle must be insured or the vehicle will not be considered legal. Therefore, a motorist cannot drive the vehicle until it is insured. Third Party vehicle insurance is included through a levy in the vehicle registration fee which is paid to the government agency Samsat (Sistem Administrasi Manunggal di bawah Satu Atap), which is responsible for cars and roads. Third-Party Vehicle Insurance is regulated under Act No. 34 Year 1964 Re: Road Traffic Accident Fund and merely covers Bodily injury, and managed by a SOE named PT. Jasa Raharja (Persero). The Indonesian government has a road insurance fund which includes life insurance for traffic accidents. The annual fee is called the Compulsory Contribution Fund for Traffic Accidents or Sumbangan Wajib Dana Kecelakaan Lalu Lintas Jalan.
Yes, motor insurance is transferable to the purchaser of the automobile. All you have to do is inform in writing about the transfer to its insurance provider. The original owner of the car needs to fill out a new proposal form. A nominal fee is charged for the insurance transfer along with the recovery of No Claim Bonus from the transfer date till policy expires on a pro-rata basis.
Teenage drivers who have no driving record will have higher car insurance premiums. However, young drivers are often offered discounts if they undertake further driver training on recognized courses, such as the Pass Plus scheme in the UK. In the US many insurers offer a good-grade discount to students with a good academic record and resident-student discounts to those who live away from home. Generally insurance premiums tend to become lower at the age of 25. Some insurance companies offer "stand alone" car insurance policies specifically for teenagers with lower premiums. By placing restrictions on teenagers' driving (forbidding driving after dark, or giving rides to other teens, for example), these companies effectively reduce their risk.
If the younger driver is covering his own costs, it might be cheaper for him to take out his own policy but include one of the older, lower risk drivers from the household as a named driver (as long as the older drive will use the car). However, if the older driver is footing the bill for both policies, multi-car insurance may still work out cheaper.
If a vehicle is to be "laid up" for whatever reason, a Statutory Off Road Notification (SORN) must be submitted to the DVLA to declare that the vehicle is off the public roads and will not return to them unless the SORN is cancelled by the vehicle's owner. Once a vehicle has been declared 'SORN' then the legal requirement to insure it ceases, although many vehicle owners may desire to maintain cover for loss of or damage to the vehicle while it is off the road. A vehicle that is then to be put back on the road must be subject to a new application for VED and be insured. Part of the VED application requires an electronic check of the MID, in this way the lawful presence of a vehicle on the road for both VED and insurance purposes is reinforced. It follows that the only circumstances in which a vehicle can have no insurance is if it has a valid SORN; was exempted from SORN (as untaxed on or before 31 October 1998 and has had no tax or SORN activity since); is recorded as 'stolen and not recovered' by the Police; is between registered keepers; or is scrapped.
Getting comparison insurance quotes is far different today than it used to be. Historically, it cost someone money to get a look at rates from a company. In their view, they were working to get you these auto insurance quote comparisons so they should be paid for this time, whether you go with a different company or not. Typically, a company would waive the quoting fee if you bought their plan.

domain: finance


In this use case, we compare two Pneumococcal Conjugate Vaccine (PCV) products that were considered for introduction into Papua New Guinea. Comparing different vaccine formulations and profiles is likely an increasing concern for countries that are graduating from Gavi, the vaccine alliance. We additionally review how the tool supports the updating of evidence over time to re-assess prioritization and to explore putative scenarios to reflect future constraints (e.g. costs or storage space requirements).
An interactive, online tool to support deliberations and decision-making using evidence-to-recommendation frameworks.
Considering New Vaccines for Introduction in Uganda


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2013 ESP Award Nominee
Stryker Endoscopy’s SDC3 is an all-in-one data management solution to increase OR workflows and efficiencies. The SDC3 features device and voice control capabilities, allowing both surgeon and staff to control devices such as the light source, arthroscopy pump, insufflator, camera, capture device, lights, and more from anywhere in the OR. The system also has the ability to save postoperative data to an iPad enabling the surgeon to send customizable surgical documents to the patient immediately following the case.
For more information, visit www.stryker.com/endoscopy

domain: medical


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domain: entertainment


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Matthew Offord, Conservative MP for Hendon, leading member of the British Committee for Iran Freedom addressed an online global conference on the conviction of Iran’s diplomat-terrorist Assadollah Assadi by a Belgian court for attempting to bomb the 2018 Free Iran gathering in Paris.
On February 4, 2021, simultaneous with the court ruling against the Iranian regime’s diplomat-terrorist, Assadollah Assadi, and his accomplices in Antwerp, Belgium, a global online conference was held, where Mrs. Maryam Rajavi, the President-elect of the National Council of Resistance of Iran (NCRI), and dozens of prominent figures from various European countries, the United States and Canada, joined thousands of Mujahedin-e Khalq (MEK/PMOI) members based in Ashraf-3 (Albania) as well as the supporters of the Iranian Resistance worldwide.
The Belgian court rejected Assadollah Assadi’s claim of diplomatic immunity and sentenced him to 20 years in prison. The three intelligence agents, Nasimeh Naami, Mehrdad Arefani, and Amir Saadouni, were sentenced to 18, 17, and 15 years in prison, respectively. Their Belgian citizenships was also revoked.
Matthew Offord, Conservative MP for Hendon, leading member of the British Committee for Iran Freedom
Dear colleagues and friends today is an important day, a historic day in the fight against terrorism. N
ow for the first time ever, an accredited diplomat of the regime in Iran, the world’s leading state sponsor of terrorism, is not only caught in the act of terrorism, but also held accountable for his crime intended to bomb the Free Iran gathering of the NCRI in Paris targeting the NCRI president-elect and opposition leader Mrs. Maryam Rajavi.
I personally attended the annual gathering in Paris in support of a free and democratic Iran in previous years, where tens of thousands of people have also participated including many parliamentarians from Europe, the United States, and across the world. So successful, the attack could have caused major bloodshed and killed many people reminiscent of an ISIS still terrorist bombing.
But luckily, the security agencies and police in France, Germany, and Belgium were able to stop the attack and prevent another terrorist tragedy in Europe. Now, with the Iranian diplomat behind bars, it would be a mistake to believe that this case is over and closed.
No. This case has only just begun because we now know that the main culprits and those ordering and truly responsible for this attack upon Iran’s organized pro-democracy opposition in Europe or in Tehran.
We know after the trial and a two-year investigation that the convicted diplomat was in reality an intelligence officer and that he did not act on his own but rather followed orders from the regime’s most senior authorities including the supreme leader.
According to reports, the decision to launch his terrorist attack was taken by the Supreme National Security Council, headed by the regime’s President Hassan Rouhani. Then it was confirmed by the Supreme Leader, Khamenei, and assigned to the Ministry of Intelligence and Security to implement the decision in close cooperation with the Ministry of Foreign Affairs, which is headed by the proposed moderator, Javad Zarif.
Iran’s state terrorism will never be stopped and defeated if these senior officials and leaders of the regime are allowed to enjoy impunity and escape accountability and justice. So while the small fish was caught, the time has come to go after the bigger fish.
The UK and its European allies has now worked together to hold the regime’s leaders in Tehran to account for their involvement in terrorism and all the other accelerated behaviors.
Also, the UK and the EU must move ahead and prescribe without delay the IRGC and the MOIS in their authority to prevent the regime and its terrorist organizations the resources and money they need to facilitate and carry out terrorist operations and to recruit agents in Europe.
I know that more and more colleagues in the UK Parliament are understanding the importance of prescribed in the IRGC and what a blow this will be for the regime’s efforts to export and support terrorism.
In this regard, I applaud my colleague Tom Tugendhat, chairman of the UK’s Parliament Foreign Affairs Committee, in joining with us in this call as suppose his latest policy recommendation to the British government to prescribe the entire IRGC and to the Foreign and Commonwealth Office to prioritize building watertight cases against human rights abuses based in Iran or acting for abroad and its next round of Magnitsky style sanctions.
I’m ending my remarks today by congratulating Mrs. Rajavi, the Iranian Resistance Movement, those of course in Ashraf 3 in Albania, and the people of Iran on this historic achievement and indeed, victory. It would have been impossible without your work and endeavors for a free and democratic Iran.
I stand with you all today and I will continue to stand with you until the day Iran is free, and I will join you in a celebration in Tehran. As I’ve always said before, and as many of you know, we just say one final word but three times, [foreign 4:37]. Thank you.

domain: news


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Fleet News has joined forces with UK100 to run two clean air zone (CAZ) roundtables in Birmingham and London, bringing together UK business fleets with council leaders and city mayors. UK100 is a local government leaders network which has pledged to shift to 100% clean energy by 2050.
Many CAZs are being taken forward in England by local and combined authorities after lengthy periods of consultation, while others are about start engagement on proposals.
Although their plans sit within the Government’s Clean Air Framework, there is a lack of consistency among authorities and a paucity of funding for initiatives that is likely to inhibit the ambitions of national fleet operators, businesses and local authorities.
Working in partnership would enable the promotion of a shared voice between local authorities, transport operators and business, and assist in creating successful air quality solutions.
While enhancing the delivery of local plans, partnership would also help promote a powerful voice to inform the Government Spending Review later this year.
Consequently, the Fleet News/UK100 CAZ roundtable discussions (sponsored by ALD, Enterprise and Geotab) have been designed with a clear objective in mind: to create political momentum for the delivery of Clean Air Zones by securing consensus on challenges and opportunities, with advocacy by national government and commitments to action from local authorities, business and transport operators.
Delegates, including UK business and local authorities, will agree to sign up to a Clean Air Zone Declaration to underline their commitment to delivering improvements in air quality.
This will result in a shared agreement between local authorities, transport operators and business on priorities that are needed locally and nationally to successfully take forward CAZs, together with commitments to action.
* Fleets interested in attending one of the CAZ roundtables (dates below) should email Fleet News editor-in-chief Stephen Briers: email@example.com
International Air Quality event
Between 22 and 25 October, London mayor Sadiq Khan will host an international air quality event and C40 air quality network meeting involving up to 15 world cities.
This four-day event will offer the opportunity for London to share its experience, expertise and influence.The event aims to support and encourage other UK and global cities and governments to develop policies and adopt legislation that will drive action towards meeting WHO-based air quality targets.
The third National Air Quality Summit, with is being arranged with UK100, is proposed to run in parallel with the international component of this event.
The added support and attention generated by the summit will reinforce pressure for the Government to adopt strong clean air legislation as it considers the Environment Bill, including adopting legally-binding WHO air quality standards for the UK.
October 8: at Fleet Live in Birmingham – for lead officers, council cabinet members and UK fleets.
October 23: in London – for council leaders, city mayors and UK fleets. This workshop will form part of the International Air Quality event hosted by the Mayor of London between October 22-25.
CAZ BRIEFING EVENT
October 9: at Fleet Live in Birmingham – for UK fleets.

domain: politics


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Dihydrodipicolinate synthase (DHDPS) catalyzes the rate limiting step in lysine biosynthesis in bacteria and plants. The structure of DHDPS has been determined from several bacterial species and shown in most cases to form a homotetramer or dimer of dimers. However, only one plant DHDPS structure has been determined to date from the wild tobacco species, Nicotiana sylvestris (Blickling et al. (1997) J. Mol. Biol. 274, 608–621). Whilst N. sylvestris DHDPS also forms a homotetramer, the plant enzyme adopts a ‘back-to-back’ dimer of dimers compared to the ‘head-to-head’ architecture observed for bacterial DHDPS tetramers. This raises the question of whether the alternative quaternary architecture observed for N. sylvestris DHDPS is common to all plant DHDPS enzymes. Here, we describe the structure of DHDPS from the grapevine plant, Vitis vinifera, and show using analytical ultracentrifugation, small-angle X-ray scattering and X-ray crystallography that V. vinifera DHDPS forms a ‘back-to-back’ homotetramer, consistent with N. sylvestris DHDPS. This study is the first to demonstrate using both crystal and solution state measurements that DHDPS from the grapevine plant adopts an alternative tetrameric architecture to the bacterial form, which is important for optimizing protein dynamics as suggested by molecular dynamics simulations reported in this study.
Citation: Atkinson SC, Dogovski C, Downton MT, Pearce FG, Reboul CF, Buckle AM, et al. (2012) Crystal, Solution and In silico Structural Studies of Dihydrodipicolinate Synthase from the Common Grapevine. PLoS ONE 7(6): e38318. https://doi.org/10.1371/journal.pone.0038318
Editor: Petri Kursula, University of Oulu, Finland
Received: January 28, 2012; Accepted: May 8, 2012; Published: June 25, 2012
Copyright: © 2012 Atkinson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding was provided by: (1) Australian Research Council for providing a Future Fellowship for Matthew A. Perugini; (2) University of Melbourne for providing a CR Roper Fellowship for Renwick CJ Dobson; (3) University of Melbourne (FRGSS 2011 Project grant) for project funding (in part); and (4) U.S. Defense Threat Reduction Agency (DTRA Project AB07CBT004) for project funding (in part). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: Please note that co-authors Ashley M. Buckle, Juliet A. Gerrard, Renwick C.J. Dobson and Matthew A. Perugini are PLoS ONE Editorial Board members. This does not alter our adherence to all the PLoS ONE policies on sharing data and materials. Also note that co-authors Matthew T. Downton and John Wagner are employed by a commercial company - IBM Research Collaboratory for Life Sciences-Melbourne. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Lysine is synthesized de novo in bacteria, plants and some fungi –. The lysine-biosynthesis pathway commences with the condensation of pyruvate and (S)-aspartate semialdehyde (ASA), to form (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid (HTPA) –. This reaction is catalyzed by dihydrodipicolinate synthase (DHDPS), which is the product of an essential gene in bacteria , , , . The structure of DHDPS has been studied extensively from a number of bacteria, including Bacillus anthracis (Ba) ,  (Fig. 1A), Corynebacterium glutamicum , Escherichia coli , , Hahella chejuensis , Methanocaldococcus jannaschii , Mycobacterium tuberculosis , Neisseria meningitides , Pseudomonas aeruginosa , Staphylococcus aureus ,  and Thermotoga maritima . Typically, bacterial DHDPS forms a tetramer of four identical (β/α)8-barrel monomers that can be described as a ‘head-to-head’ dimer-of-dimers (Fig. 1A). The tetramer contains four active sites, one per monomer, that are located at the ‘tight’ dimer interface (i.e. ab or cd, Fig. 1A). Each ‘tight’ dimer unit associates via noncovalent interactions at the ‘weak’ dimer interface (i.e. ac or bd, Fig. 1A) to form the homotetrameric structure. The ‘tight’ dimer interface also contains a cleft that binds the allosteric inhibitor, lysine, which mediates feedback inhibition in DHDPS enzymes from Gram-negative bacteria and plants –. However, DHDPS from Gram-positive species, including Ba , ,  and S. aureus ,  do not bind lysine and are thus insensitive to feedback inhibition. Given that the structural requirements for catalysis, and where appropriate allostery, are encoded by the ‘tight’ dimer unit, it is not obvious why the enzyme adopts a dimer-of-dimers. Interestingly, recent studies show that dimeric mutants of DHDPS from E. coli ,  and Ba  possess significantly attenuated catalytic activity. Loss of function of the dimeric mutants is attributed to excessive dynamics or ‘breathing motion’ at the ‘tight’ dimer interface, which compromises the integrity of the active sites , . Accordingly, the buttressing of two dimeric units together to form the homotetrameric structure is thought to stabilize the tight dimer interface, including the key active site residues , , .
Dihydrodipicolinate synthase from (A) B. anthracis (PDB ID: 3HIJ ) and (B) N. sylvestris . Structural coordinates of the N. sylvestris DHDPS were kindly provided by Prof Robert Huber (Max Planck Institute for Biochemistry).
By contrast, structural characterization of DHDPS from plants is limited to a single study of the enzyme from the wild tobacco plant, Nicotiana sylvestris . This study shows that N. sylvestris DHDPS also forms a homotetramer, but in a ‘back-to-back’ arrangement (Fig. 1B) opposite in orientation to the typical bacterial tetrameric form (Fig. 1A). Consequently, the allosteric sites that bind lysine and mediate feedback inhibition  are located in the interior of the tetramer (Fig. 1B) rather than on the outside of the structure as observed for E. coli DHDPS . However, the N. sylvestris enzyme  is the only plant DHDPS structure determined to date. The unique quaternary architecture observed in the crystal structure of N. sylvestris DHDPS has not yet been confirmed in other plant species or validated in aqueous solution; and surprisingly, the structural coordinates of the N. sylvestris enzyme are not available in the Protein Data Bank (PDB). Studies validating the quaternary structure of plant DHDPS will thus offer insight into the molecular evolution of this important oligomeric enzyme.
Therefore, the aim of this study was to determine the quaternary structure of DHDPS from the agriculturally-important species, Vitis vinifera (Vv) or the common grapevine. Here, we present a thorough characterization of the structure of Vv-DHDPS both in aqueous solution and the crystal state compared to Ba-DHDPS, an example of the typical bacterial tetramer (Fig. 1A). We show that Vv-DHDPS adopts a ‘back-to-back’ dimer-of-dimers consistent with the structure reported for N. sylvestris DHDPS (Fig. 1B), and subsequently demonstrate using molecular dynamics (MD) simulations that the ‘back-to-back’ architecture is important for stabilizing protein dynamics of the ‘tight’ dimer unit. This study suggests that DHDPS from plants adopt an alternative quaternary architecture to the typical bacterial form, thus offering insight into the molecular evolution of an important oligomeric enzyme.
Results and Discussion
Vv-DHDPS is Folded and Active
Recombinant Vv-DHDPS was expressed and purified to homogeneity as described previously . Circular dichroism (CD) spectroscopy shows that the recombinant enzyme is folded (Fig. S1A) and is comprised of a similar proportion of α-helical and β-strand secondary structure as observed for DHDPS from other species , , , . The CD spectrum of Vv-DHDPS was also obtained in the presence of the substrate, pyruvate. However, no significant change in secondary structure is observed in the presence of this ligand relative to the apo form (Fig. S1A). To confirm that the recombinant enzyme is active, the kinetic properties of Vv-DHDPS were determined using the quantitative coupled assay employing NADPH-dependent dihydrodipicolinate reductase (DHDPR) . Initial rates (ΔAbs340 min−1) were measured at varying concentrations of both DHDPS substrates, pyruvate and ASA, using excess amounts of E. coli DHDPR and NADPH. The resulting Michaelis-Menten curves (Fig. S1B, symbols) were globally fitted to various bi-substrate kinetic models, namely the ternary complex, Ping-Pong and Ping-Pong with substrate inhibition models. The Ping-Pong model (without substrate inhibition) provided the global best fit (Fig. S1B, solid lines), which is consistent with the mechanism observed for other DHDPS enzymes , , , , . The fit resulted in a R2 value of 0.98 and yielded KM constants of 1.02 mM and 0.180 mM for pyruvate and ASA, respectively, and a Vmax of 160 µmol min−1 mg−1 (kcat = 45 s−1) (Fig. S1B). These kinetic constants are similar to those determined for other DHDPS enzymes , , .
Vv-DHDPS is a Tetramer in Solution
To characterize the quaternary structure of Vv-DHDPS in aqueous solution, sedimentation velocity experiments were conducted in the analytical ultracentrifuge at an initial enzyme concentration of 13 µM in the presence and absence of pyruvate. The absorbance versus radial position profile for Vv-DHDPS in the absence of pyruvate is plotted in Fig. 2A. These data show a distinct sedimenting boundary consistent with the presence of a single species. This assertion is supported by 2-dimensional spectrum analysis (2DSA) , which decomposes the velocity experimental data into a sum of non-interacting finite element solutions and provides information on sedimentation and shape. The resulting analysis shows Vv-DHDPS (Mr = 37,876.5) sediments predominantly as a single species with a standardized weight-average sedimentation coefficient (s20,w) of 7.3 S (Fig. 2B), molecular weight of 153 kDa (Fig. S2), and a frictional ratio (f/f0) of 1.35 (Table S1). These data demonstrate that Vv-DHDPS exists as a tetramer in aqueous solution. By contrast, Ba-DHDPS (Mr = 31,233), which also forms a tetramer in solution and the crystal state , sediments with a s20,w of 6.4 S (Table S1). Similar sedimentation velocity data were also obtained for Ba-DHDPS and Vv-DHDPS in the presence of saturating amounts of pyruvate (Fig. S3), which indicates that at an initial protein concentration of 13 µM the substrate does not alter the tetrameric quaternary structure of the enzymes. The hydrodynamic properties of Vv-DHDPS compared to Ba-DHDPS are summarized in Table S1.
(A) Absorbance at 280 nm measured as a function of radial position from the axis of rotation (cm) for Vv-DHDPS (13 µM) centrifuged at 40,000 rpm. The raw data are presented as open symbols plotted at time intervals of 10 min overlaid with the 2DSA fit shown in panel B. (b) Pseudo-3D plots of solute distributions for 2DSA Monte Carlo of Vv-DHDPS using a grid resolution of 10,000 solutes. The colour scale represents the signal of each species in optical density units.
Crystal Structure of Vv-DHDPS Reveals Alternative Tetrameric Architecture
To further investigate the quaternary structure and shape of the Vv-DHDPS tetramer, the crystal structure of the enzyme in complex with pyruvate was determined to a resolution of 2.2 Å (PDB ID: 3TUU) (Fig. 3A). The asymmetric unit contains two tetramers comprised of four identical subunits. Each tetramer can be described as a dimer-of-dimers, with the monomers within the two dimeric units ab and cd (Fig. 3A) tightly bound to each other, and weaker interactions between monomers ac and bd. The quaternary architecture of the Vv-DHDPS tetramer is identical to the N. sylvestris structure (Fig 1B) and quite distinctive from the typical bacterial tetramer (Fig 1A). Indeed, when crystal packing is investigated using symmetry operations, it can be seen that the orientation of the dimeric units are incompatible with formation of the bacterial head-to-head tetramer (Fig. S4).
(A) Crystal structure of Vv-DHDPS (PDB ID: 3TUU) showing the position of the active site lysine residue (yellow spheres) in each monomer and the self-association interfaces. Two monomers come together at the tight dimer interface to form the dimeric unit, which dock at the weak dimer interface to form a homotetramer. The asymmetric unit contained eight monomers assembled as two homotetramers. (B) Active site residues of Vv-DHDPS overlaid with E. coli DHDPS (cyan). Pyruvate is shown in yellow. Tyr132 (orange) from the adjacent monomer interdigitates across the tight interface and is overlaid with the equivalent residue in E. coli DHDPS (green).
Each monomer comprises an N-terminal (β/α)8-barrel domain and a C-terminal domain consisting of 3 α-helices and 2 short β-strands (Figs. S5A & S5B). The active site centers around Lys184, which forms a Schiff base with pyruvate and is located at the center of each monomeric unit (Fig. 3B). Three hydroxyl-containing amino acids, namely Tyr132, Thr69 and Tyr156, form the conserved catalytic triad with Tyr132 contributed from the adjacent monomer across the ‘tight’ dimer interface (i.e. interface between subunits ab or cd, Fig. 3A). The spatial orientation of the catalytic triad residues (Fig. 3B), as well as Arg161 and Ile223 that are also important for catalytic activity, is consistent with that observed in the active sites of other DHDPS structures , , . Examination of potential interfaces within the enzyme using the Protein Interfaces, Surfaces and Assemblies (PISA) program  show that each of the two tight dimer interfaces (ab and cd) bury approximately 1790 Å2 per monomer, which corresponds to ∼14% of the total surface area of the monomer. By contrast, the interfaces between monomers a & c and b & d, bury a surface area of approximately 630 Å2 per monomer.
SAXS Analyses Confirms Alternative Quaternary Structure of Vv-DHDPS
SAXS was employed to validate the analytical ultracentrifugation studies in solution (Fig. 2) and the crystal structure of Vv-DHDPS (Fig. 3). Scattering data for Vv-DHDPS were collected in the presence of pyruvate (Fig 4A), compared to Ba-DHDPS (Fig. S6A).The radius of gyration (Rg) for Vv-DHDPS was determined by Guinier analysis to be 35.2 Å. The pair distance distribution function [P(r)] was calculated using the indirect Fourier transform method (Fig. 4B). The Rg from the P(r) analysis plot was calculated to be 34.3 Å, and the maximum dimension of the scattering particle (Dmax) to be 100 Å, which is in close agreement with the crystal structure (Fig. 3A, Dmax is 100 Å). By comparison, Ba-DHDPS has a Rg of 30.9 Å determined by Guinier analysis and a Rg of 31.7 Å with a Dmax of 90 Å determined from P(r) analysis (Fig. S6C). Again, this is in close agreement with the crystal structure (PDB ID: 3HIJ, Dmax is 85 Å). The scattering profile data for Ba-DHDPS is consistent with the tetrameric form of the protein observed in the crystal structure and in solution (Fig. S6A) .
(A) Theoretical scattering profiles from Vv-DHDPS (solid line) and Ba-DHDPS (dashed line) and the raw SAXS data (○).Theoretical scattering profiles were generated from crystallographic coordinates using CRYSOL. (B) P(r) plots of Vv-DHDPS from experimental data (black) and SOMO bead model (red) using ULTRASCAN. (C) SOMO bead model of Vv-DHDPS. The various colored beads represent acidic (green), hydrophobic (cyan), polar (red), basic (yellow) and non-polar (magenta) side-chains. Blue beads represent the protein main-chain and brown indicates buried beads.
Direct comparisons of both the crystal structures and SAXS profiles of Vv-DHDPS and Ba-DHDPS (PDB ID: 3HIJ ) were performed using CRYSOL . The scattering profile of Vv-DHDPS fits that calculated for the crystal structure of the enzyme (Fig. 4A) [reduced chi-squared (χ2v) = 1.5]. This represents a statistically better fit to the data than the fit to the Ba-DHDPS CRYSOL profile [(χ2v = 7) (PF(F;v1,v2) <8.7×10−34 (F >4.5, v1 = 278, v2 = 278)] . The crystal structure and theoretical scattering profile of Vv-DHDPS do not exactly overlay, which is indicative that there are some differences between the protein in solution compared to the crystal state. Indeed, crystal packing (Fig. S4) and the high concentrations of polyethylene glycol and salt employed for crystallization  may account for this difference. In addition, the lack of electron density observed for the N-terminal region (including the His-tag) indicates that this region is highly flexible. Thus, CORAL  rigid body modeling was performed using the Vv-DHDPS PDB coordinates (3TUU) with the 30 missing N-terminal residues added. This yielded a significantly better fit (Fig. S7). Conversely, the Ba-DHDPS scattering data fit more closely to the theoretical profile calculated from the crystal structure of Ba-DHDPS (3HIJ) when analyzed by CRYSOL (χ2v = 1.2), compared to that for Vv-DHDPS (χ2v = 6.3) (Fig. S6A). In addition, the Vv-DHDPS and Ba-DHDPS crystal structures were also used to construct bead models (Fig. 4C and Fig. S6B) using the program SOMO in the ULTRASCAN software package . The resulting theoretical P(r) distributions fit well to the experimental P(r) distributions (Fig 4B and Fig. S6C). The SOMO bead models were also used to predict the hydrodynamic properties from the crystal structures of Ba-DHDPS (Fig. 1A) and Vv-DHDPS (Fig. 3A), which agree well with those determined by analytical ultracentrifugation (Table S1). The higher than predicted frictional ratio determined by analytical ultracentrifugation for Vv-DHDPS (Table S1) is likely to be due to the N-terminal region of the enzyme that is disordered and thus absent in the crystal structural model shown in Fig. 3A. Nevertheless, analytical ultracentrifugation, X-ray crystallography and SAXS analyses together demonstrate that Vv-DHDPS forms a ‘back-to-back’ tetrameric architecture (Figs. 2, 3, and 4), compared to the ‘head-to-head’ conformation observed for the typical bacterial tetramer (Fig. 1A & Fig. S6).
Molecular Dynamics Simulations of Vv-DHDPS
To gain insight into the importance of tetramerization to the Vv-DHDPS structure, molecular dynamics (MD) simulations were performed. MD simulations were conducted on the Vv-DHDPS tetramer (i.e. chains a, b, c & d, Fig. 3A) compared to the ‘tight’ dimer unit (i.e. chains ab, Fig. 3A). The MD simulations show that the majority of residues in the Vv-DHDPS ‘tight’ dimer have significantly greater root mean square fluctuations (RMSFs) (Fig. 5, red line) compared to the wild-type tetramer (Fig. 5, black line). The larger RMSFs observed for the dimer include the key catalytic residues Thr69, Tyr156 and Lys184, as well as Tyr132, which interdigitates across the ‘tight’ dimer interface to form part of the active site of the adjacent monomer (Fig. 5). These data suggest that the dimer possesses greater conformational flexibility than the wild-type tetramer, and thus formation of the ‘back-to-back’ dimer-of-dimers functions to attenuate protein dynamics. Indeed, similar MD simulation results have recently been reported for bacterial DHDPS , which in turn, support biophysical analyses of mutant dimers and wild-type tetramers from the bacterial species Ba  and E. coli , . Taken together, the results presented here and those reported in previous studies , , , , suggest that plant and bacterial DHDPS enzymes evolved to form homotetramers, albeit with different quaternary architectures, as a means to attenuate ‘breathing motion’ of the ‘tight’ dimer unit. This study therefore offers further insight into the molecular evolution at the quaternary structure level of an important bacterial and plant enzyme.
Simulations were analyzed by aligning chain A from all frames of the trajectories, and computing the root mean squared fluctuations (RMSF) of chain B, the monomer on the opposite side of the ‘tight-dimer’ interface. Shown are the RMSF values by residue number for the dimer (red) and tetramer (black). The inset shows 75 frames of the aligned dimer at 1 ns intervals.
In this study, we show for the first time that DHDPS from the plant Vitis vinifera forms a homotetramer or dimer-of-dimers both in solution and the crystal state (Figs. 2, 3, and 4). Consistent with previous studies of DHDPS from Nicotiana sylvestris , we demonstrate that Vv-DHDPS adopts a ‘back-to-back’ dimer-of-dimers compared to the ‘head-to-head’ architecture observed for DHDPS from most bacterial species. We subsequently show using MD simulations that tetramerization of Vv-DHDPS is important for attenuating protein dynamics of the ‘tight’ dimer unit, which offers insight into the molecular evolution of an important bacterial and plant enzyme.
Materials and Methods
Cloning, Expression and Purification of Vv-DHDPS
The dapA gene encoding Vv-DHDPS was purchased from Geneart and cloned into the pET28a expression vector as described elsewhere . Recombinant protein was produced in the host strain E. coli BL21-DE3 via induction by IPTG at 16°C. Cells were harvested following overnight IPTG treatment and then resuspended in 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole, before lysis by sonication. Vv-DHDPS was subsequently isolated by metal-affinity liquid chromatography as described previously .
Circular Dichroism Spectroscopy
Circular dichroism (CD) spectra of Vv-DHDPS (4 µM) were recorded using an Aviv Model 410-SF CD spectrometer. Wavelength scans were performed between 198 and 250 nm in 20 mM Tris, 150 mM NaCl, pH 8.0 in 1.0 mm quartz cuvette as reported previously , , . Data were analysed using the CDSSTR algorithm from the CDPro software package  incorporating the SP22X database.
DHDPS-DHDPR Coupled Enzyme Kinetic Assay
Kinetic analyses of Vv-DHDPS were performed using the DHDPS-DHDPR coupled assay as previously described , using E. coli DHDPR. Assays were routinely conducted in triplicate at a constant temperature of 30°C with reaction mixtures allowed to equilibrate in a temperature-controlled Cary 4000 UV-visible spectrophotometer for 10 min before initiating the reaction with 60 nM DHDPS. Prior to the experiment, pyruvate and ASA concentrations were routinely quantified by the addition of limiting amounts of substrate by measuring the oxidation of NADPH spectrophotometrically at 340 nm. Initial rate data were analyzed using the ENZFITTER program available from Biosoft. Data were fitted to the various models, including the bi-bi ping-pong substrate model that yielded the best fit as assessed by Sigma values and the lowest standard error associated with the kinetic constants.
Sedimentation velocity experiments were performed in a Beckman Coulter model XL-I analytical ultracentrifuge. Double sector quartz cells were loaded with 400 µL of buffer and 380 uL of Vv-DHDPS or Ba-DHDPS at an initial concentration of 13 µM. The cells were loaded into an An50-Ti rotor and left to equilibrate at 30°C. The rotor was accelerated to 40,000 rpm and absorbance readings were collected continuously at 280 nm and 30°C using a step size of 0.003 cm without averaging. Initial scans were carried out at 3,000 rpm to determine the optimal wavelength and radial positions for the high speed experiment. Samples of Vv-DHDPS monitored in the presence of pyruvate contained ligand in both the reference and sample channels. Solvent density, solvent viscosity, and estimates of the partial specific volume of Vv-DHDPS (0.7386 ml/g) and Ba-DHDPS (0.7463 ml/g) at 30°C were calculated using SEDNTERP . Data were analyzed via 2-Dimensional Spectrum (2DSA) Monte Carlo analysis  incorporated in the ULTRASCAN software package , , which can be downloaded from www.ultrascan.uthscsa.edu.
Crystallization of Vv-DHDPS and X-ray Diffraction Data
Vv-DHDPS was crystallized as described previously using sitting- and hanging-drop vapor diffusion . For X-ray data collection, crystals were transferred to reservoir solution containing 20% (v/v) glycerol and directly flash frozen in liquid nitrogen. Intensity data were collected at the Australian Synchrotron using the MX2 beamline as described in . Diffraction data sets were processed and scaled using the package MOSFLM  and SCALA , . Molecular replacement was performed using PHASER  with E. coli DHDPS (PDB ID: 1YXC ) as the search model. CHAINSAW  from the CCP4 suite  was used to prepare the model of E. coli DHDPS, omitting waters and reducing it to its monomeric form. Structural refinement of the resulting 8 monomers was performed using REFMAC5 , with iterative model building using COOT . In the first steps of refinement, non-crystallographic restraints were applied, followed by simulated annealing using PHENIX . The structure was validated using the MolProbity Server . Refinement statistics are given in Table 1. Ramachandran statistics showed 98% of the residues in the most favored region, 1.6% in the additionally allowed regions and 0.4% (a single residue) in the disallowed region, namely Tyr 132, which is consistent with the equivalent Tyr residues observed in DHDPS structures from other species , .
Small Angle X-ray Scattering
Small-angle X-ray scattering (SAXS) data were collected at the Australian Synchrotron, Clayton on the SAXS/WAXS beamline. The X-ray beam size at the sample was 250 µm horizontal, 80 µm vertical and data were collected using a Pilatus 1M detector positioned 900 mm from the sample, giving a q range of 0.01–0.6 Å−1 (wavelength, 1.0332 Å). The protein sample analyzed was subjected to in-line size exclusion chromatography on a Superdex 200 5/150 GL gel-filtration column (GE Healthcare) with a bed volume of 3 ml equilibrated with buffer at a flow rate of 0.2 ml.min−1. 50 µl Vv-DHDPS at 234 µM was injected and the fractionated sample flowed through a 1.5 mm quartz capillary where it was exposed to the X-ray beam. 600 detector images of sequential 5 s exposures were collected at 298 K, corresponding to a total elution volume of 4.2 ml. Radial averaging, background subtraction and image-series analysis were performed using SAXS15ID software (Australian Synchrotron). Eight sequential detector images were averaged to generate each SAXS data set for subsequent analysis using the ATSAS (v.2.3) software . The region of the gel filtration chromatogram used for analysis is shown in Fig. S8. Guinier fits were made using PRIMUS  and P(r) distribution analyses performed using GNOM . Theoretical scattering curves were generated from atomic coordinates and compared with experimental scattering curves using CRYSOL  and CORAL . Statistical analysis was performed as described previously . Briefly, for each fit was calculated from , the parameter reported by CRYSOL.where N is the number of data points and m the number of fitting parameters. To analyze a change in between the Ba-DHDPS and Vv-DHDPS fits, the statistic was calculated. The F distribution was then integrated to yield the probability the two values are equal, , where indicates significance. Bead models and subsequent P(r) plots were generated using the Solution Modeler (SOMO) software  in the ULTRASCAN suite , .
Molecular Dynamics Simulations
Molecular dynamics simulations were performed with the native Vv-DHDPS tetramer from the crystal structure (PDB ID: 3TUU) and a dimer formed from two monomers joined at the ‘tight’ dimer interface. In both cases, the substrate, pyruvate, was removed and the structures were solvated using the TIP3P water model. The CHARMM force field  and the molecular dynamics package NAMD  were used. After allowing 25 ns for the structure to equilibrate at constant temperature of 293K and atmospheric pressure, trajectories of 75 ns were generated at 0.01 ns intervals.
Secondary structure and enzyme kinetic analyses of Vv-DHDPS. (A) CD spectra of Vv-DHDPS at 0.2 mg/ml recorded in 0.5 nm increments with a 2 s averaging time from 198 to 250 nm. Samples were prepared in standard buffer and analyzed in a 1 mm pathlength quartz cuvette. Raw data without pyruvate (circles) and with 5 mM pyruvate (triangles) were fitted by nonlinear least squares regression (solid lines) using the CDPro software package and employing the CDSSTR algorithm with the SP22X reference set . The nonlinear best fit resulted in a RMSD of 0.145 and structural composition of 30% α-helix, 20% β-strand, 28% turn and 22% unordered structure for the absence of pyruvate and a RMSD of 0.121 and structural composition of 31% α-helix, 18% β-strand, 24% turn and 27% unordered structure in the presence of pyruvate. (B) Michaelis-Menten analyses of Vv-DHDPS. The initial velocity at 0.1–3.0 mM pyruvate plotted as a function of ASA concentration (dots). A global best-fit to a bi-bi Ping Pong model without substrate inhibition using the ENZFITTER software package (BioSoft) with an R2 of 0.98 and p>F of 9.37×10−39.
Sedimentation velocity molecular weight analysis of Vv-DHDPS. Pseudo 2DSA plot of f/f0 versus molecular weight of Vv-DHDPS using the data shown in Figure 2A. A grid resolution of 10,000 solutes was employed . The colour scale represents the signal of each species in optical density units.
Sedimentation velocity analyses of Ba-DHDPS and Vv-DHDPS in the presence of pyruvate. Shown are the pseudo-3D plots for solute distributions for 2DSA Monte Carlo analyses of Ba-DHDPS (panel A) and Vv-DHDPS (panel B) at an initial protein concentration of 13 µM in the presence of 5 mM pyruvate. A grid resolution of 10,000 solutes was employed in the analyses . The colour scale represents the signal of each species in optical density units.
Crystal lattice of Vv-DHDPS (PDB ID: 3TUU). Vv-DHDPS crystal packing generated using symmetry operations. The orientation of the dimeric units is incompatible with formation of the head-to-head tetramer commonly observed in bacteria DHDPS (Fig. 1A).
Tertiary structure of Vv-DHDPS (PDB ID: 3TUU). (A) View looking down the (β/α)8-barrel and C-terminal domain. The active site is defined by the position of Lys184 (stick view). (B) Side view of the (β/α)8-barrel and C-terminal domain.
SAXS analyses of Ba-DHDPS. (A) Fits of theoretical scattering profiles from Vv-DHDPS (dashed line) and Ba-DHDPS (solid line) to the SAXS data (▴). Theoretical scattering profiles were generated from crystallographic coordinates and fitted to the Ba-DHDPS SAXS data using CRYSOL . (B) SOMO bead model of Ba-DHDPS. The various colored beads represent acidic (green), hydrophobic (cyan), polar (red), basic (yellow) and non-polar (magenta) side-chains. Blue beads represent the protein main-chain and brown indicates buried beads. (C) P(r) plots of Ba-DHDPS from experimental data (black) and SOMO bead model shown in panel B (blue) (37) using ULTRASCAN , .
Comparison of experimental and theoretical SAXS data of Vv-DHDPS. Theoretical scattering profile from Vv-DHDPS (solid line) with 30 N-terminal residues modeled using CORAL (36) to the SAXS data (•). This fit (reduced chi-squared (χ2v) = 1.0). This represents a statistically better fit to the data than the fit to the CRYSOL profile [(χ2v = 1.5) (PF(F;v1,v2) <5.1 × 10−11 (F >2.2)].
SAXS gel filtration chromatogram. Fraction employed in SAXS analysis is highlighted in pink.
Hydrodynamic properties of Vv -DHDPS and Ba -DHDPS.
We would firstly like to acknowledge the support and assistance of the friendly staff at the Bio21 Collaborative Crystallographic Centre at CSIRO Molecular and Health Technologies, Parkville, Melbourne and the beamline scientists at the Australian Synchrotron, Victoria, Australia. We would also like to thank all members of the Perugini laboratory for helpful discussions during the preparation of this manuscript.
Conceived and designed the experiments: SCA JW MAP. Performed the experiments: SCA MTD. Analyzed the data: SCA CD MTD CFR AMB RCJD. Contributed reagents/materials/analysis tools: FGP JAG. Wrote the paper: SCA MTD JW MAP.
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- 15. Devenish SR, Huisman FH, Parker EJ, Hadfield AT, Gerrard JA (2009) Cloning and characterisation of dihydrodipicolinate synthase from the pathogen Neisseria meningitidis. Biochim Biophys Acta 1794: 1168–1174.
- 16. Kaur N, Gautam A, Kumar S, Singh A, Singh N, et al. (2011) Biochemical studies and crystal structure determination of dihydrodipicolinate synthase from Pseudomonas aeruginosa. Int J Biol Macromol 48: 779–787.
- 17. Burgess BR, Dobson RC, Bailey MF, Atkinson SC, Griffin MD, et al. (2008) Structure and evolution of a novel dimeric enzyme from a clinically important bacterial pathogen. J Biol Chem 283: 27598–27603.
- 18. Girish TS, Sharma E, Gopal B (2008) Structural and functional characterization of Staphylococcus aureus dihydrodipicolinate synthase. FEBS Lett 582: 2923–2930.
- 19. Pearce FG, Perugini MA, McKerchar HJ, Gerrard JA (2006) Dihydrodipicolinate synthase from Thermotoga maritima. Biochem J 400: 359–366.
- 20. Frisch DA, Gengenbach BG, Tommey AM, Sellner JM, Somers DA, et al. (1991) Isolation and characterization of dihydrodipicolinate synthase from maize. Plant Physiol 96: 444–452.
- 21. Dereppe C, Bold G, Ghisalba O, Ebert E, Schar HP (1992) Purification and characterization of dihydrodipicolinate synthase from pea. Plant Physiol 98: 813–821.
- 22. Laber B, Gomis-Ruth FX, Romao MJ, Huber R (1992) Escherichia coli dihydrodipicolinate synthase. Identification of the active site and crystallization. Biochem J 288: 691–695.
- 23. Domigan LJ, Scally SW, Fogg MJ, Hutton CA, Perugini MA, et al. (2009) Characterisation of dihydrodipicolinate synthase (DHDPS) from Bacillus anthracis. Biochim Biophys Acta 1794: 1510–1516.
- 24. Griffin MD, Dobson RC, Gerrard JA, Perugini MA (2010) Exploring the dihydrodipicolinate synthase tetramer: how resilient is the dimer-dimer interface? Arch Biochem Biophys 494: 58–63.
- 25. Blickling S, Beisel HG, Bozic D, Knablein J, Laber B, et al. (1997) Structure of dihydrodipicolinate synthase of Nicotiana sylvestris reveals novel quaternary structure. J Mol Biol 274: 608–621.
- 26. Atkinson SC, Dogovski C, Newman J, Dobson RC, Perugini MA (2011) Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from the grapevine Vitis vinifera. Acta Crystallogr Sect F Struct Biol Cryst Commun 67: 1537–1541.
- 27. Dobson RC, Valegard K, Gerrard JA (2004) The crystal structure of three site-directed mutants of Escherichia coli dihydrodipicolinate synthase: further evidence for a catalytic triad. J Mol Biol 338: 329–339.
- 28. Karsten WE (1997) Dihydrodipicolinate synthase from Escherichia coli: pH dependent changes in the kinetic mechanism and kinetic mechanism of allosteric inhibition by L-lysine. Biochemistry 36: 1730–1739.
- 29. Kumpaisal R, Hashimoto T, Yamada Y (1987) Purification and characterization of dihydrodipicolinate synthase from wheat suspension cultures. Plant Physiol 85: 145–151.
- 30. Dobson RC, Devenish SR, Turner LA, Clifford VR, Pearce FG, et al. (2005) Role of arginine 138 in the catalysis and regulation of Escherichia coli dihydrodipicolinate synthase. Biochemistry 44: 13007–13013.
- 31. Brookes E, Cao W, Demeler B (2010) A two-dimensional spectrum analysis for sedimentation velocity experiments of mixtures with heterogeneity in molecular weight and shape. Eur Biophys J 39: 405–14.
- 32. Dobson RC, Griffin MD, Devenish SR, Pearce FG, Hutton CA, et al. (2008) Conserved main-chain peptide distortions: a proposed role for Ile203 in catalysis by dihydrodipicolinate synthase. Protein Sci 17: 2080–2090.
- 33. Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372: 774–97.
- 34. Svergun D, Barberato C, Koch MHJ (1995) CRYSOL - a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Cryst 28: 768–773.
- 35. Mills RD, Trewhella J, Qiu TW, Welte T, Ryan TM, et al. (2009) Domain organization of the monomeric form of the Tom70 mitochondrial import receptor. J Mol Biol 388: 1043–1058.
- 36. Petoukhov MV, Svergun DI (2005) Global rigid body modeling of macromolecular complexes against small-angle scattering data. Biophys J 89: 1237–1250.
- 37. Rai N, Nöllmann M, Spotorno B, Tassara G, Byron O, et al. (2005) SOMO (SOlution MOdeler): differences between X-Ray- and NMR-derived bead models suggest a role for side chain flexibility in protein hydrodynamics. Structure 13: 723–734.
Reboul CF, Porebski BT, Griffin MDW, Dobson RCJ, Perugini MA, et al. (2012) Structural and dynamic requirements for optimal activity of the essential bacterial enzyme dihydrodipicolinate synthase. PLoS Comp Biol PCOMPBIOL-D-11–01947–10.1371/journal.pcbi.100253739.
- 39. Davis AJ, Perugini MA, Smith BJ, Stewart J, Ilg T, et al. (2004) Properties of GDP-mannose pyrophosphorylase, a critical enzyme and drug target in Leishmania mexicana. J Biol Chem 279: 12462–12468.
- 40. Sreerama N, Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287: 252–260.
Laue TM, Shah BD, Ridgeway TM, Pelletier SL (1992) Computer-aided interpretation of analytical sedimentation data for proteins. In: Harding SE, Rowe AJ, Horton JC, editors. pp. 90–125. Cambridge: The Royal Society of Chemistry.
Demeler B (2005) ULTRASCAN a comprehensive data analysis software package for analytical ultracentrifugation experiments. In: Scott DJ, Harding SE, Rowe AJ, editors. pp. 210–229. Cambridge: Royal Society of Chemistry.
- 43. Demeler B Rashidi H, Buehler L, editors. (2005) Hydrodynamic methods. CRC Press LLC: 226–255. Bioinformatics basics: applications in biological science and medicine.
Leslie AGW, Powell HR (2007) Processing diffraction data with Mosflm. In: Read RJ, Sussman , J L, editors. pp. 41–51. Dordrecht: Springer.
- 45. Evans P (2006) Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr 62: 72–82.
- 46. Collaborative Computational Project Number 4 (1994) The CCP4 suite: Programs for protein crystallography. Acta Crystallographica Section D 50: 760–763.
- 47. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, et al. (2007) PHASER crystallographic software. J. Appl. Cryst. 40: 658–674.
- 48. Stein N (2008) CHAINSAW: a program for mutating pdb files used as templates in molecular replacement. J Appl Cryst 41: 641–643.
- 49. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53: 240–255.
- 50. Emsley P, Cowtan K (2004) COOT: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126–2132.
- 51. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, et al. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66: 213–221.
- 52. Chen VB, Arendall WB 3rd, Headd JJ, Keedy DA, Immormino RM, et al (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66: 12–21.
- 53. Konarev PV, Petoukhov MV, Volkov VV, Svergun DI (2006) ATSAS 2.1, a program package for small-angle scattering data analysis. J Appl Cryst 39: 277–286.
- 54. Konarev PV, Volkov VV, Sokolova AV, Koch MHJ, Svergun DI (2003) PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J Appl Cryst 36: 1277–1282.
- 55. Svergun D (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Cryst 25: 495–503.
- 56. MacKerell AD, Bashford D, Bellott, Dunbrack RL, Evanseck JD, et al. (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins†. J Phys Chem B 102: 3586–3616.
- 57. Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, et al. (2005) Scalable molecular dynamics with NAMD. J Comput Chem 26: 1781–1802.

domain: biology


text:
Obong Akan Udofia, APC governorship candidate, Akwa Ibom State.
By Abasifreke Effiong, Uyo.
A former member of the House of Assembly in Akwa Ibom state, Obong Kufreabasi Etuk, has said that the All Progressives Congress, will win the 2023 governorship election in the state with the pedigree of its candidate, Obong Akan Udofia.
Addressing supporters of the APC in Uyo at the 53rd birthday celebration of Udofia, Etuk said Akan Udofia is the governorship candidate of the APC in the State.
“Obong Akanimo Udofia is the candidate of the APC in Akwa Ibom State. People can say what they like, but you see the Akwa Ibom State government house, we are taking over in 2023. The problem that we have is that Akan Udofia is so popular such that the government in power is so much intimidated and the only thing they can do is to shout.”
“We have told our people not to join issues with persons and institutions who are arguing back and forth over the party’s nomination. We must not attack them, but we must show them that we are going to win them.”
“I can tell us that Akwa Ibom State will be governed by APC in 2023. I am telling members and supporters of the APC that this time around, we must work and win. We will not allow what happened in 2015 and 2019 to happen again. Don’t come to Uyo to say I will do this and that for Obong Akan Udofia, go and work in your unit. There is no structure or unit in Uyo, everyone must go to their units. We have made a decision, no elder will be allowed to go to Obong Akanimo Udofia’s house to declare that he is going to support him, if you want to support him, go to your village and unit, and declare it there. If all of us own our units, no one will rig us out.”
Friends of Obong Akan Udofia cutting his 53rd birthday cake, on Saturday 18th June, 2022.
Etuk, who is at the forefront of mobilising support for Akan Udofia and the APC in the State said Udofia will create wealth and unlock the numerous potentials of the State especially its mineral resources.
He said Udofia has shown sufficient commitment to creating wealth and empowering people in the state long before his entrance into the 2023 governorship race.
The former lawmaker said through Udofia’s magnanimity, he was able to employ 150 Akwa Ibom people in 2004 to work offshore.
“I have known Obong Akan Udofia for over 30 years. The prosperity message you are hearing today didn’t start today, that has been his character. I am introducing a man that can never change, he cannot change because he has seen all. That is why I can stand here to tell you that Obong Akanimo Udofia is the best man to follow.”
“When my contract with Shell finished, I went to him one day and told him to get a job for me. He made a phone call that got me an offshore job with a French company. Through that job, I employed over 150 people in Akwa Ibom State. With Obong Akan Udofia as Governor, Akwa Ibom state would be sure of large-scale employment opportunities beyond the civil service, our people will have better opportunities in the oil and gas sector. Some of our youths need opportunities to work elsewhere other than scrambling over themselves seeking to become councillors.”
“With our mineral resources, there are so many areas we can create wealth and one person who can unlock that sector, create wealth and lead Akwa Ibom into having access to the opportunities and potentials in solid mineral sector is Obong Akanimo Udofia.”
“I am here at the forefront of this project because I believe in Akan Udofia”.
Etuk said friends of Obong Akan Udofia decided to use the occasion of his birthday to support select start-ups and micro scale businesses in the state, saying that the gesture was to support the wealth creation and prosperity agenda of Obong Udofia.
18 micro-scale business owners were given grant, while 20 others received startup packs which included set of sewing machines and salon equipment.
Obong Kufreabasi Etuk, pictured with the starter packs donated to beneficiaries in celebration of Akan Udofia’s 53rd birthday.
Speaking on behalf of Obong Udofia, his younger brother and Managing Director/CEO of Desicon Engineering Limited, Mr. Ubong Udofia, described Akan Udofia as the best that can ever happen to Akwa Ibom State, owing to his peaceful disposition and desire to impact positively on people lives.
He thanked friends and supporters of Obong Akan Udofia for coming together to celebrate his brother’s 53rd birthday and using the occasion to give business support to people.
One of the beneficiaries, Mrs. Nkoyo Nyong Otoyo who hails from Udung Uko Local Government Area said the sewing machine she received came at a time she was struggling with the bad one in her shop.
She reasoned that if Etuk and others could empower people they had not met before as a mark of celebrating Udofia’s birthday, there were strong convictions that the government of Udofia would provide more empowerment programmes for the prosperity of the people across all strata of the society.
“People should support Akan Udofia. Imagine what he has done today. He has not become the governor yet, but he is doing all these. It means if he gets there, he will really bring the real prosperity that we want”, she said.


text:
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If there was any doubt about Houston’s prominence as an emerging technology hub, the Houston Technology Center shattered many suspicions May 10 at its 2012 gala, “A Celebration of Entrepreneurs.”
During the event, HTC recognized six of the most celebrated entrepreneurs in their fields, all of whom started successful companies in the Houston area. The honorees ranged from Chris Coker, the president and co-founder of Oxane Materials Inc., an energy-focused nanotechnology company, to Beth Williams, the CEO of TechTrans International, which provides international integration and support such as translations and language instruction.
One of the most moving speeches from the night's winners came from Dr. S. Ward Casscells, a professor of medicine and public health at University of Texas Health Science Center at Houston, president of Know Med Inc., a senior scholar at Texas Heart Institute and a member of numerous other esteemed organizations and companies. Casscells made it a point to accept his award at the gala, even though he is undergoing treatment at a local hospital.
During his speech, he said his award proves that “one does not have to be in San Diego or San Francisco or Austin,” to launch a successful startup company. Houston is just as accommodating to startups, he said, and he applauded HTC for “doing heroic work” by incubating these companies.
However, Casscells has participated in some pretty heroic work himself. In 2006, he volunteered for medical military service in Iraq and subsequently served the U.S. as the assistant secretary of defense for health affairs from 2007 to 2009.
Molly Ryan covers manufacturing, technology, the Port and logistics for the Houston Business Journal. For her breaking stories and industry insights, follow her on Twitter.
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Dresden Nuclear Power Station, Unit 2 issued the following announcement on Oct. 10.
The Nuclear Regulatory Commission is now distributing publicly available fuel cycle
facility correspondence originating from the agency’s headquarters in Rockville, Md., and the
Region II office in Atlanta through an electronic mailing Listserv system.
Correspondence related to operating reactors, decommissioning reactors and select waste
programs is already available through an electronic distribution process. The process distributes
public documents generated by the NRC staff to those registered to receive Listserv
communications. The Listserv does not provide notice of documents generated by external parties,
or NRC documents that contain proprietary, security-related, safeguards, or other information that is
withheld from public disclosure.
Individuals who would like to receive licensing and inspection correspondence for fuel cycle
“Email Updates” at the top of the page, next select “Fuel Cycle Facilities Correspondence” under
the heading “Lyris Subscription Services.” To complete the process, an email address should be
entered, the facility or facilities of interest should be selected and lastly, individuals must click the
“Subscribe” button to be added to the list.
Each subscriber will receive an email confirmation with instructions for managing the NRC
Listserv subscription and will be responsible for ensuring their email address is current.
Original source can be found here.


text:
(Blood Cleaner) E= Frequency equivalent
This Product is FREE of Alcohol, Fillers, Chemical, Synthetics & Preservatives
Supplied in spray bottle.
Our Genopathic® Remedies are formulated to use topically (External only). These take 5-10 seconds for the frequency to enter the bloodstream. They work very fast. Do not overdose. Keep away from children.
Our Genopathic® Remedies require only 1 spray a day.
These are rubbed into your wrist or the back of the hand.
The essence of active ingredients in MSM-E, if appropriately prescribed, may assist patients suffering from the following conditions: (this statement does not imply or make a claim for a cure for these disorders).
MSM-E = Sulphur Blood Cleaner,
This product may help with the following:
• The assimilation of essential amino acids
• Protein catalysts and enzymes
• Pain and inflammation reduction
• Back pain
• Leg pain
• Increasing mental awareness
• Improving concentration
• Mood elevation
• The effects of aging
• Insulin production
• Improving liver functions
• Enhancing antioxidant levels
• Metabolising carbohydrates
• Healing injuries
• Cellular detoxifying
• To promote gastrointestinal health
• To improve immune Function
Best used in combination with A.I.R-E, Lacto-E

domain: health


text:
URBANA – During the first three months of the pandemic, the number of U.S. adults dying increased compared to previous years, according to a recent study led by University of Illinois researchers involving data from the U.S. Centers for Disease Control and Prevention.
The statistically significant increase in deaths remained even after deaths attributed to COVID-19 were removed from the data. The trend holds for women, age 25 to 44, and men, age 15 to 54.
The study is published in the journal Public Health.
The findings suggest the pandemic is causing ancillary deaths, which are not directly caused by the coronavirus but are a consequence of the changes that have occurred as a result of the pandemic.
Internal medicine professor Janet Jokela and computer science professor Sheldon Jacobson conducted the research.
The study did not examine what is causing the uptick in deaths, but Jokela says she suspects it could be the result of patients delaying treatment for other conditions — like cancer or heart disease — or people not getting help for mental health issues or addiction.
“We’ve documented that this occurs,” she says. “I think it’s imperative that more research is done to try to understand why this is happening.”
One surprising finding: females between the ages of 5 and 14 saw a decrease in deaths. Jacobson says this can be explained by recognizing that accidents are one of the leading causes of death for that demographic — so the stay-at-home orders that took effect in the spring seems to have had a protective effect on young girls.
Jacobson says the findings suggest COVID-19 is not just a public health issue but a societal issue affecting all aspects of life, including the economy, education, manufacturing, services.
“And as a result of that, when you have such a large entity as our nation being shifted with an event like COVID-19, there are going to be unintended consequences,” he says. “And some of these are showing up in fatalities.”
Jacobson says we may never know the true explanation for these excess deaths.
“But as time goes on — and we collect more data and analyze it — we will hopefully be able to tease out some of these insights,” he says.
Jacobson and Sheldon say their findings are consistent with a recent CDC analysis, which determined nearly 200,000 more Americans died between late January through early October of 2020 compared to the same timeframe in 2019.
The CDC’s report finds the largest percentage increases were seen among adults aged 25 to 44 and among Hispanic or Latino people.


With two wins at home, New York is on the verge of advancing to the second round of the playoffs.
The New York Knicks won 102-93 in a game against the Cleveland Cavaliers in Game 4 of the 1st round of the 2022-2023 NBA playoffs held at Madison Square Garden in New York, USA on the 24th (hereinafter Korean time).
New York went 37-45 last season and failed to make the playoffs. And in the off-season, they recruited Jaylen Brunson (188cm, G), but were evaluated as ‘overpay’. However, once the season started, all these evaluations disappeared. New York with Brunson recorded 47 wins and 35 losses with a strong defense.
New York, which advanced to the playoffs, met the Cleveland Cavaliers. New York is ahead with a record of 3 wins and 1 loss in the regular season. And this momentum continued in the playoffs.
New York won a pleasant victory with Brunson, who scored 27 points from the first game. Although they lost the second game, it was encouraging to record 1 win and 1 loss in their away matches. And the third game was played at home in New York.
New York, who played the game at home, showed off their long-term skill, salty water defense. The opponent’s field goal success rate was tied at 38%. Only 79 goals were lost. In particular, Cleveland’s young players were overwhelmed by the atmosphere of New York’s Madison Square Garden.
The 4th game was played like that. This time, it was New York’s home. New York led from the first quarter. Although they allowed a turnaround midway through the third quarter, Brunson took the lead again by scoring 6.2 seconds before the end of the quarter. And in the 4th quarter, the players played evenly and won the game. In the match that day, New York showed a strong defense, tying the opponent to 93 points. 안전놀이터
After the game, New York coach Tim Thibodeau gave the approval of the game. “Our team has a secret weapon,” said coach Thibodeau. “The atmosphere at Madison Square Garden is incomparable to other stadiums. Opponents are overwhelmed by this atmosphere. This is why we are stronger at home,” he said, thanking the home fans.
In fact, New York’s Madison Square Garden fans have a passion comparable to any other stadium, and this atmosphere continues even in the playoffs. New York, which overpowered the atmosphere, won 2 at home and made the series 3-1. Now, if you add one more win, you will succeed in advancing to the second round of the playoffs in 10 years.


text:
Jack Attack comes to life in Alice Springs
The Memorial Bowls Club, Alice Springs has been the first club in the NT to take up the barefoot bowls game of “Jack Attack”. Jack Attack – “The New ^SOCIAL^ Way To Play Bowls. Short, Sharp, Snappy”. The Memorial Bowls Club, Alice Springs, Inc has been the first club in the NT to take up the new barefoot bowls initiate.
The Memorial Bowls Club had a very successful running barefoot bowls night league already in place under the careful guidance of Letitia Baldwin and Helen Siganto that had many players returning each season to socialise with mates, having a few drinks and playing some bowls. The level of competition and skill level of the players could be considered semi-serious, playing traditional fours. With Letitia and Helen stepping aside in 2015, to travel. Club President Ray Dienelt proposed that for the club to stay with the times and move forward the club needed to embrace the new Bowls Australia concept “Jack Attack”. The club committee voted in favour to take on the new initiate, while risky potentially losing our valued returning social Thursday night league bowlers, new leadership in Jack Attack Co-ordinator David Knight and Club President Ray Dienelt have pushed on. While the concept of moving to a more basic version and simplified rules of the game is very appealing to new bowlers, we have had some of our regular bowlers step back. Teaming up with Sourthern Cross TV’s Mez Elliot we developed an advertising campaign to get the word out. Facebook has been a great tool to reach a younger and community audience. We had articles about Jack Attack in the local newspaper. Ray has talked on Radio about the concept numerous times. Mez has also buffed the profile on radio, and has had great results so far. We are looking into TV advertising, to further reach members of the community.
Where we differ though, is we have sought community business houses to sponsor a team of 3 entry fee. The business may choose clients, employees or anyone to come and have a game. Each week we have more and more people telling us they never knew how much fun it was or how addictive it was to try and draw a shot. We have had a couple of false starts trying to get the competition off the ground, but with the hard work of Ray Dienelt and David Knight, the benefits are starting to come through. Players love the new short version, socialising while cheering on their teams. One player commented that they forgot the town they were in, and enjoyed walking up and down on the green barefoot totally relaxing.
Players enjoy a FREE sausage sizzle every game night, with prizes donated by CUB, like stubby holders, t-shirts, hats and towels etc. Our biggest problem is only having one green. As the competition continues to grow we can only accommodate 16 teams playing in 2 sections of 8 in a seven week round robin with a grand final of the two section winners playing off in the final. From humble beginnings, with music creating the atmosphere, and support from our club members. We are positive this is the way forward for people to enjoy lawn bowls in a social way, with many becoming hooked and looking forward to Thursday night bowls between 6.30 – 8pm at the Memorial Bowls Club, Alice Springs.
For more information on how to play Jack Attack at Alice Springs, Click Here.
For information on how to start your own Jack Attack Competition at your local bowls club, Click Here

domain: hobby


text:
GRAND FALLS-WINDSOR, NL – The Montreal Canadiens Alumni Tour was hoping to bring one of its 50 annual charity hockey games to Grand Falls-Windsor.
The group reached out to local organizations, as well as the town of Grand Falls-Windsor, to see if they wanted to take on this opportunity as a fundraiser.
The tour was left with a gap in its Newfoundland and Labrador schedule when one community backed out, leaving March 16 open.
“It’s a little bit late in the game to try and make it work,” said Brian Da Silva, sports coordinator, sales and events with Montreal Canadiens Alumni.
“From all the feedback that we got there wasn’t enough time or interest to get it going within a month. I don’t believe we will be able to do a game in Grand Falls-Windsor for this year.”
The Town of Grand Falls-Windsor initially declined the offer to host a charity hockey game because historically, these events have not gone over well in the town, said Deputy Mayor Mike Browne.
The last two occasions the town has had alumni hockey teams visit, the municipality lost money because of poor attendance.
The town was also not interested in paying the $20,000-price tag to host the event, plus hotel accommodations for the players.
“We did offer the organizer the building for free and offered them 100 per cent of the gate proceeds,” said Browne.
This offer came after the organization went back to the town a second time after being told Grand Falls-Windsor council was not interested.
The Montreal Canadiens Alumni Tour is a charity event whereby the organization paying them to come to town sets up a match with a local team, sets the ticket prices, sells the tickets and takes 100 per cent of the proceeds from the event.
Da Silva said typically tickets range in price from $15 to $25. With 973 seats in the Joe Byrne Arena and room for 400 standing, if tickets were sold at $25 a seat the organization hosting would stand to make a little over $34,000, before costs.
But as history has shown, these events are not ones to sell out.
Aside from the $20,000-price tag, the event was too short notice for the town, and it would have been a scramble to advertise it properly, said Browne.
The Newfoundland portion of the tour will bring alumni including Steve Shutt (1972-1985), Patrice Brisebois (1991-2009) and Stephane Richer (1984-2005) to Conception Bay South on March 15 and Fogo Island on March 17, towns that have already confirmed hosting the events.
The Montreal Canadiens Alumni Tour has reached out to other towns besides Grand Falls-Windsor to try to fill the gap in their schedule, including Bonavista –
home of Montreal Canadiens Alumni player Michael Ryder – St. Albans, Wesleyville, Gander and Corner Brook.
“It’s a little bit late in the game to try and make it work but we’re still working on it,” said De Silva.


text:
Contact Sam Chhoeun for more Information Samctcr@gmail.com (585) 381-2529 ext.114
Pickleball 101 [Beginner Level]
A Co-Ed group class for players new to the game. You will learn basic skills & strokes: underhand serve, return, forehand/backhand drive, dink shots, volleys, & overheads. We will also show you how to score & explain the rules of the game.
- Wednesdays 9:00-10:00am
- Thursdays 5:30-6:30pm
- Fridays 9:00-10:00am
Pickleball 201 [Intermediate Level]
A Co-Ed group class for players who know how to keep score and are looking to advance onto soft shot game. Players will learn proper techniques on different shots, consistency on ball rally, cross court attack dinks, important third shot from the baseline, and doubles strategies. You will develop hitting patterns and footwork movement on the No Volley Zone (Kitchen Area).
- Wednesdays 10:00-11:00am
- Thursdays 6:30-7:30pm
- Fridays 10:00-11:00am
TCR Pickleball 301 [Advance]
A Co-Ed group class for tournament level players. Players will develop 3rd shot strategies, tactical play, control points with spin. Players will learn to use drop shots, lobs, and fast-paced groundstrokes to win points.
- Wednesdays 5:30-6:30pm
Sam is available for one on one or private group lessons Monday through Saturday.
60 Min: $60 member
45 Min: $50 member
30 Min: $40 member
A $5 fee is applied to private lessons for non-members
Pickleball Lesson Information
MUST register in advance
If the 3-person minimum has not been met by 7pm the night before, the clinic will be canceled
24 hour cancellation – please notify us if you cannot make it to avoid being charged the full amount
For more information and to enroll in lessons, contact Sam Chhouen at (585) 381-2529 ext. 114 or by email.