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250-421 Design of DP Solutions for UNIX using NBU 5.0

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250-421 exam Dumps Source : Design of DP Solutions for UNIX using NBU 5.0

Test Code : 250-421
Test name : Design of DP Solutions for UNIX using NBU 5.0
Vendor name : Symantec
: 110 real Questions

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Symantec Design of DP Solutions

Cloud-primarily based ERP solutions are available for smaller businesses | killexams.com real Questions and Pass4sure dumps

No influence discovered, are trying original keyword!All of here is, of course, proper, however the advanced and infrequently-puzzling veracity of embracing specific technology options will besides exist overwhelming ... when you will gain investments within the planning and design of your ...

Cadence (CDNS) Beats on this tumble earnings & Revenues, publications strong | killexams.com real Questions and Pass4sure dumps

Cadence Design methods, Inc. CDNS delivered robust fourth-quarter 2018 results, wherein both the top and final analysis outpaced the respective Zacks Consensus Estimate. The figures besides occupy been larger than administration’s respective guided ranges.

Cadence delivered non-GAAP profits of fifty two cents per share for the fourth quarter, surpassing the Zacks Consensus evaluate by passage of 4 cents. management had anticipated profits between 46 cents and forty eight cents. The device surged 33.three% from the year-ago determine of 39 cents.

beneath ASC 606, the industry pronounced revenues of $569.8 million simply beating the Zacks Consensus evaluate of $551 million. additionally, the precise line turned into better than the extreme conclusion of management’s expectation of $545-$555 million. The device advanced 13.6% on a 12 months-over-year foundation.

powerful adoption of the business’s digital and signoff, customized and analog, IP options, and an ever increasing customer ground and start of hardware systems drove year-over-year increase.

Shares of Cadence are up 8.9% in the after-hours buying and selling. this can primarily exist attributed to wonderful fourth-quarter results and strong outlook. especially, Cadence stock has back 34.1% in previous twelve months, substantially outperforming the industry’s rally of 18.three%.

Quarter in aspect

under ASC 606, Product & upkeep revenues got here in at approximately $534.four million and accounted for basically 93.8% of total revenues. The determine became neatly ahead of Zacks Consensus evaluate of $515 million.

under ASC 606, services revenues of $35.4 million contributed 6.2% to complete revenues, lagging the Zacks Consensus evaluate of $36.5 million.

Geographically, Americas, Asia, Europe, hub East and Africa (EMEA) and Japan contributed forty four%, 31%, 17% and 8%, respectively to the total revenues under the brand original accounting ordinary.

The company suggested non-GAAP working margin of 31% during the quarter under evaluation.

Product-clever, practical Verification, Digital IC & signoff, customized IC design, methods Interconnect & evaluation and IP, comprised 25%, 28%, 25%, 9% and 13% of the overall revenues, respectively per ASC 606 ordinary.

IP segment witnessed strong quarter driven by using effective adoption of the company’s PCIe and DDR items.

The enterprise more advantageous Tensilica respond with DNA 100 Processor, a profound neural-community primarily based accelerator. The accelerator will enrich effectivity and efficiency for rising applications in drones, Intenet of things (IoT), automobile sensor fusion, surveillance, amongst others.

all over the stated quarter, the industry additionally extended alliance with Samsung by passage of robust adoption of its digital, custom and verification items. It besides multiplied lengthy-term partnership with Analog instruments for the building of mixed signal solutions for IoT, automobile, clinical and industrial functions, including the adoption of several of recent digital and verification items.

Traction witnessed by passage of Xcelium Parallel Simulator and Palladium Z1 drove revenues in system Design and Verification solutions. Traction witnessed through Palladium Z1 on the returned of sturdy demand for transforming into hardware capability was notable.

management is besides elated on the order power in Palladium Cloud respond which presents cloud-primarily based emulation potential as per the shoppers’ demand.

in the Digital and Signoff house, the industry introduced its newest DDR5 verify chip. Cadence taped-out greater than eighty 7-nanometer (nm) designs within the quarter by leveraging Innovus. administration mentioned that around 50 clients occupy chosen Innovus.

Story continues

The industry is still confident about its ongoing collaboration with Taiwan Semiconductor Manufacturing company. notably, Cadence garnered 4 companion of the yr awards at TSM’s Open Innovation Platform, comprising 5 nm design architecture collaboration.

moreover, Cadence collaborated with Microsoft’s Azure, Amazon’s Amazon net functions (“AWS”) and Google Cloud platform to enable antiseptic design development of electronic techniques and semiconductors. administration is elated with the sturdy pipeline of the enterprise’s ingenious cloud-able options.

With Cadence Cloud, the enterprise aims to present a complete cloud portfolio enabling the building of semiconductors and different digital methods.

balance Sheet & money movement

The enterprise ended the said quarter with money and money equivalents of approximately $533.three million in comparison with the outdated quarter’s device of $550 million.  Cadence’s lengthy-time age debt as on Dec 29, 2018, changed into $345.three million in comparison with $345.1 million, as on Sep 29, 2018.

The company generated operating cash circulation of well-nigh $132 million within the quarter in comparison with previous quarter’s stated device of $110 million.

The industry repurchased shares value about $100 million in the fourth quarter.

assistance

For first-quarter 2019, Cadence expects total revenues under ASC 606 in the latitude of $565-$575 million and non-GAAP salary in the attain of forty eight-50 cents per share.

The Zacks Consensus Estimates for revenues and salary are pegged at $547.5 million and 46 cents, respectively.

The enterprise offered 2019 outlook. Revenues are now projected within the attain of $2.270-$2.310 billion. Non-GAAP income are now guided within the latitude of $1.97-$2.07 per share.

The Zacks Consensus evaluate for revenues and earnings are pegged at $2.24 billion and $1.95 per share, respectively.

extra, non-GAAP operating margin for 2019 is anticipated at 30-31%. operating cash movement is expected in the latitude of $640-$690 million.

Zacks Rank and stocks to accept as true with

Cadence presently consists of a Zacks Rank #3 (hold).

Some better-ranked shares within the broader know-how sector are Symantec service provider SYMC, salesforce.com, inc. CRM and Fortinet, Inc. FTNT, each and every wearing a Zacks Rank #1 (amazing buy). that you would exist able to see the complete checklist of nowadays’s Zacks #1 Rank stocks right here.

Symantec, salesforce and Fortinet occupy a protracted-term income boom charge of seven.9%, 24.2% and 16.8%, respectively.

Zacks' ample 10 stocks for 2019

in addition to the stocks mentioned above, would you enjoy to know about their 10 ultimate purchase-and-holds for the yr?

Who would not? Their annual top 10s occupy beaten the market with surprising regularity. In 2018, while the market dropped -5.2%, the portfolio scored neatly into double-digits generic with individual shares rising as extreme as +61.5%. And from 2012-2017, whereas the market boomed +126.three, Zacks' ample 10s reached an even greater sensational +181.9%.

See latest shares nowadays >>

need the newest thoughts from Zacks investment analysis? today, which you could download 7 gold standard stocks for the next 30 Days. click to obtain this free record Fortinet, Inc. (FTNT) : Free inventory evaluation record Cadence Design techniques, Inc. (CDNS) : Free inventory analysis report salesforce.com, inc. (CRM) : Free inventory analysis report Symantec organisation (SYMC) : Free stock evaluation record To read this text on Zacks.com click right here. Zacks investment analysis


Workhuman adding a hundred and fifty original Jobs At Dublin, eire HQ | killexams.com real Questions and Pass4sure dumps

Workhuman®, previously Globoforce, has announced an immense growth of its operations with the introduction of one hundred fifty original jobs and the opening of its newly expanded $four.5 million headquarters in Dublin. The Irish expertise company additionally announced that it has rebranded as Workhuman. The transformation acknowledges each the effectiveness and traction of its Workhuman® Cloud platform and demand from progressive international organizations seeking to cheer and empower their americans.

Dublin(supply: Workhuman)

“Our major expansion of operations demonstrates the striking demand for their Workhuman Cloud platform and underlines their commitment to eire into the long-term,” eminent Dublin endemic Eric Mosley, co-founder and CEO of Workhuman. “We created the market category of sociable focus to back world organizations align their americans and lifestyle to a shared intention. Now, twenty years on from their founding, we're assisting shape the future of travail with the emerging human applications class. Making travail greater human has at complete times been their mission and now we’re delighted to promulgate the herbal evolution of Workhuman as the identify of their business.”

Headquartered in Dublin and Massachusetts, Workhuman is the world’s quickest transforming into integrated sociable attention® and continuous efficiency management platform. The industry has helped some of the world’s biggest agencies expend gratitude to combine their people and tradition to a shared goal, including LinkedIn, Symantec Corp., Cisco, Eaton, Baker Hughes, Intuit, and The Hershey business. these days, four million people around the world are on the platform throughout a hundred and sixty international locations.

the brand original 150 extremely-skilled roles to exist filled over the subsequent three years may exist within the fields of technology, finance, HR, product, eCommerce, and operations. The Dublin office is core to Workhuman’s product construction with its Irish-primarily based technology groups using and possessing the design and building of the cloud application creation. Workhuman has invested more than $60 million in research and construction over the ultimate five years to carry original innovations to its award‐profitable expertise platform. The company currently employs greater than 500 americans globally, with 270 working in Dublin. Workhuman has additionally been named a superb vicinity to travail in eire for six consecutive years.

Dublin(supply: Workhuman)

With its $four.5 million funding, Workhuman has taken over a complete screen at Park West company Park in Dublin 12. The newly elevated headquarters points interactive digital partitions, flexi desks with the option to stand while working, collaboration areas, practising, wellbeing and relaxation rooms for onsite activity courses, in addition to a excellent flooring innovation hub with panoramic metropolis views. the original house is a physical manifestation of the train to create an environment the residence people can execute their most excellent work. participating with leading workplace designers, the workplace is designed to fulfill many distinctive travail styles and americans wants.

“Workhuman’s announcement of one hundred fifty original jobs is a pretty ample example of a extremely creative Irish company this is making wonderful strides globally while investing in its Irish headquarters for the lengthy-time period,” eminent Minister for enterprise, enterprise and Innovation, Heather Humphreys TD. “The investment by using the company in R&D and product building at its Park West ground is an extra boost to eire’s booming indigenous tech sector, and i am delighted that the govt can pilot its operations via commercial enterprise eire.”

“we now occupy been working with Eric and the team in Workhuman from the starting,” talked about Julie Sinnamon, CEO, enterprise ireland. “Workhuman is likely one of the world’s fastest-transforming into efficiency management structures for industry and is the ultimate illustration of an Irish technology industry with international ambition. Their purpose is to aid greater Irish organizations enjoy Workhuman to compete and win sustained company in far places markets. enterprise eire looks forward to working with the group to continue to aid their growth ambitions as they expand their attain even additional in international markets, strengthening their consumer ground internationally and continuing to create jobs right here in ireland.”


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Design of DP Solutions for UNIX using NBU 5.0

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NMR structure of the C-terminal domain of TonB protein from Pseudomonas aeruginosa | killexams.com real questions and Pass4sure dumps

Introduction

The periplasmic space resides between the outer membrane (OM) and cytoplasmic membrane (CM) of Gram-negative bacteria. The OM protects Gram-negative bacteria from environmental hazards such as antibiotics and detergents. At the very time, Gram-negative bacteria require rare essential nutrients such as iron, vitamins that are present in the extracellular environment at very low concentrations. Gram-negative bacteria occupy evolved active acquisition systems to pass the essential nutrients through OM and CM. Since there is no electrochemical gradient to power the active transport at the OM and no ATP in the periplasmic space, these transporters must extract energy from the CM. These transporters in the OM are termed TonB-dependent transporters (TBDT) because they presumably extract the energy from the proton-motive force (pmf) of the CM via the trans-periplasmic protein, TonB protein (Fig. 1A). The energy transduction is assumed to grasp residence via the TonB involved anchored in CM and consisting of TonB, ExbB, and ExbD proteins. ExbB and ExbD are accessory proteins anchored in the CM that convey the pmf across the CM to TonB (Celia et al., 2016; Clarke, Tari & Vogel, 2001; Krewulak & Vogel, 2008; Postle & Larsen, 2007).

Figure 1: TonB-dependent energy transduction system and structures of differently dissected E. coli TonB. (A) A schematic model of the TonB-dependent energy transduction system, which transduces the proton motive force of the CM to TonB-dependent transporters (TBDT) via TonB. (B) Sequence alignment presenting the full-length E. coli TonB sequence and differently dissected CTDs used for the structure determination (labeled with PDB IDs). CTDs are highlighted in gray, and the TM region is indicated by a bar. (C) Ribbon drawings of previously reported E. coli TonB CTD structures. The ultimate β-strands at the C-terminal region with the largest conformational differences among the reported structures are colored in red. 1IHR forms intertwined dimers (Chang et al., 2001) and 1U07 forms dimers connected via the C-terminal extended strand (Ködding et al., 2005) in crystals.

TonB protein mediates the energy transduction from the CM to TBDTs. TonB has an N-terminal transmembrane (TM) domain anchored in the CM (Fig. 1). The TM domain is followed by the central region mostly consisting of Pro-Glu and Pro-Lys repeats (Fig. 1B). Extended conformation of the central region allows the protein to span the periplasmic space between OM and CM (Domingo Köhler et al., 2010). The C-terminal domain (CTD) of TonB protein has a globular structure and interacts with a conserved TonB box motif located at the N-terminus of TBDTs (Cadieux & Kadner, 1999; Pawelek et al., 2006; Peacock et al., 2005; Shultis et al., 2006). Despite a wide variety of models for the energy transduction mechanism by TonB, it is still unclear how TonB protein works in the energy transduction that presumably causes the structural changes of the plug domain in TBDTs to facilitate the transport (Celia et al., 2016; Chimento, Kadner & Wiener, 2005; Gresock et al., 2011; Klebba, 2016; Letain & Postle, 1997; Sverzhinsky et al., 2015; White et al., 2017). The crystal structures of the complexes with two different outer membrane receptors from Escherichia coli BtuB and FhuA suggests direct interactions between β-sheets in the CTD of TonB protein and the TonB box (Shultis et al., 2006; Pawelek et al., 2006). Currently proposed models to induce the structural changes of the plug domain comprise the mechanical pulling of the plug domain via the interaction with TonB box and CTDs of TonB, which are supported by the atomic force microscopy and molecular dynamics (MD) simulation studies suggesting that the interaction is strong enough to remain stable during the mechanical unfolding of the plug domain (Chimento, Kadner & Wiener, 2005; Gumbart, Wiener & Tajkhorshid, 2007; Hickman et al., 2017). Furthermore, the interaction between a highly conserved positive freight at Arg166 of EcTonB and the negative freight of Glu56 of FhuA receptor is proposed to exist involved in the disruption of the plug domain (Pawelek et al., 2006).

Interestingly, several groups occupy reported divorce conformations of the differently dissected CTD of TonB protein from E. coli solved by X-ray crystallography or NMR spectroscopy (Fig. 1C). The crystal structures of E. coli TonB-CTD consisting of the ultimate 85 or 77 residues were composed of an intertwined dimer conformation with three β-strands and one α-helix (PDB codes: 1IHR and 1QXX, respectively) (Chang et al., 2001; Koedding et al., 2004). Whereas a longer construct consisting of the ultimate 92 residues (EcTonB-92) was monomeric in solution, the crystal structure revealed dimerization via β-strands in the C-terminus (PDB: 1U07) (Ködding et al., 2005). Furthermore, TonB protein from E. coli (EcTonB-137, PDB code: 1XX3) was monomeric as observed in solution NMR experiments (Peacock et al., 2005). CTDs of EcTonB bound to outer membrane receptors BtuB and FhuA grasp a monomeric conformation (Shultis et al., 2006; Pawelek et al., 2006). Therefore, the biological relevance of the intertwined dimers create in the crystal structures of the shorter fragments of CTDs manner the ultimate 85 or 77 residues has not been pellucid (Ködding et al., 2005; Koedding et al., 2004; Postle et al., 2010). In contrast, Electron Spin Resonance (EPR) experiments suggested that a significant population of TonB exists as a dimer in solution while the monomeric figure is bound to a TBDT (Freed et al., 2013). The major differences among the monomeric structures of EcTonB are observed around the residues 235–239 (highlighted in red) at the C-terminal region (Fig. 1C). Whereas these residues in the NMR structure of EcTonB-137 fold to an additional β-strand (β6) and forming an anti-parallel sheet with the preceding β5-strand, the very C-terminal End is either extended or largely invisible in the crystal structures (Fig. 1C). Intriguingly, β5-strand (residues 226–231) in EcTonB-CTD directly interacts with TonB box of TBDTs in the crystal structures of EcTonB-CTD/TBDT complexes (Pawelek et al., 2006; Shultis et al., 2006). The β5-strand is not accessible for the proposed interaction in the NMR structures of EcTonB-137 (Peacock et al., 2005) due to the additional β6-strand. Therefore, β6-strand in EcTonB-137 NMR structure has to exist exchanged with the strand in TonB box when interacting with TBDTs (Peacock et al., 2005). In contrast, the recent solution NMR structure of the ultimate 92 residues from Helicobacter pyroli TonB (HpTonB-92) showed the disordered C-terminal region and the absence of β6-strand (Ciragan et al., 2016), which is more in line with the crystal structures of EcTonB-CTD reported previously (Ködding et al., 2005; Shultis et al., 2006;Pawelek et al., 2006).

Here, they report the NMR structure and MD simulations of a CTD of TonB protein from Pseudomonas aeruginosa (PaTonB-96) to investigate whether the plasticity of the C-terminal region observed for the reported structures of EcTonB is a common feature of TonBs across different organisms.

Materials and Methods NMR sample preparation

The C-terminal 96 residues of a TonB protein from P. aeruginosa (UniProt: Q51368) was cloned from genomic DNA (ATCC-47085) as an N-terminal SUMO fusion protein, which was previously termed PsTonB-96 (Guerrero, Ciragan & Iwaï, 2015). The construct expresses a His-tag SUMO fusion protein that produces a 99-residue protein after the SUMO tag removal, which they termed PaTonB-96. PaTonB-96 contains the ultimate 96 residues of PaTonB (247–342) and three residues (SHM) at the N-terminal End from the cloning site. The plasmid (pFGRSF15) coding the gene of PaTonB-96 was transformed into E. coli ER2566 strain (New England Biolabs, Ipswich, MA, USA) for production of doubly 15N, 13C-labeled samples. The transformed E. coli cells were grown overnight at 30 °C in 50 mL LB medium supplemented with 25 μg/mL kanamycin. The cells were spun down at 900 × g for 15 min and gently re-suspended in two L pre-warmed M9 medium supplemented with 25 μg/mL kanamycin, containing 15NH4Cl and 13C6-D-glucose as sole nitrogen and carbon sources, respectively. The cells were grown at 37 °C until an OD600 of 0.6. Then, the temperature was lowered to 30 °C for protein expression. The protein expression was induced with a final concentration of one mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The cells were incubated for additional 5 h before harvesting by centrifugation at 4,900 × g at 4 °C for 15 min. The labeled PaTonB-96 was purified following the previously published protocol (Guerrero, Ciragan & Iwaï, 2015). The purified protein was dialyzed against 20 mM sodium phosphate buffer (pH 6.0). The protein solution of PaTonB-96 was concentrated to one mM for NMR analysis using a centrifugal device. The final sample volume was 250 μL containing 10% D2O.

NMR measurements

NMR measurements for the structure determination were recorded on Varian INOVA 800 MHz equipped with a cryogenically cooled five mm probe head. For the sequential backbone assignment, a standard set of double and triple resonance NMR spectra were recorded at 25 °C, including [1H, 15N]-HSQC, HNCO, HNCA, HNCACB, HN(CO)CA, HN(CA)CO, and CBCA(CO)NH (Sattler, Schleucher & Griesinger, 1999). The aliphatic side-chain assignment was carried out using [1H, 13C]-HSQC, HCCH-COSY, ct-[1H, 13C]-HSQC, HBHA(CO)NH, H(CCCO)NH, (H)CC(CO)NH, 15N-resolved [1H, 1H]-TOCSY, and 15N-edited [1H-1H]-NOESY spectra. The assignments for the aromatic side-chains were based on the spectra of (HB)CB(CGCD)HD, (HB)CB(CGCDCE)HE, aromatic region ct-[1H, 13C]-HSQC and 13C-edited [1H,1H]-NOESY. Data was acquired using VnmrJ (Varian Inc., Palo Alto, CA, USA) and data were processed using the NMRpipe software (Delaglio et al., 1995).

NMR measurements for the backbone dynamics were recorded on a Bruker Avance 850 MHz equipped with a cryogenically cooled probe head. The longitudinal (T1) and transverse (T2) relaxation rates and 15N{1H}-heteronuclear nuclear Overhauser effects (NOEs) for backbone 15N atoms were determined at 25 °C using the well-established NMR experiments (Barbato et al., 1992; Kay, Torchia & Bax, 1989). T1(15N) and T2(15N) relaxation times were determined using the following retard times: 10, 50, 100, 200, 300, 500, 800, 1,000, 1,200, and 2,000 ms for T1 and 16, 64, 96, 128, 156, 196, 224, and 256 ms for CMPG pulse train with one ms interval for T2 relaxation rates, respectively. Relaxation times were obtained by fitting a single exponential decay to peak intensity values: I(t) = I0 × exp (–t/T1) or I0 × exp (−t/T2), where I(t) is the peak volume at a time t. Heteronuclear 15N{1H}-NOEs were obtained with a relaxation retard of 5 s with or without saturation of protons. Heteronuclear 15N{1H}-NOEs (η) were determined from the volumes of the HSQC signals using the ratio of η = I/I0. The relaxation data were processed and analyzed using Bruker Dynamic hub (Version 2.1.8; Bruker Inc., Billerica, MA, USA).

Solution NMR structure determination

The sequence-specific resonance assignment was performed with standard methods using triple resonance NMR experiments and CcpNmr Analysis software (version 2.4.1) (Vranken et al., 2005). The chemical shift values from the sequential resonance assignment were used together with NOE peak lists for the structure calculation with the program CYANA 3.0 (Mumenthaler et al., 1997; Güntert & Buchner, 2015). NOE distance restraints were obtained from 3D 15N- and 13C-edited [1H, 1H]-NOESY spectra with 80-ms mixing time. The conformations of prolines were checked based on CB-CG chemical shift before the structure calculation (Schubert et al., 2002). complete prolines were predicted to exist in trans conformation except P299 that was set to cis-conformation. The three-dimensional NMR conformers were generated using CYANA 3.0, based on the automated NOESY cross peaks assignment (Güntert, 2004; Güntert, Mumenthaler & Wüthrich, 1997). The restrained energy minimization of the final 20 best conformers was performed using AMBER 14 (Pearlman et al., 1995), and the structures were validated with PSVS 1.5 (Bhattacharya, Tejero & Montelione, 2007). The structural statistics are summarized in Table 1.

Table 1:

Structural statistics of the energy-minimized NMR structure of PaTonB-96.

PaTonB-96a Completeness of resonance assignments (%)b Backbone 98.5 Side chain, aliphatic 97.0 Side chain, aromatic 88.0 Distance restraints Total 1,698 Intraresidue (i = j) 465 Sequential (|i−j| = 1) 483 Medium attain (1 < |i−j| < 5) 198 Long attain (|i−j| ≥ 5) 552 No. of restraints per residue 17.2 No. of long-range restraints per residue 5.6 Residual restraint violations Average no. of distance violation per structure 0.1–0.2 Å 1 >0.2 Å 0 (max. 0.14) Average no. of dihedral angle violations per structure >2.5° 0 Model qualityc Rmsd backbone atoms (Å) 1.0 Rmsd cumbersome atoms (Å) 1.6 Rmsd bond lengths (Å) 0.014 Rmsd bond angles (°) 2.1 MolProbity Ramachandran statisticsc Most favored regions (%) 97.4 Allowed regions (%) 2.5 Disallowed regions (%) 0.1 Global attribute scores (raw/Z score)c Verify3D 0.34/−1.93 ProsaII 0.41/−0.99 PROCHECK (ϕ–ψ) −0.27/−0.75 PROCHECK (all) −0.23/−1.36 MolProbity clash score 0.18/1.49 Model contents Ordered residue ranges 247–258, 261–320, 323–338 Total no. of residues 99 BMRB accession number 34235 PDB ID code 6FIP MD simulation

The MD simulations were performed with Gromacs5 (Abraham et al., 2015) by using Amber ff99SB-ILDN force province and tip4p water model (Jorgensen et al., 1983; Lindorff-Larsen et al., 2010). The first 10 ns from total 400-ns simulation was considered to exist the equilibrium age by monitoring the protein root-mean-square-deviation, inertia tensor eigenvalues, and rotation angles. The leisure was used for the analysis since the first 10 ns of simulation trajectories was sufficiently enough to remove the significant fluctuations in these parameters (Ollila, Heikkinen & Iwaï, 2018). The NMR structure determined in this travail was used as the initial structure and the secondary structures remained unchanged during the entire simulation period. The temperature was coupled to 25 °C with v-rescale thermostat (Bussi, Donadio & Parrinello, 2007), and the pressure was isotropically set to one bar using Parrinello-Rahman barostat (Parrinello & Rahman, 1981). The time-step was two fs, Lennard-Jones interactions were cut-off at one nm, PME (Darden, York & Pedersen, 1993; Essmann et al., 1995) was used for electrostatics, and LINCS (Hess, 2008) was used to constrain complete bond lengths. The simulation data is available from the Zenodo repository (Ollila, 2018a).

The analysis and interpretation of 15N spin relaxation times are described in detail elsewhere (Ollila, Heikkinen & Iwaï, 2018). Briefly, rotational correlation functions of the backbone N–H bonds were calculated from the simulation data and the spin relaxation times were calculated using the Redfield equations (Abragam, 1961; Kay, Torchia & Bax, 1989; Ollila, Heikkinen & Iwaï, 2018). Before the spin relaxation time calculation, the overestimated overall rotational diffusion in the MD simulation due to the water model was corrected by dividing the rotational diffusion coefficients around complete inertia axes with a factor of 1.2, assuming that the protein rotates as an anisotropic rigid body. This scaling factor was create to exist capable of reproducing the 15N spin relaxation times in ample agreement with the experimental data of PaTonB-96 for tip4p water model (Ollila, Heikkinen & Iwaï, 2018). The computer codes used for the analysis and the related data are available (Ollila, 2018b, 2018c).

Results NMR solution structure of PaTonB-96

The protein consisting of the C-terminal 96 residues of TonB from P. aeruginosa (PaTonB-96) was previously identified as a minimal domain that was soluble when expressed using an E. coli expression system, while other shorter fragments were not soluble even with several different fusion partners (Guerrero, Ciragan & Iwaï, 2015). This fragment has 45% identity (40/88 residues) to EcTonB-92, suggesting a similar structure to EcTonB. [1H, 15N]-HSQC spectrum of PaTonB-96 shows the well-dispersed NMR signals for amide groups (Fig. 2), indicating a globular folded conformation. complete of the main-chain chemical shifts were assigned except for S244 and HN of H245. 95.2% of the expected side-chains were assigned. The assigned chemical shifts were used for the automatic analysis of NOE peaks with the CYANA to cipher the NMR structure. device 3 shows the lowest energy solution NMR structure of PaTonB-96 with the secondary structure elements and a superposition of the 20 lowest energy conformers. The structural statistics of the 20 NMR conformers are summarized in Table 1. PaTonB-96 adopts a mixed α/β structure with the topology of βαββαββ. The structure consists of a β-sheet composed of three anti-parallel β-strands (β2, β3, and β5), a β-sheet composed of two short β-strands (β1 and β4) and two short α-helices (αI and αII). The structural coordinates and the chemical shifts occupy been deposited in the Protein Data Bank (http://www.rcsb.org/, PDB code: 6FIP) and BMRB (http://www.bmrb.wisc.edu/, accession number: 34235).

Figure 2: Two-dimensional [1H, 15N]-HSQC spectrum of one mM PaTonB-96. The spectrum was recorded at the 1H frequency of 800 MHz at 25 °C. The residue number and single letter code for amino acid types indicate the assignments. Trp HNe, Gln and Asn side chain amide resonances are marked by “sc.” Figure 3: NMR structures of PaTonB-96. (A) Ribbon drawings of the lowest energy conformer of the PaTonB-96 structures showing the secondary structure elements. (B) Stereoview of an ensemble of the 20 lowest energy NMR conformers. Red and blue color are used for α-helices and β-sheets, respectively. N and C indicate N- or C-termini, respectively. Figures are generated with PyMol (Schrodinger, 2015). Comparison of the NMR structures between E. coli and P. aeruginosa

The primary and secondary structures of PaTonB-96 were compared with the structure of EcTonB-137 (PDB: 1XX3) (Peacock et al., 2005) (Fig. 4). The length of PaTonB-96 is similar to that of the globular portion of EcTonB-137 (88 residues). Notable structural differences are observed around the C-terminal end. The C-terminal End of EcTonB-137 forms an anti-parallel β-strand (β6) with β5-strand to constitute a β sheet. In contrast, the C-terminal End in PaTonB-96 is unstructured and exhibits an extended resilient conformation as besides seen from the spin relaxation data (Fig. 5). The contrast can exist easily explained by the shorter C-terminal End after the β5-sheet in PaTonB-96, being too short to figure an additional β-strand. The shorter C-terminal End in PaTonB-96 is compensated by the longer loop between β4 and β5-strands with additional five residues, compared with the structured 88-residue region of EcTonB-137. Higher mobility of this longer loop is besides confirmed by the 15N relaxation measurement and MD simulation (Fig. 5). Interestingly, the extended and disordered conformation of the C-terminal End in PaTonB-96 more closely resembles the crystal structures of EcTonB-92 (PDB: 1U07) or the structures from TonB/TBDT complexes (PDB: 2GRX and 2GSK) (Fig. 1). The interaction between β5-strand of TonB and the TonB box in TBDTs has been proposed to exist essential for the TonB-mediated energy transfer (Pawelek et al., 2006; Shultis et al., 2006). Such interaction would exist hindered by the additional β6-stand create in EcTonB-137. Thus, their results hint that β5-strand is more accessible for the proposed interactions with TonB box in PaTonB-96 where the β6-strand is absent.

Figure 4: Comparison between PaTonB-96 and EcTonB-137. (A) The sequence comparison between PaTonB-96 and the ultimate 90 residues of EcTonB-137 with the secondary structure elements. (B) Stereoview of the superposition of the 20 NMR structures of PaTonB-96 (gray) and residues 151–239 of the 20 NMR structures of EcTonB-137 (PDB: 1XX3, blue). N and C stand for the N- and C-termini, respectively. Figure 5: MD simulation of PaTonB-96 and comparison with the experimental data. (A) The comparison of the experimental and simulated 15N relaxation parameters and the internal motions presented by S2 and te obtained from the MD simulation. The regions for β-sheets, αI, and αII are highlighted in blue, purple, and orange, respectively. (B) A superposition of snapshots of the structure from the MD simulation trajectory indicating conformational fluctuations during the MD simulation. The residues with enhanced flexibilities are colored in yellow. The aI-helix with orientation fluctuations is colored in purple. Rotational diffusion coefficient values obtained from the MD simulation of the experimental spin relaxation data are, Dxx = 1.51 ± 0.01, Dyy = 1.72 ± 0.03, and Dyy = 3.79 ± 0.03 (rad2·107/s). These result in Dav = (Dxx + Dyy + Dzz)/3 = 2.3 ± 0.02 (rad2·107/s), τc = (6Dav)–1 = 7.2 ± 0.02 (ns), A = 2Dzz−(Dyy + Dxx) = 4.4 and R = Dyy−Dxx = 0.2, where A is the axiality and R is the rhombicity. The overlay of the trajectories was produced by vmd (Humphrey, Dalke & Schulten, 1996). Structural motions in PaTonB-96

Fast dynamics (picosecond to nanosecond) of proteins has been commonly investigated by measuring the spin relaxation times (T1, T2, and heteronuclear NOEs) of backbone 15N atoms (Jarymowycz & Stone, 2006; Van Den Bedem & Fraser, 2015). Here, they characterize the structural dynamics of PaTonB-96 by combining T1, T2, and heteronuclear NOEs for backbone 15N atoms and MD simulations (Ollila, Heikkinen & Iwaï, 2018). The measured spin relaxation times for PaTonB-96 are shown together with the MD simulation results (Fig. 5A). The ample agreement with the experimental data allows us to expend the MD simulation trajectory to interpret the timescales for the overall rotational diffusion (τc), effective correlation times for internal mobility (τeff), and order parameters (S2). The overall rotational diffusion coefficients around inertia axes (see the caption of Fig. 5) demonstrate that PaTonB-96 has high axiality (A = 4.4), but with a rather low rhombicity (R = 0.2) suggesting that the protein has an approximately ellipsoidal shape. The MD simulation analysis estimates τc = 7.2 ns for the timescale of unbiased overall rotational diffusion. This is in line with 6.9 ns estimated from the T1/T2 values using the model-free approach (Kay, Torchia & Bax, 1989; Ollila, Heikkinen & Iwaï, 2018) as well as with the values in the literature for monomeric proteins with the similar molecular weight (Krishnan & Cosman, 1998). They thus conclude that PaTonB-96 is monomeric in solution as was the previously published NMR structure for EcTonB-137 (Peacock et al., 2005).

The results divulge that there are four regions with enhanced internal motions in PaTonB-96 (Fig. 5). The regions with notable conformational fluctuations are colored in yellow and purple in the overlaid snapshots from the MD simulation (Fig. 5). The first few N-terminal residues exhibit low order parameters and long effective correlations times related to the enhanced conformational fluctuations, suggesting that the N-terminal region is already the soar of the resilient central region of the TonB protein that connects between TM region and CTD. The MD simulation revealed orientational fluctuations in αI helix (purple in Fig. 5B). This fluctuation besides influenced the order parameters and effective correlation times of the neighboring residues in the MD simulation (Fig. 5A). However, the changes were not observed in the 15N spin relaxation analysis, presumably because the 15N relaxation is not sensitive to the time scale of the fluctuation. Residues 320–326 in the loop between β4 and β5-strands indicate some enhanced conformational fluctuations, which were besides detectable by the 15N spin relaxation analysis and order parameters but does not affect the effective correlation times (Fig. 5A). The ultimate five residues of the C-terminal End (residues 338–342) showed the enhanced conformational fluctuations, characterized with lower order parameters, long effective correlation times, and changes in the 15N spin relaxation rates, which are consistent with the experimental 15N spin relaxation data. The resilient residues at the C-terminal End are in immediate contact with the αI helix, indicating orientational fluctuations as described above.

Modelling of PaTonB and TonB box interactions

It is believed that TonB conveys the CM chemical potential to TBDTs by structural changes via the interaction between CTD of TonB and TonB box, a short stretch of peptide chain in the N-terminus of plug domain of TBDTs (Cadieux & Kadner, 1999; Pawelek et al., 2006; Peacock et al., 2005; Shultis et al., 2006) (Fig. 1A). In the crystal structure of EcTonB-93/BtuB complex, EcTonB orients the αI helix against the plug domain and forms a parallel β-sheet interaction with the TonB box of BtuB (Shultis et al., 2006) (Fig. 6A). They created a hypothetical model of a involved between PaTonB-96 and TBDT by superimposing PaTonB-96 to residues 153–233 of EcTonB in the crystal structure of the involved because no structure of TBDTs from P. aeruginosa is available (Fig. 6B). β5-strand (residues 226–231) of EcTonB-93 and TonB box of BtuB forms the parallel β-strands, of which interactions are mostly hydrophobic (Fig. 6C). The modeled structure of PaTonB-96/BtuB involved indeed resembles the conserved TonB box interactions observed with the EcTonB/BtuB complex, supporting that the modeled structure is plausible. The highly conserved Val10 in the TonB box is located next to phenylalanine in both TonB structures (Phe230 and Phe336 in EcTonB-93 and PaTonB-96, respectively), suggesting that this hydrophobic interaction might exist captious for the TonB/TBDT complexes (Fig. 6C).

Figure 6: Models for interactions between TonB and TBDTs. (A) The crystal structure of EcTonB-93 (cyan) bound to BtuB transporter (green, PDB: 2GSK). (B) A cartoon model of the involved of E. coli BtuB transporter and PaTonB-96 (gray) created by the superposition of CTDs of the TonB proteins. (C) Illustrations depicting the interactions between β5-strand of EcTonB-93 (cyan) or PaTonB-96 (gray) with the TonB box of BtuB transporter from E. coli (green). The consensus TonB box sequences based on 57 sequences from UniProtKB/Swiss-Prot (PROSITE: PS00430) is shown at the top. (D) Two hypothetical interaction models for PaTonB/TonB box from probable P. aeruginosa BtuB (Uniprot: Q9I473). White circles and dotted black circles indicate exposed and buried sides of the model, respectively. Discussion

We are interested whether the structural features create in TonB and TonB/TBDTs complexes from E. coli are shared among other Gram-negative organisms. In the modeled structure of PaTonB-96/EcBtuB complex, the positive freight at the soar of β5-strand (Lys332 in PaTonB-96) could complement the negative freight of Asp6 in the TonB box, of which negative freight can exist create in the half of the TonB box consensus motif from various Gram-negative organisms (Fig. 6C). This might hint that the positive-negative freight interaction could play an valuable role in the specific recognition of TonB box. However, putative BtuB sequences from P. aeruginosa execute not contain the exact consensus TonB box motif shown in Fig. 6C. Based on the observation from the model of PaTonB-96/EcBtuB complex, they searched a potential TonB box sequence in a putative BtuB sequence (PaBtuB) (Uniprot: Q9I473). They create a stretch of the sequence “DQVVTATR” (residues 29–36) at the N-terminal region of the workable plug domain region, with which PaTonB might interact, and hypothesized two interaction models (Fig. 6D). In the model A, they assumed Val32 in the PaBtuB as the highly conserved Val in the TonB box motif. In this model, the negative freight of Asp29 could exist located in the vicinity of Lys332 and the sequence of “VVTA” is identical to the PaTonB-96/EcBtuB model. In the other model (model B), they considered Asp29 as the key residue in the alignment. The highly conserved Val in the TonB box is now replaced by Ala in this model, but the negative freight at Glu339 in PaTonB could exist better compensated by Arg36 in PaBtuB if the extended the ultimate β-sheet of PaTonB.

It is noteworthy that similar interactions are not credible for the solution NMR structure of EcTonB-137 due to the steric hindrance by the presence of an additional β6-strand. The β6-strand needs to exist disrupted and exchanged to the TonB box for the similar interaction as observed in the crystal structures of the two TonB/TBDT complexes (Pawelek et al., 2006; Shultis et al., 2006). The solution NMR structure of PaTonB-96 elucidated in this travail is accessible to the TonB box without the strand exchange, because the disordered and extended C-terminal End does not interfere with the interaction site. This open conformation at the C-terminus is besides observed in the NMR structures of HpTonB-92 (Ciragan et al., 2016) and TonB-like protein, HasB from Serratia marcescens (Amorim et al., 2013). Thus it might exist a more common structure among CTDs of TonB proteins across different organisms.

Our hypothetical model of PaTonB-96 interactions with the TonB box and crystal structures of TonB/TBDT complexes suggests that αI–helix positions toward the plug domain located in the TBDT barrel (Fig. 6A) (Pawelek et al., 2006; Shultis et al., 2006). It was previously proposed that the positively charged Arg166 residue in αI–helix of EcTonB-CTD could interact with the negatively charged Glu56 in plug domain of FhuA receptor and disrupt its structure, thereby lowering the energy barrier required for the pore opening (Pawelek et al., 2006). Interestingly, the MD simulation detected orientational fluctuations of αI–helix in PaTonB-96 (Fig. 5), which besides contains positively charged Arg271. Such fluctuations might further facilitate the proposed disruption due to the interactions between Arg271 in PaTonB and the negatively charged residues in the plug domain of TBDTs. The resilient loop between β4 and β5 strands create in PaTonB-96 is located distantly from BtuB (Fig. 6B). Therefore, the modeled involved of PaTonB/BtuB does not directly indicate any structural role for this resilient loop.

Conclusions

We report the solution structure of PaTonB-96, which is largely similar to the previously reported monomeric NMR structure of EcTonB-137 (PDB code: 1XX3) (Peacock et al., 2005) except for the C-terminal end. Whereas the C-terminal region of NMR structures of EcTonB-137 formed an additional anti-parallel β6-strand with β5-strand, the C-terminal End of PaTonB-96 has extended resilient conformation, which resembles the crystal structures of TonB proteins interacting with TBDTs (Pawelek et al., 2006; Shultis et al., 2006). The absence of the ultimate β6 strand in PaTonB-96 structure suggests that the β5-strand is more accessible for the interaction with TonB box than in EcTonB-137, for which the strand exchange is required. Furthermore, the structural model suggests that the electrostatic interactions between PaTonB-96 and TonB box might exist more favorable than for EcTonB. Based on the structural model, they identified a potential TonB box sequence in PaBtuB. They besides speculate that the orientation fluctuations observed in the αI–helix of PaTonB-96 detected in MD simulation could lower the energy barrier of the suggested channel opening by disrupting the plug domain structure within the TBDT barrel when the C-terminus of TonB is bound to TonB box. The NMR structures and PaTonB-96 could thus pilot further experimental analysis to unveil the structural basis of the mechanism of TonB-dependent energy transduction.

Supplemental Information

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Press Release Summary:

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Original Press Release: Ricoh Introduces the Aficio SP C420DN Advanced Color Laser Printer

New Aficio SP C420DN Offers high Color Print Productivity and Lower Cost of Ownership

WEST CALDWELL, N.J., June 4 /-- Ricoh Americas Corporation, a leading provider of digital office equipment, today introduced the Aficio(R) SP C420DN color laser printer. Designed for businesses in color-intensive environments, the SP C420DN combines high productivity printing with precipitate and a simple user interface to provide a low total cost of ownership.

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"A major goal of Ricoh is to improve the overall efficiency of their customers' industry operations and provide easy-to-use products and solutions to improve their bottom line," said Hede Nonaka, executive vice president, marketing, Ricoh Americas Corporation. "The Aficio SP C420DN satisfies the market's demand for a speedy and dependable laser color printer, offering a wide variety of printing capabilities that fulfill a number of printing needs."

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About Ricoh Americas Corporation

Ricoh Americas Corporation, headquartered in West Caldwell, N.J., is a subsidiary of Ricoh Company Ltd., the 72-year-old leading supplier of office automation rig and electronics, with fiscal year 2007 sales in excess of $19.4 billion, a 7.3 percent enlarge over the previous year.

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Ricoh Americas Corporation directly or through its network of authorized dealers markets and distributes products in North, Central and South America.

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Ricoh Aficio SP C420DN succession Specifications

Engine Specifications

Configuration DesktopTechnology Laser beam scanning, Electrophotographic, Dual component toner, 4tandem drum methodResolution (dpi) 600 x 600 dp(default)1200 x 600 dpi1200 x 1200 dpiPrinting precipitate 31 pages-per-minute in fullcolor and B&WFirst Print precipitate Color: 15 secondsB&W: 10 secondsWarm-Up Time Less than 30 secondsDimensions (W x D x H) 17.6" x 23.2" x 19.2"Weight 110. lbs. or lessPower Source 120V, 60HzMaximum Power Consumption 990W or lessEnergy Saver Mode 6W or lessMaximum Paper Capacity Up to 2,300 sheetsStandard Paper Capacity 550 sheetsBypass Tray 100 sheetsOptional Paper Feed unity 550 sheets (up to three paper feed units can exist added)Automatic Duplex standard (prints at nearly100% simplex precipitate through the standard tray)Output Capacity 500 sheets, countenance downPaper Sizes (All Sources) letter (8.5" x 11"), A4(8.3" x 11.7"), A5 (210 x297mm), EXE (7.25" x 10.5"),B5 (6.9" x 9.8"), Legal(8.5" x 14")Custom Sizes: standard PaperTray and Optional Paper FeedUnits:(width 3.9" - 8.5', length5.8" - 14") Bypass Tray(width 2.8" - 8.5", length5.5" - 35.4")Paper Weight (All Sources) 14lb. - 57lb. Bond / 120lb.Index (52-216 g/m2)Duplexing: 16 lb. - 43 lb.Bond/ 90 lb. Index (60-163 g/m2)Acceptable Paper Types Thick Paper/ Card Stock (Up(All Sources) to 57 lb./ 120 lb. Index/216 g/m2), plain Paper/Recycled Paper, LaserPrinter QualifiedTransparencies, Envelopes(Com 10, Monarch, C6, C5, DL)Safety Regulations UL UL60950, FCC Part15 ClassB device, Energy Star Tier 2 compliant

Controller SpecificationsCPU PMC-Sierra R7935-835L800 MHzPrinter Languages Adobe PostScript3, PCL 5c,PCL 6, Ricoh RPCS, PDFDirect, PictBridge(TM)(optional)Fonts 136 PostScript 3 fonts, 45PCL fonts, 13 International fontsMemory (RAM) 256 MB RAM standard; 512 MBRAM maximumHard Disk Drive 60 GB optional (40GBreserved for file storage)Standard Interfaces 10/100Base-TX Ethernet, USB2.0, USB Host I/FOptional Interfaces IEEE 1284, IEEE 802.11a/gWireless LAN, BluetoothWireless, Gigabit EthernetNetwork Protocols Simultaneous auto-sensing/auto switching TCP/IP,Novell IPX/SPX, AppleTalkOperating Systems Windows 2000/XP/Server2003/Vista; NetWare v. 3.12,3.2, 4.1, 4.11, 5.0, 5.1, 6,6.5; Macintosh OS 8.6 -9.2x, OS X 10.1, 10.2, 10.3or later; UNIX (using RicohUNIX filter aspect 11) SunSolaris, HP-UX, Red HatLinux, SCO Open Server, IBMAIX (for UNIX support, visitwww.ricoh-usa.com/downloads); SAP R/33.x or laterDrivers RPCS/PCL 5c/PCL 6: Windows2000/XP/Server2003/Vista;XPS: Windows Vista (viadownload only); PS: Windows2000/XP/Server2003/Vista,Mac OS 8.6 - 9.2x, OS X 10.1or later; UNIX: Sun Solaris,HP-UX, Red Hat Linux, SCOOpenServer, IBM AIXPrint Utilities SmartDeviceMonitor for Admin, WebSmartDeviceMonitor, Web Image Monitor,DeskTopBinder V2 Lite, Printer Utilityfor V2 Lite, Printer Utility forMax, Agfa Font Manager 2000,DeskTopBinder Professional(optional)

Paper Handling AccessoriesPaper Feed Unit nature 4000: portion # 420165Paper Size Standard: letter (8.5" x11"), A4 (8.3" x 11.7"), A5(210 x 297 mm), EXE (7.25" x10.5"), B5 (6.9" x 9.8"),Legal (8.5" x 14") Custom:(width 3.9" - 8.5", length5.8" - 14")Free Paper Weights 14 - 57 lb. Bond/ 120lb.Index (52 - 216 g/m2)Capacity 550 sheets; up to 3 units may exist added

Ricoh Web Site: www.ricoh-usa.com

CONTACT: Russell Marchetta of Ricoh Americas Corporation, +1-973-882-2075, russell.marchetta@ricoh-usa.com

Web site: www.ricoh-usa.com/

http://www.ricoh-usa.com/downloads

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