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250-351 Administration of HA Solutions for(R) Windows using VCS 5.0

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250-351 exam Dumps Source : Administration of HA Solutions for(R) Windows using VCS 5.0

Test Code : 250-351
Test denomination : Administration of HA Solutions for(R) Windows using VCS 5.0
Vendor denomination : Symantec
: 253 real Questions

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Symantec Administration of HA Solutions

Symantec Ghost retort Suite up to three.3 DLL privilege escalation | real Questions and Pass4sure dumps

A vulnerability become present in Symantec Ghost solution Suite up to three.three (working gadget). It has been declared as troublesome. This vulnerability affects a code secrete of the component DLL Handler. The manipulation with an unknown input leads to a privilege escalation vulnerability. The CWE definition for the vulnerability is CWE-269. As an influence it is accepted to influence confidentiality, integrity, and availability.

The debilitated spot changed into introduced 02/08/2019. The advisory is purchasable at This vulnerability turned into named CVE-2018-18364 in view that 10/15/2018. local entry is required to route this attack. A separate authentication is captious for exploitation. The technical particulars are unknown and an exploit is not attainable. The constitution of the vulnerability defines a likely finances of USD $5k-$25k for the time being (estimation calculated on 02/09/2019).

applying the patch 3.three RU1 is capable of rep rid of this issue.

CPE CVSSv3 VulDB Meta groundwork score: 5.3VulDB Meta Temp rating: 5.1

VulDB groundwork rating: 5.3VulDB Temp score: 5.1VulDB Vector: 🔒VulDB Reliability: 🔍

CVSSv2 VulDB groundwork ranking: 🔒VulDB Temp rating: 🔒VulDB Reliability: 🔍Exploiting category: Privilege escalation (CWE-269)local: YesRemote: No

Availability: 🔒

fee Prediction: 🔍existing fee Estimation: 🔒

threat Intelligence hazard: 🔍Adversaries: 🔍Geopolitics: 🔍economic system: 🔍Predictions: 🔍movements: 🔍 Countermeasures informed: PatchStatus: 🔍0-Day Time: 🔒

Patch: 3.3 RU1

Timeline 10/15/2018 CVE assigned02/08/2019 Advisory disclosed02/09/2019 VulDB entry created02/09/2019 VulDB ultimate replace resourcesAdvisory:

CVE: CVE-2018-18364 (🔒)

accessCreated: 02/09/2019Complete: 🔍

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Symantec Unveils modern options | real Questions and Pass4sure dumps

Symantec Corp. has introduced Veritas Storage groundwork 5.0 inordinate Availability (HA) for windows, and Symantec protection tips supervisor 4.5.

The Veritas Storage groundwork delivers information and application availability for MS home windows environments. It combines two options- Storage groundwork for windows and Veritas Cluster Server. It helps control explosive facts boom, optimize storage hardware investments, provide software availability, and pressure down operational fees by means of a set of typical tools for home windows, Linux, and UNIX environments.

Rob Soderbery, Sr. VP, Symantec’s Storage groundwork neighborhood, noted, “Standardization on Storage basis HA for windows allows customers to beget more flexibility in their storage hardware choices and drives down operational prices by enabling them to consume a separate device. This liberate has furthered the ROI of standardization with the aid of cutting back the can impregnate of deploying Storage basis on every server and enabling consumers to beget visibility and centralized control of storage management, inordinate availability, and catastrophe recovery capabilities throughout their entire records middle.”

Veritas Cluster Server introduces aspects designed to expand manageability and in the reduction of administration burden of offering tall availability. Its at ease, web-based mostly Cluster administration Console simplifies the assignment of managing, monitoring, and configuring dissimilar clusters for windows, Linux, and UNIX, operating in distinctive statistics centers. It significantly reduces operational costs by using proposing finished insurance policy throughout actual and virtual server environments including home windows, VMware, and Microsoft digital Server.

Symantec safety counsel manager four.5 offers automation of the incident management lifecycle. It assists consumers in mitigating IT risk through reporting on the effectiveness of IT security controls and enabling its directors to instantly reply to protection threats and incidents in network environments.

It supplies integrated adventure archiving and administration, compliance reporting, and administration of industry deployments. It offers shoppers long-term retention of logs for forensic and compliance mandates. It additionally offers an infrastructure monitoring person interface and consumer administration via lively directory integration and roles management.

Symantec protection suggestions manager four.5 is obtainable in an equipment profile aspect via its worldwide network of price-brought resellers, distributors, and programs integrators.

Symantec's Veritas Cluster Server 5.0 for VMware ESX offers inordinate Availability and disaster recovery for physical and digital Server Environments | real Questions and Pass4sure dumps

supply: Symantec

November 07, 2006 08:00 ET

Veritas Cluster Server for VMware ESX Simplifies Cluster Administration and Automates Failover for VMware virtual Servers across Heterogeneous Networks

CUPERTINO, CA -- (MARKET WIRE) -- November 7, 2006 -- Symantec Corp. (NASDAQ: SYMC) today unveiled Veritas™ Cluster Server (VCS) 5.0 for VMware ESX, bringing inordinate availability and catastrophe recovery to heterogeneous information facilities operating virtual server application. VCS for VMware ESX automates far off failover for catastrophe recovery and gives administration of clustered digital and physical servers. excellent to evade downtime in case of utility, digital computer, community hyperlink, or server screw ups, VCS for VMware ESX centralizes cluster management in a separate ESX server or throughout a campus or WAN. VCS is a key ingredient of Veritas Server foundation, a suite of items which enables commercial enterprise shoppers to find in detail what's running on the servers of their statistics center, actively control and administer those servers, and ensure that mission necessary purposes running on these servers are at everything times available. Symantec will exist demonstrating VCS for VMware ESX on the VMworld 2006 conference being held in l. a. this week.

"VMware administrators are seeking tools that not most efficient automate disaster recovery however aid them minimize the vulnerabilities linked to working numerous digital servers on the equal actual server," pointed out Poulomi Damany, director of product administration for Symantec's information core administration group. "Veritas Cluster Server for VMware ESX solves these problems by means of combining catastrophe restoration and tall availability, and consolidating handle of both digital and actual servers and their dependencies."

VCS for VMware ESX enhances Symantec's clustering solutions for home windows, Linux and UNIX structures. Symantec is the market leader in cross-platform server clustering, in line with the 2006 edition of the IDC global Clustering and Availability utility file(1). With delivered champion for VMware ESX, the market's most frequent digital server platform, VCS for VMware ESX gives a separate solution to consolidate management of VMware digital servers in heterogeneous records middle environments.

complete tall Availability and catastrophe recovery

VCS for VMware ESX offers inordinate availability and catastrophe recovery for physical and virtual servers. by means of simplifying and automating far flung failover for VMware virtual server environments, VCS for VMware ESX provides introduced insurance arrangement towards digital machine or utility failures, including:

-- utility and useful resource monitoring, as well as server monitoring, which offers a higher degree of availability; -- computerized healing from software, community storage, virtual aid, digital server, and actual server disasters; -- Centralized administration of digital and physical resources and servers from a separate console; -- complete trying out for catastrophe recuperation integrating both utility failover and statistics replication to permit organizations to observe at various disaster recovery with out disrupting production environments. "because it managers are attempting to rein in server sprawl and expand resource utilization across the business, they are faced with the challenge of deploying assorted statistics availability and management solutions to handle and protect an ever-becoming population of digital servers," eminent Brian Babineau, Analyst, enterprise route neighborhood. "With VCS for VMware ESX, Symantec has simplified the assignment for VMware shoppers through offering a separate platform that can preclude downtime of mission essential applications operating in digital and physical server environments across any distance and any platform."

New assist for VMware ESX

VCS for VMware ESX also allows for customers to maximise the superior features of VMware through recognizing and seamlessly interoperating with VMware's VMotion and allotted aid Scheduler (DRS). If a digital computing device is moved from one server to yet another for deliberate maintenance using VMotion, the circulate might exist identified by means of VCS and VCS will entangle the essential action to update the cluster fame for that reason. it's additionally compatible with disbursed resource Scheduler (DRS), VMware's workload optimization characteristic.

automatic disaster recovery checking out

pleasing to VCS is fireplace Drill, a function of VCS that provides an added layer of insurance arrangement for virtual servers. With fire Drill, organizations can check their catastrophe recovery arrangement and configuration with out impacting the creation environment. In virtual environments where server places change frequently, hearth Drill helps computer screen and song cellular servers, their configuration and dependency hyperlinks.

price and Availability

Veritas Cluster Server for VMware ESX is scheduled to exist released within the first quarter of 2007. Pricing for VCS for VMware ESX begins at $1,995 per server.

About Symantec

Symantec is the zone leader in proposing solutions to champion people and enterprises guarantee the security, availability, and integrity of their assistance. Headquartered in Cupertino, Calif., Symantec has operations in 40 international locations. greater counsel is accessible at

(1) IDC, worldwide Clustering and Availability utility 2005 dealer Shares, Doc #203676, October, 2006

word TO EDITORS: if you'd relish more information on Symantec corporation and its items, gratify contend with the Symantec advice room at everything expenses eminent are in U.S. bucks and are legitimate only in the u.s..

Symantec and the Symantec emblem are trademarks or registered trademarks of Symantec employer or its associates within the U.S. and different countries. different names could exist emblems of their respective house owners.

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Administration of HA Solutions for(R) Windows using VCS 5.0

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RIM introduces BlackBerry Mobile Fusion - The Next Generation Enterprise Mobility Solution for BlackBerry, Android and iOS | real questions and Pass4sure dumps




Press Release 

RIM Announces BlackBerry Mobile Fusion - The Next Generation Enterprise Mobility Solution for BlackBerry, Android and iOS Smartphones and Tablets 

Simplifies Management of Smartphones and Tablets for industry and Government

Waterloo, ON - Research In Motion (RIM) (NASDAQ: RIMM; TSX: RIM) today introduced BlackBerry® Mobile Fusion - the Company's next-generation enterprise mobility solution and RIM's entry into the multi-platform Mobile Device Management (MDM) marketplace. structure on years of leading enterprise mobility management solutions from RIM, BlackBerry Mobile Fusion will simplify the management of smartphones and tablets running BlackBerry®, Google® Android® and Apple® iOS® operating systems.

"We are pleased to interject BlackBerry Mobile Fusion - RIM's next generation enterprise mobility solution - to Make it easier for their industry and government customers to manage the diversity of devices in their operations today," said Alan Panezic, Vice President, Enterprise Product Management and Marketing at Research In Motion. "BlackBerry Mobile Fusion brings together their industry-leading BlackBerry Enterprise Server technology for BlackBerry devices with mobile device management capabilities for iOS and Android devices, everything managed from one web-based console. It provides the necessary management capabilities to allow IT departments to confidently oversee the consume of both company-owned and employee-owned mobile devices within their organizations."

RIM is the leading provider of enterprise mobility solutions with over 90 percent of the Fortune 500 provisioning BlackBerry devices today. The enterprise market for smartphones and tablets continues to grow in both the company-provisioned and employee-owned (Bring Your Own Device or BYOD) categories. BYOD in particular has led to an expand in the diversity of mobile devices in consume in the enterprise and modern challenges for CIOs and IT departments as they struggle to manage and control wireless access to confidential company information on the corporate network. This has resulted in increased claim for mobile device management solutions.

BlackBerry Mobile Fusion brings together the market-leading BlackBerry® Enterprise Server (version 5.0.3) for BlackBerry smartphones; modern management capabilities for BlackBerry PlayBook tablets built on BlackBerry Enterprise Server technology; and mobile device management for smartphones and tablets running Android and iOS operating systems.

BlackBerry Mobile Fusion will provide the following mobile device management capabilities for everything supported mobile devices*:

• Asset management• Configuration management• Security and policy definition and management• Secure and protect lost or stolen devices (remote lock, wipe)• User- and group-based administration• Multiple device per user capable• Application and software management• Connectivity management (Wi-Fi®, VPN, certificate)• Centralized console• tall scalability

BlackBerry smartphones will continue to profit from the many advantages of the end-to-end BlackBerry solution including the very advanced IT management, security and control available with BlackBerry Enterprise Server 5.0.3, which is partake of BlackBerry Mobile Fusion. These advanced features comprise BlackBerry® BalanceTM technology supporting the consume of a separate device for both drudgery and personal purposes without compromising the organization's exigency to secure, manage and control confidential information; over 500 IT policies; over-the-air app and software installation and management; tall availability; and much more. BlackBerry Mobile Fusion will also interject modern self-service functionality for employees to secure lost or stolen BlackBerry smartphones and BlackBerry PlayBook tablets.

BlackBerry Mobile Fusion is currently in early beta testing with select enterprise customers. RIM is now accepting customer nominations for the closed beta program which will start in January, and universal availability is expected in late March.

For more information, visit

* Device security, manageability and controls will continue to vary according to the inherent capabilities of the individual device operating systems.

About Research In Motion

Research In Motion (RIM), a global leader in wireless innovation, revolutionized the mobile industry with the introduction of the BlackBerry® solution in 1999. Today, BlackBerry products and services are used by millions of customers around the world to tarry connected to the people and content that matter most throughout their day. Founded in 1984 and based in Waterloo, Ontario, RIM operates offices in North America, Europe, Asia Pacific and Latin America. RIM is listed on the NASDAQ Stock Market (NASDAQ: RIMM) and the Toronto Stock Exchange (TSX: RIM). For more information, visit or

Architecture of the complete oxygen-sensing FixL-FixJ two-component signal transduction system | real questions and Pass4sure dumps

Signal relay through a two-component system

In two-component systems, a sensor histidine kinase undergoes autophosphorylation and transfers a phosphate group to its cognate response regulator, which then mediates cellular responses by binding to DNA, performing enzymatic reactions, or interacting with other proteins. In the FixL-FixJ two-component system of the plant root nodule symbiont Bradyrhizobium japonicum, the histidine kinase FixL undergoes autophosphorylation only when it is not bound to oxygen. This ensures that its cognate response regulator FixJ stimulates the expression of genes required for nitrogen fixation only under low-oxygen conditions. Wright et al. combined high- and low-resolution structural analyses with modeling techniques and functional analysis to generate a model of signal relay through the FixL-FixJ two-component system. The model shows how the dissociation of oxygen from FixL stimulates FixL autophosphorylation and phosphotransfer from FixL to FixJ.


The symbiotic nitrogen-fixing bacterium Bradyrhizobium japonicum is captious to the agro-industrial production of soybean because it enables the production of tall yields of soybeans with shrimp consume of nitrogenous fertilizers. The FixL and FixJ two-component system (TCS) of this bacterium ensures that nitrogen fixation is only stimulated under conditions of low oxygen. When it is not bound to oxygen, the histidine kinase FixL undergoes autophosphorylation and transfers phosphate from adenosine triphosphate (ATP) to the response regulator FixJ, which, in turn, stimulates the expression of genes required for nitrogen fixation. They purified full-length B. japonicum FixL and FixJ proteins and defined their structures individually and in complicated using small-angle x-ray scattering, crystallographic, and in silico modeling techniques. Comparison of energetic and dormant forms of FixL suggests that intramolecular signal transduction is driven by local changes in the sensor domain and in the coiled-coil region connecting the sensor and histidine kinase domains. They also create that FixJ exhibits conformational plasticity not only in the monomeric status but also in tetrameric complexes with FixL during phosphotransfer. This structural characterization of a complete TCS contributes both a mechanistic and evolutionary understanding to TCS signal relay, specifically in the context of the control of nitrogen fixation in root nodules.


Two-component systems (TCSs) are widely distributed in bacteria, fungi, and higher plants. They facilitate cellular adaptation in response to environmental change and are considered capable targets for the development of novel antibiotics and plant growth modulators because of their conspicuous absence in metazoans (1–3). TCSs are generally composed of two types of multidomain proteins: sensory histidine kinases (HKs) and response regulators (RRs). The TCSs can exist classified into three classes, based on their domain architectures. Class I HKs consist of an N-terminal stimulus-specific sensor domain and a C-terminal HK module. The latter comprises the dimerization and histidine phosphotransfer (DHp) and catalytic adenosine triphosphate (ATP)–binding (CA) domains. Class II HKs, which are specific for chemotaxis (4, 5), hold an N-terminal histidine-containing phosphotransfer (HPt) domain and a C-terminal HK module. Class III HKs beget features of both class I and class II HKs, combining the class I HK’s sensor domain and the HPt domain and HK module of the class II HKs (5, 6). RRs hold a conserved N-terminal receiver (REC) domain, which is connected to diverse C-terminal effector domains. In response to an environmental stimulus sensed by the HK sensor domain, the CA domain catalyzes the autophosphorylation of a specific histidine residue in the DHp (class I) or HPt (class II and class III) domain. That phosphoryl group is subsequently transferred to a conserved aspartate residue in the REC domain of the cognate RR. This phosphotransfer activates the RR to promote DNA or RNA binding, enzymatic reactions, or protein interactions that are mediated by the C-terminal effector domain (7). When autophosphorylation activity is turned off, HKs often act as a phosphatase of their cognate phosphorylated RRs (8), thus contributing to shutting down the pathway.

The key questions of TCS signaling are how HKs are activated by stimuli and how they interact with the RRs. The answers to these questions beget been hampered by the exigency of molecular- and atomic-level structural information on intact, full-length HKs in both the kinase-active and kinase-inactive forms and in complicated with RRs. Here, they portray the structural characteristics of the oxygen (O2)–sensing FixL and FixJ (FixL-FixJ) TCS of the rhizobium species Bradyrhizobium japonicum, a root nodule, nitrogen-fixing bacterium that forms symbiotic relationships with leguminous plant such as soybean. B. japonicum FixL is a class I HK that senses the O2 tension in the cytoplasm through a heme-containing Per-Arnt-Sim (PAS) domain and transfers phosphate from ATP by sequential autophosphorylation and phosphotransfer reactions in its C-terminal effector modules to the RR FixJ (Fig. 1). O2 association to and dissociation from the heme-PAS domain of FixL trigger intra- and intermolecular signaling mechanisms such that the deoxy (O2-unbound) profile of FixL is energetic for autophosphorylation and phosphotransfer, whereas the oxy (O2-bound) profile does not undergo autophosphorylation and exhibits phosphatase activity toward FixJ (9, 10). As a result, the rhizobial FixL and FixJ system stimulates the expression of genes required for nitrogen fixation only when O2 concentrations in the plant root nodules are low because the energetic heart of nitrogenase is O2-labile (11).

Fig. 1 Schematic representation of the domain structures of full-length and truncated versions of B. japonicum FixL and FixJ.

Full-length Bradyrhizobium japonicum FixL comprises two N-terminal Per-Arnt-Sim (PAS) domains, PAS-A and PAS-B, and C-terminal dimerization and histidine phosphotransfer (DHp) and catalytic adenosine triphosphate (ATP)–binding (CA) domains. His200 in the PAS-B domain is captious for binding to heme; His291 in the DHp domain is the site of autophosphorylation; and Asp431-Val467 of the CA domain constitutes the ATP-binding site. Full-length B. japonicum FixJ contains an N-terminal receiver (REC) domain and a C-terminal effector domain that binds to DNA. FixJ is activated by FixL-mediated phosphorylation at Asp55. Structures of the truncated FixL and FixJ proteins FixLPAS-PAS and FixJN used in this study are indicated. The residues that define the boundaries of these domains are noted. Domain structures were generated using the SMART utensil (

Although most TCS HKs, including FixL homologs from most other species, are integrated into the membrane for sensing extracellular stimuli, B. japonicum FixL is a water-soluble, cytoplasmic sensor. The vicissitude of purifying integral membrane TCS HKs in combination with their multidomain configurations and structural flexibility has impeded structure-function studies. Thus, everything previous structural studies of TCS HKs, and even those of FixL, were performed with truncated, rather than full-length, proteins. They beget isolated full-length B. japonicum FixL and FixJ proteins at tall purity and obtained structural information on FixL in both the kinase-active (deoxy) and kinase-inactive (oxy) forms, FixJ, and the FixL-FixJ complicated by combining size exclusion chromatography–integrated small-angle x-ray scattering (SEC-SAXS), x-ray crystallography, and molecular modeling techniques. This analysis provides insights into how microorganisms and plants accommodate to environmental change at the molecular smooth and elucidates details of a microbial signaling pathway that facilitates the agro-industrial production of soybeans for human food, livestock feed, and biofuel with only limited exigency for nitrogenous fertilizers.

RESULTS Autophosphorylation and phosphotransfer activities of full-length FixL and FixJ

We prepared recombinant full-length B. japonicum FixL and FixJ with very tall purity (fig. S1, A and B). B. japonicum FixL is a naturally occurring soluble FixL, in contrast to membrane-anchored FixLs, such as that from Sinorhizobium meliloti. B. japonicum FixL comprises N-terminal tandem PAS domains, PAS-A and PAS-B, followed by C-terminal DHp and CA domains (Fig. 1). The PAS-B domain senses O2 through a heme b cofactor (10, 12). The role of the N-terminal PAS-A domain in B. japonicum FixL is not clear, but some biological studies of the water-soluble FixL from Rhizobium etli hint that the PAS-A domain influences the oxygen affinity of the heme in the PAS-B domain (13).

We tested the phosphotransfer activity of their highly purified full-length FixL in several heme iron oxidation and ligation states. These included oxy (Fe2+-O2), deoxy (Fe2+), ferric cyanide–bound (cyanomet; Fe3+-CN−), and ferric ligand–free (met; Fe3+) forms (fig. S2, A and B). Phosphotransfer from FixL to FixJ was suppressed in the oxy and cyanomet forms, whereas the activities of these reactions were fully restored in the deoxy and met forms (Table 1). These results expose that their preparation of full-length FixL and FixJ was successful and that signal transduction by the FixL and FixJ system could exist controlled by ligand (O2 or CN−) binding to the sensor domain of FixL, irrespective of the ferrous or ferric oxidation status of the heme iron. Because of the low affinity of O2 for FixL (Table 1) and relatively snappily autoxidation (τ1/2 ~ 15 min) of oxy FixL (12), they used the met and the cyanomet forms of the full-length FixL as analogs for the deoxy and oxy states, respectively, as samples for the SAXS measurements. They also create that CN− binding to the heme iron of the FixL sensor domain in the met profile suppressed the autophosphorylation activity (fig. S3A) but promoted the phosphatase activity toward phosphorylated FixJ (fig. S3B). On the basis of these experimental data, they expected that the tertiary and quaternary structures of the full-length FixL are equivalent between ferric CN−– and ferrous O2–bound forms.

Table 1 Phosphotransfer activities of full-length FixL to FixJ by ATP-NADH coupled assay (42). Molecular architecture of FixL

Very minuscule amounts of aggregated protein can adversely influence SAXS measurements (14). Despite the tall monodispersity of full-length B. japonicum FixL, minuscule amounts of aggregation often resulted in poor SAXS data from static measurements (table S1). To overcome this effect, they performed SEC-SAXS, where the data collection was performed in-line with protein purification, both at the RIKEN beamline BL45XU (15) at the SPring-8 synchrotron in Japan and at the sway beamline (16) at the SOLEIL synchrotron in France. The SECs of FixL in the met profile with SAXS parameters plotted together with absorbance at 280 and 398 nm showed that the aggregated species were eliminated before the SAXS measurements (fig. S4A). Using this method, they obtained high-quality SAXS data from full-length FixL in both the met and the cyanomet forms. The plot of log q versus log I(q) obtained by SEC-SAXS at BL45XU (Fig. 2A) and the SEC-SAXS parameters are summarized in Table 2 and table S2. The data measured at SPring-8 and SOLEIL showed a tall degree of reproducibility. Radii of gyration (Rg) of 49.7 ± 0.1 Å and 48.4 ± 0.2 Å for met and cyanomet FixL, respectively, were lower than those collected by static SAXS measurements (Table 2 and table S2) and reflect the aptitude of SEC-SAXS to seclude scattering from the species of interest. Molecular mass estimations from experimental SEC-SAXS data forecast protein masses of 140.1 ± 1.9 kDa and 136.3 ± 2.3 kDa for the met and cyanomet forms, respectively, indicating that FixL is homodimeric in solution. These observations are consistent with the structural characteristics of other HKs, in which two helices of the DHp domain from each monomer interact to profile a stable four-helix bundle in the homodimer (17, 18). The distance distribution function, P(r), for each FixL status gave information on the maximum dimension (Dmax) and the tolerable electron distribution (Fig. 2B). The Dmax was calculated at 163 ± 4 Å and 158 ± 3 Å for the met and cyanomet forms, respectively. CN− binding to the heme group of FixL suppressed the kinase activity, which was reflected in a slight subside in Rg (48.4 Å versus 49.7 Å; Table 2) and Dmax (163 Å versus 158 Å; Table 2).

Fig. 2 SEC-SAXS profiles of full-length FixL, FixJ, and FixL-FixJ complexes.

(A) Log-log plots of x-ray scattering for the met and cyanomet forms of full-length FixL, full-length FixJ, the FixL-FixJ complex, and the FixL-FixJN complex, q = 4πsinθ/λ, where 2θ is the scattering angle and λ is the wavelength of incident x-rays. I(q) is the measured scattered intensity at a given value of 2θ. (B) Corresponding pair distance distribution functions for the indicated proteins and protein complexes. P(r) is the frequency of r intramolecular distances. (C) Dimensionless Kratky plots, which compare the compactness of a protein, for full-length FixL (met form), the truncated FixLPAS-PAS (met form), full-length FixJ, and the full-length FixL-FixJ complex. qRg = q, as defined above, multiplied by the radius of gyration of the protein. A compact protein has a peak maximum at √3 and 1.2 on the abscissa and ordinate, respectively. Movement of the peak further into the positive quartile of the graph indicates unfolding and conformational flexibility. Data were collected on the BL45XU beamline at the SPring-8 synchrotron. n > 3 independent protein preparations and data collections.

Table 2 Structural parameters for FixL and FixL-FixJ complexes determined by SEC-SAXS experiments at BL45XU in SPring-8.

Using the FixL SEC-SAXS data, they constructed an ab initio model of full-length FixL in the met state. FixL exhibits an extended and club-like profile (fig. S5, A and B). Correspondingly, dimensionless Kratky plot analysis of full-length FixL describes a protein that does not adopt either a compact or globular conformation (Fig. 2C). For comparison, they also collected SEC-SAXS data for a truncated profile of FixL that contains only the PAS-A and PAS-B domains (FixLPAS-PAS; Fig. 1 and fig. S6, A and B). The Kratky plot analysis of FixLPAS-PAS is much different from that of full-length FixL and shows a tall degree of globularity (Fig. 2C). Using this information as a guide, they constructed a pseudoatomic model of full-length FixL, incorporating a combination of previously published crystallographic domain structures [Protein Data Bank (PDB) identifiers (IDs): 3MR0 for PAS-A domain, 1DRM (9) for PAS-B domain, and 4GCZ (19) for HK module], homology modeling, a priori structure prediction, and refinement against the SAXS data, and compared this model with the space-filling model (Fig. 3A). The length of the dimer axis in the model is identical to the experimentally determined Dmax value (Table 2), and its calculated SAXS profile (fig. S7, A and B) has a χ value of 2.57 when compared with the experimental data, indicating the internal consistency of their proposed model and that it is a reasonable snapshot of FixL in solution.

Fig. 3 Space-filling and pseudoatomic models of full-length FixL.

SAXS-based models of the met profile of full-length FixL showing the overall shape and domain arrangement.

Our model for FixL displays an elongated structure in which the tandem PAS domains (PAS-A-A and PAS-B-B) and the DHp domains homodimerize. There is no interaction between the PAS-A dimer and PAS-B dimer or between either the PAS-A or PAS-B dimer and the CA domain. They note that the PAS-B sensor domain connects with the kinase module only through a coiled-coil linker region. This architecture of full-length FixL is comparable to an “in-line” model proposed for transmembrane and membrane-associated HKs (17, 18, 20).

Our constructed model has an asymmetric DHp domain (Fig. 3A). To test the possibility that FixL homodimers beget a symmetric four-helix bundle, they assembled models of FixL based on the HKs VicK [PDB ID: 4I5S (21)] (fig. S8A), DesKC [PDB ID: 3GIE (22)] (fig. S8B), and CckA [PDB ID: 5IDJ (23)] (fig. S8C). This approach did not succumb a better fortunate to the experimental data than that provided by their proposed structure of intact, asymmetric full-length FixL.

Phosphotransfer activities of FixL mutants

In their proposed model, they create that the sensor PAS-B domains are followed by the coiled-coil linker region and then the four-helix bundle, but there is no direct interaction between the PAS-B and the catalytic kinase domains. From this observation, as one of the feasible mechanisms for intermolecular signal transduction of the O2 sensor FixL, they could submit that conformational change in the PAS-B domain is propagated to the DHp domain through the coiled-coil linker region (see Discussion). To examine the consequence of the coiled-coil linker region in transducing the signal from the sensor domain to the kinase domain to stimulate autophosphorylation, they prepared 12 proteins presence substitution mutations of the coiled-coil region by site-directed mutagenesis (R254A, T257A, E258A, E258Q, Q261A, T262A, T262S, Q263A, R265A, L266P, Q267A, and L269P) and measured their autophosphorylation activities in both the met and cyanomet forms (fig. S9, A and B). Except for the E258Q mutation, which was tolerated and responded to the CN− binding, each mutation significantly reduced the phosphorylation activity in the met form, indicating that the dimerization of the coiled-coil region might exist liable for activation of the kinase activities of the HK module in FixL. In addition, they also eminent that the activity of R254A mutant FixL showed a fivefold lower activity in the met profile and qualify impairment in the cyanomet form. The Arg254 residues on the coiled-coil helices near the terminate of the PAS-B domains might profile a hydrogen bonding interaction between the helices to couple CN− binding to kinase activity (fig. S9).

Crystal and solution structures of full-length FixJ

One of the most intriguing features of TCSs is the intermolecular communication that facilitates the transfer of a phosphoryl group from the kinase domain to the RR. They analyzed full-length FixJ alone with SEC-SAXS and crystallographic techniques. Full-length FixJ was crystallized in space groups C2221 (one chain in the asymmetric unit) and P212121 (five chains, A to E, in the asymmetric unit) (Fig. 4, A and B, and table S3). Crystallographic analyses revealed that FixJ is a two-domain protein, comprising an N-terminal α/β-type REC domain with a phosphorylation site (Asp55) and a C-terminal everything α-type effector domain with a helix-turn-helix DNA binding motif connected by a helical linker (Fig. 4A). The N-terminal REC domain and the C-terminal effector domain each exhibits the very fold as those reported for other DNA binding RRs (24). The linker region contained two α helices (α6A and α6B) in the C2221 data. Chains A to D in the P212121 data represented similar overall conformations with one another, whereas the chain E adopted a different conformation in the linker region compared to chains A to D. Helices α6A and α6B observed in the C2221 data were fused to a separate helix, α6, in the chains A to D of the P212121 data, whereas the helix α6A was transformed to a taut curve structure in the chain E of the P212121 data, indicating that the helix α6A is conformationally flexible. Structural comparison of FixJ crystal structures with those of other full-length RRs shows that the α6A helix in the linker region is labile and acts as a hinge enabling TCS RRs, including FixJ, to adopt a variety of overall molecular shapes.

Fig. 4 Crystal and SAXS solution structures of full-length FixJ.

(A) Crystal structure of full-length FixJ (the C2221 data) showing the relative positions of the N-terminal REC (pink), linker (cyan), and C-terminal effector (green) domains. The phosphorylation site, Asp55, is indicated as a stick model with carbon and oxygen atoms indicated in yellow and red, respectively. (B) Comparison of the crystal structures of FixJ in space groups C2221 (blue) and P212121 [yellow (chain A as a representative of chains A to D) and magenta (chain E)]. The view is in the very orientation as (A). (C) Space-filling and pseudoatomic models of full-length FixJ. The ribbon model was colored by temperature factors (b factors). Low and tall temperatures are represented in colder and warmer colors, respectively. (D) Comparison of FixJ crystallographic and SAXS models against experimental SAXS data. Experimental SAXS data are shown in black; the calculated scattering curve of the SAXS FixJ model in (C) is shown in red; the calculated scattering curves of the crystal structures of FixJ are shown in other colors as indicated. q = 4πsinθ/λ as defined in Fig. 2.

The SEC-SAXS parameters of B. japonicum FixJ (Table 2), that is, Rg = 22.3 ± 0.2 Å, Dmax = 66 ± 2 Å, and molecular mass = 24.5 ± 0.7 kDa, are comparable with those of the S. meliloti ortholog (25) and betoken that FixJ in the nonphosphorylated status is monomeric in solution. An ab initio model constructed from experimental scattering data showed that FixJ in solution exhibits an ellipsoidal shape (Fig. 4C and Table 2), and dimensionless Kratky plot analysis (Fig. 2C) also indicated that FixJ does not leisure in an extended conformation in solution. No crystallographic conformer fitted the experimental SAXS data well, indicating that the tolerable conformation of FixJ in solution is not fully represented by the crystal structures. They were able to refine the FixJ structure against the SAXS data to create a pseudoatomic model that fits the experimental data with a χ value of 2.5 (Fig. 4D). In solution, the N-terminal REC domain and the C-terminal effector domain are able to fold back upon one another because of the kinked linking α helix, thus allowing a more compact structure. The estimated Dmax (66 Å) from the SAXS data is smaller than the longitudinal length of ~78 Å for a dumbbell-like shape of the crystal structures. This dissimilarity may arise from the conformational flexibility in the linker region, which confers conformational plasticity in solution.

Solution structure of the full-length FixL-FixJ complex

To promote FixL-FixJ complicated formation, they performed SEC of FixL in a buffer containing 40 μM FixJ. This concentration is 10-fold greater than the FixL-FixJ dissociation constant (Kd), which has been reported as 0.8 to 4.0 μM in the absence of ATP analogs and Mg2+ (26). Under this condition, the FixL-FixJ complicated is expected to predominate over that of homodimeric FixL in solution. When the FixJ concentration in the loading buffer was reduced to 20 μM, the FixL-FixJ complicated was not well separated from homodimeric FixL by SEC.

FixJ-saturated FixL elutes earlier than FixL homodimers (fig. S4B). Under these conditions, they collected SEC-SAXS data for the FixL-FixJ complicated with the met profile of FixL (Fig. 2). Size parameters were create to exist 53.1 ± 0.1 Å for Rg, 160 ± 5 Å for Dmax, and 185.4 ± 1.5 kDa for molecular mass (Table 2). The Rg value was larger than that of FixL alone, and the molecular mass was increased by 45.3 kDa, indicative of two molecules of FixJ (24.5 kDa) binding to each FixL homodimer. These parameters betoken that phosphotransfer from FixL to FixJ is facilitated through homodimeric FixL binding to two FixJ molecules to profile a heterotetramer. In addition, the SAXS data did not betoken large domain rearrangements upon complicated formation, and dimensionless Kratky plot analysis (Fig. 2C) showed that the noncompact FixL conformation is conserved after forming a complicated with two molecules of FixJ.

On the basis of this observation, they docked two FixJ monomers onto their FixL pseudoatomic model (Fig. 3B). In this construction, the phosphodonor 3-phospho-His291 in the FixL DHp domain and the phosphoacceptor Asp55 of FixJ are within phosphotransfer distance (~3 Å). The overall fortunate of this model to the experimental SAXS data is good, with a χ value of 2.71 (fig. S7B), thus validating the proposed model. To their knowledge, this is the first complete model of the complicated formed by an intact full-length sensor HK and its full-length cognate RR that mediates TCS transduction.

To compare the kinase-active FixL-FixJ complicated with the dormant complex, they also measured the SEC-SAXS of the complicated in the cyanomet form, whose parameters are shown in Table 2. Their values, especially Rg and Dmax, were the very as those of the energetic met FixL-FixJ complex, suggesting that the ligand (CN−) binding to the PAS-B sensor domain of FixL, which suppressed the kinase activity, does not strongly influence the overall molecular shape of the FixL-FixJ complex.

In the proposed FixL-FixJ complex, the N-terminal REC domain of FixJ, as determined by their FixJN structure, fits well within the SAXS envelope model, whereas the C-terminal effector domain of FixJ, as determined by their structure of FixJC, does not. They hypothesize that, given the conformational flexibility of monomeric FixJ, the C-terminal domain is also able to adopt multiple conformations while in complicated with FixL due to the dynamic nature of the linker region observed in the crystallographic structure of FixJ (Fig. 4B). To test this possibility, they also measured and analyzed the SEC-SAXS of FixL complexed with FixJN. The SAXS parameters of the FixL-FixJN complicated showed that the Dmax (170 ± 3 Å) of this complicated is similar to that of the FixL-FixJ complex, whereas the molecular mass (153.6 ± 1.4 kDa) and Rg (48.9 ± 0.1 Å) values were smaller, as expected (Table 2). The absence of the FixJ C-terminal domain was reflected in the changes in mass and Rg but did not influence the maximum dimension of the FixL-FixJ complex. In addition, ab initio models of the FixL-FixJ and FixL-FixJN complexes were very similar (fig. S5B). These results hint that the C-terminal domain of FixJ does not contribute to the FixL-FixJ interaction and is instead free to scurry in solution.


Intra- and intermolecular signal transduction through TCSs occur in everything domains of life except metazoans. These systems facilitate survival by allowing rapid adaptation to environmental changes. To understand the molecular mechanism of TCSs in detail, structural information about TCS HKs, RRs, and the complexes they profile beget been generated. However, many structural studies on TCSs beget relied on breaking the protagonists down into biochemically tractable domains. Here, they beget investigated the structure of a complete TCS using the full-length forms of components of the B. japonicum O2-sensing system: FixL, FixJ, and the FixL-FixJ complex. The information gained for the kinase-active and kinase-inactive forms of full-length FixL and of full-length FixL in complicated with FixJ is particularly valuable because it enabled us to investigate the modular structures that facilitate signal relay in this agriculturally necessary TCS. To validate their model of full-length FixL, which used the asymmetric four-helix bundle structure of the FixL DHp domain conjoined to a light sensor domain as a template (19), they created homology models of FixL based on other HK DHp domains. These structures exhibited consistently worse fortunate to the experimental SAXS data than did the FixL model they proposed.

With respect to the intramolecular signal transduction from the sensor domain to the CA domain of FixL, two feasible mechanisms beget been proposed so far. Sousa and co-workers proposed a “globular” model based on biochemical studies of the full-length, cytoplasmic R. etli FixL protein (13), which has a similar domain organization as B. japonicum FixL. In the R. etli FixL, interactions between this protein’s cytoplasmic PAS domains (PAS-A and PAS-B) in homodimers or between the PAS homodimers and the CA domain are colorable in the globular form. Therefore, it was proposed that changes to these interactions would exist involved in the intramolecular signal transduction stimulated by the association of O2 with or dissociation from the sensor PAS domain. On the other hand, the crystal structure of the transmembrane Thermotoga maritima ThkA, the separate PAS domain of which exhibits 24% primary structure similarity to that of FixL PAS-B, displays no interaction between the PAS domains in the homodimeric form, but rather direct interaction of the PAS domains with the CA domains through hydrogen bonding (27). These models, proposing interactions between the PAS domain and the CA domain, were based on the assumption that the mechanism of the intramolecular signal transduction is not necessarily similar between the water-soluble and membrane-integrated TCS HKs. The former HK functions as a sensor of a stimulus in the cytoplasm, whereas the sensor domain of the later HK detects an extracellular stimulus and transfers information into the cytoplasm across the membrane for cellular adaptation. However, the proposed architectures of R. etli FixL and ThkA are incongruent with their present SAXS data of full-length B. japonicum FixL, deduced parameters, and low-resolution models. These structural inconsistencies hint that neither the globular model nor the direct transfer of the signal from the sensor to the CA domains is possible.

Our results lead us to submit an in-line model for full-length FixL with dimerization through PAS-A–PAS-A, PAS-B–PAS-B, and DHp-DHp interactions, but no direct interaction of the heme-containing PAS-B domain with the CA domain (Fig. 3A). Their model of intact full-length FixL provides us with a structural basis from which to contend the molecular activation mechanism of TCS HK modules by autophosphorylation in response to stimuli. The full-length FixL met (active) and cyanomet (inactive) forms (Table 2) expose that the overall molecular conformation is not largely changed upon activation. Correspondingly, their SAXS envelope models are also unaltered by CN− binding (fig. S5A). Thus, it is likely that the conformational changes that control kinase activity after ligand binding are localized, causing subtle changes in the interaction between the sensor domain and the HK domain. They submit that, upon O2 dissociation from the heme iron in the PAS-B domain, a local conformational change in this domain, previously observed in high-resolution structures and spectroscopic characterization of FixL sensor domains (28–32), is propagated to the DHp domain through the coiled-coil linker region. These structural changes intuition ATP bound to the CA domain and adjust the position of the His291 residue of DHp, resulting in phosphotransfer (Fig. 6).

Our thought was supported by the results of site-directed mutagenesis experiments, in which mutations in the coiled-coil linker region inhibited the phosphotransfer activity of FixL (fig. S9). The results are consistent with their previous drudgery on chimeric sensor proteins, in which the FixL PAS-B sensor domain was fused with the HK domain of T. maritima ThkA at the coiled-coil region (33). The kinase activities of the chimeric proteins were impaired, maintained, or enhanced by ligand binding, depending on where in the coiled-coil region the two functional domains were fused. everything these results champion their proposal that the coiled-coil linker region between the PAS-B and DHp domains functions as a key modulator of the autophosphorylation activity of FixL.

This proposal, the so-called in-line mechanism, is comparable to those given from previous studies of membrane-integrated or membrane-associated TCS HKs such as VicK and DesKC. The structures of truncated forms of these HKs, in which the transmembrane region or the extracellular domain or both were deleted (18, 20–23, 34), were the basis of this proposal. However, there was no structural comparison between the kinase-active and kinase-inactive forms of either. The only full-length structure of an HK in the literature is an engineered, chimeric HK called YF1, which is energetic in the unlit and dormant by stimulation with blue light (19). YF1 was constructed by fusing the FixL HK module (DHp + CA domains) with the light-sensitive light-oxygen-voltage (LOV) domain of Bacillus subtilis YtvA (19). On the basis of the structures of YF1 and the energetic and dormant forms of the isolated LOV domain, it was suggested that the coiled-coil linker region between the sensor and the DHp domain promotes the symmetry and asymmetry transitions of the four-helix bundle of the DHp domain to adjust the orientation between the DHp and the CA domains. The effects of site-directed mutagenesis of the coiled-coil linker region on the kinase activity of YF1 (19) are comparable to their observations of the effects of similar mutations on the kinase activity of FixL (fig. S9). Therefore, their present study using the intact and full-length FixL establishes the intramolecular signal transducing mechanism of HK.

Autophosporylation of His291 in the FixL DHp domain is followed by the phosphotransfer reaction, in which the phosphoryl group is transferred to Asp55 in the N terminus of FixJ. Formation of a transitory FixL-FixJ complicated facilitates phosphotransfer. To gain insight into this transitory status in solution, they collected SEC-SAXS data under FixJ-saturated conditions and observed SAXS parameters indicating a stoichiometry of 2:2 for the FixL and FixJ complex. This result is consistent with structural data for complexes of truncated HK and truncated RRs: HK853-RR468 (20), ThkA-TrrA (27), DesK-DesR (22), and Spo0B-Spo0F (34). Stranava and co-workers (35) recently reported that AfGcHK and its cognate RR predominantly profile a complicated with 2:1 stoichiometry, but they did not obtain a 2:1 complicated under the condition of excess FixJ in their SEC-SAXS study.

Our SAXS data betoken that FixJ binding induces no large FixL conformational change (Fig. 4, A and B). This observation is consistent with previous crystallographic studies of the truncated HK and RR complexes mentioned above (20, 22, 34), in which a slight rotation of the CA domain was observed, but there were no major changes in the overall structure of the HK (Fig. 6). In addition, the volume of the N-terminal FixJ REC domain is located close to the DHp-CA regions of the space-filling model (Fig. 3B). Because a phosphotransfer reaction from the HK to its cognate RR is common in TCSs, specific recognition of the REC domain by the kinase domain, such as that observed in the full-length FixL-FixJ complex, is almost certainly a universal feature of TCSs. After phosphotransfer to the Asp residue in the REC domain of the RR, which activates the RR and releases it from the HK, the C-terminal effector domain of the RR dimerizes and then interacts with its specific target. Phosphorylated FixJ promotes the expression of NifA and FixK by binding to elements upstream of the promoters of these genes, which encode proteins necessary for nitrogen fixation. Throughout TCSs, the function of the RR is highly variable and may mediate DNA binding (69.4%), RNA binding (1%), protein binding (1.7%), or enzymatic activity (8.1%) (36). Such functional diversity is reflected in the structural diversity of the C-terminal effector domains of RRs. There are at least 35 classes of RR, with each class including anywhere from 1 protein up to roughly 25,000 proteins (36–38). It appears to us that a conformationally supple RR, as described here for FixJ, very effectively limits the role that it can play in forming complexes with the cognate HK (Figs. 5 and 6). This is necessary to ensure that structurally disparate effector domains can exist activated by a universal TCS phosphotransfer mechanism to the REC domain of the RR. Thus, modularity ensures functionality.

Fig. 5 Space-filling and pseudoatomic models of the FixL-FixJ complex.

SAXS-based models of the FixL-FixJ complicated showing the overall shape, domain arrangement, and mode of complicated formation. The C-terminal effector domain of FixJ is not present in the model of the FixL-FixJ complicated because it does not profile partake of the complicated interface and is allowed conformational license by the linker connecting the N-terminal REC domain to the effector domain.

Fig. 6 Schematic representation of the FixL-FixJ TCS.

Our SAXS results hint that there are no large changes of the overall shape of full-length FixL upon O2 dissociation from the heme group. However, the orientation of the coiled-coil helices between the heme-containing PAS-B (pink) and DHp (green) domains may change. Such localized structural change could alter the distance between the ATP-binding site in the CA domain (orange) and the autophosphorylation site at His291 in the DHp domain. In the full-length FixL-FixJ complex, a phosphorylation site (Asp55) in the FixJ REC domain approaches His291 of the FixL DHp domain, which mediates phosphotransfer. The C-terminal DNA binding domain of FixJ is connected to the REC domain by a supple linker, allowing the effector domain to exhibit multiple conformations.

The O2 sensor FixL is categorized as a class I HK, in which the DHp domain is directly adjacent to the CA domain (34, 35). Although everything HKs discussed here (VicK, DesK, CckA, EnvZ, HK853, and ThkA) are in this very category, the domain architecture of FixL is the simplest among them. On the other hand, FixJ, which consists of a REC domain and a helix-turn-helix DNA binding domain (effector domain) through which it acts as a transcriptional activator, belongs to the NarL-like superfamily (39), which is the largest family of RRs. For the class I HKs, the REC domain is a common component for receiving a phosphate group. Therefore, the full-length architecture of the FixL-FixJ TCS is expected to portray the universal mechanism of intra- and intermolecular signal transduction in TCSs composed of class I HKs. It is feasible for the in-line mechanism supported by their findings to operate in everything class I HKs, irrespective of whether the HK sensor is water-soluble (cytosolic) or membrane-integrated. The in-line mechanism also does not require that the effector domain of the RR interact with the HK. In addition, the present study provides fundamental lore for understanding the physiological, biochemical, and biological consequence of TCS, particularly those involving class I HKs, including their molecular evolution (40) or protein engineering for synthetic biological systems for the development of antibiotics and plant growth effectors.

The rhizobia are a group of nitrogen-fixing bacteria that are adept at establishing symbiotic relationships with leguminous plants, a global staple food group. This relationship provides nitrates to the host and a boost to growth and plant survival with benefits to agricultural productivity. The model organism B. japonicum is of particular interest in this esteem because it resides in the root nodules of the soybean plant Glycine max, which provides more protein per hectare cultivated than any other food source. B. japonicum is sprayed onto soybean seed stock on an industrial scale to entangle handicap of this relationship. Their results open the path for genetic modification of this rhizobial TCS to better crop yields.

MATERIALS AND METHODS Preparation of recombinant FixL and FixJ proteins

B. japonicum FixL and FixJ genes encoding full-length FixL and FixJ and truncated proteins [FixLPAS-PAS (residues 1 to 275) and FixJN (residues 1 to 124)] were separately amplified by polymerase chain reaction (PCR) with PfuTurbo DNA polymerase (Agilent Technologies). The PCR fragments were cleaved by Bsr GI and Avr II for FixL, Bsr GI and Nhe I for FixJ (New England Biolabs), and cloned into 5′Bsr GI–3′Avr II sites of pET-47b(+) vector (Novagen) for expression with hexa-His tag, followed by the HRV3C protease cleavage site in the N terminus of those proteins. Site-directed mutagenesis of the coiled-coil region in full-length FixL was performed by QuikChange protocol (41) using pET-47b(+) vector inserted in the full-length FixL gene as a template.

Escherichia coli BL21(DE3) cells (Nippon Gene) carrying these plasmids were inoculated in terrific broth (TB) containing kanamycin (50 μg/ml; Wako) and 1% glucose for 4 hours at 37°C with shaking at 150 rpm. One milliliter of the preculture solution was inoculated into 300 ml of TB medium containing kanamycin [50 μg/ml; plus 250 μM 5-aminolevulinic acid (Cosmo Energy Holdings) only for the expression of FixL]. The cultivation was done at 37°C with shaking at 120 rpm. After 4 hours of cultivation, expression of FixL or FixJ was induced with 0.3 or 0.2 mM isopropyl-β-d-thiogalactopyranoside, respectively, and cultivation was allowed to continue for another 15 hours at 23°C with shaking at 80 rpm. The cells were harvested by centrifugation at 4000g for 10 min, and the cells were washed in 30 mM tris-HCl (pH 8.0) twice.

Purification of FixL and FixJ was performed by the following very steps at 4°C. Harvested cells were resuspended in a lysis buffer [50 mM tris-HCl (pH 8.0), 300 mM NaCl, 10% (w/v) glycerol, and one tablet of cOmplete EDTA-free protease inhibitor cocktail (Roche)]. The lysate was mixed with lysozyme (0.1 mg/ml; Sigma-Aldrich), deoxyribonuclease I (0.05 mg/ml; Sigma-Aldrich), and 5 mM MgCl2 for 30 min and disrupted by Microfluidizer M-110Y (Microfluidics). Cell debris was removed by ultracentrifugation at 40 krpm for 1 hour. The supernatant was loaded onto a HisTrap HP (GE Healthcare) column equilibrated with buffer A [50 mM tris-HCl (pH 8.0), 300 mM NaCl, 10% (w/v) glycerol, 10 mM imidazole/HCl (pH 8.0)]. FixL or FixJ was eluted by an imidazole concentration gradient (0 to 300 mM). The eluted fractions were treated with N-terminal 6×His-tagged HRV3C protease (produced in-house) to remove the 6×His-tag from the recombinant FixL and FixJ proteins, and the solution was dialyzed with buffer B [40 mM tris-HCl (pH 8.0), 150 mM NaCl, 10% (w/v) glycerol]. After the dialysis, the protein solution was loaded to HisTrap FF (GE healthcare) equilibrated with buffer B to remove the HRV3C protease and remaining His-tagged proteins, and the flowthrough was collected. The collected solution was concentrated by Amicon Ultra-15 (Merck Millipore) and centrifuged at 15 krpm for 20 min. The supernatant was loaded to HiLoad 16/600 Superdex 200 (GE healthcare) equilibrated with buffer B. The purity of FixL or FixJ was checked by SDS–polyacrylamide gel electrophoresis (PAGE). The purified samples were mixed with 2× SDS-PAGE buffer containing 125 μM tris-HCl (pH 6.8), 4% SDS, 20% (w/v) sucrose, 0.01% (w/v) bromophenol blue (BPB), and 10% (v/v) 2-mercaptoethanol and boiled at 95°C for 10 min before the electrophoresis. NuPAGE Bis-Tris gels (10%; Thermo Fisher Scientific) were used for the electrophoresis with NuPAGE Mops SDS running buffer (Thermo Fisher Scientific) for FixL and NuPAGE MES SDS running buffer (Thermo Fisher Scientific) for FixJ. The gels were stained by EzStain AQua (ATTO). In FixL, the highly purified fractions with an Rz (A398nm/A280nm) value of >1.3 were used for SAXS studies. These spectra were measured in 40 mM tris-HCl (pH 8.0), 150 mM NaCl, and 10% (w/v) glycerol at 20°C by NanoDrop 2000c spectrophotometers (Thermo Fisher Scientific). Because His-tagged full-length FixL showed the kinase inhibition upon cyanide binding to the heme and nearly the very phosphotransfer activity as the His-tag–removed protein, they used the His-tagged full-length FixL proteins in the phosphorylation activity assays of the coiled-coil mutants (fig. S9).

Autophosphorylation, phosphotransfer, and phosphatase activity assay of purified FixL and FixJ proteins

Autophosphorylation of the FixL protein was monitored by radioactivity of phosphorylated FixL by 32P. Reaction mixtures contained 2 or 6 μg of FixL in 7.5 μl of 50 mM tris-HCl (pH 8.0), 50 mM KCl, 1 mM MgCl2, 50 μM MnCl2, and 5% (w/v) glycerol with or without 5 mM KCN. The reactions were started with the addition of the compund of ATP (10 or 25 nM) and γ-32P-ATP (4 or 10 μCi) (PerkinElmer), incubated the reaction compund at 23°C for 10 min, and stopped with one-fourth volume of SDS-PAGE sample buffer. After SDS-PAGE, phosphorylated proteins were visualized with an imaging analyzer BAS-1800 on an imaging plate (Fujifilm).

Phosphotransfer activities from FixL to FixJ were determined using an enzymatic assay in which ATP hydrolysis is coupled to NADH (reduced profile of NAD+) oxidation using lactate dehydrogenase and pyruvate kinase (42). To measure basal activity, the reaction buffer [50 mM tris-HCl (pH 8.0), 50 mM KCl, 3 mM phosphoenolpyruvic acid (Wako), 0.3 mM NADH (Sigma-Aldrich), 8 U of lactate dehydrogenase (Toyobo), 25 U of pyruvate kinase (Sigma-Aldrich), 5 mM MgCl2, and 5 mM ATP] was equilibrated at 20°C for 5 min. The reaction was started by the addition of 1 μM purified FixL and 10 μM purified FixJ, and the time course of A340nm was monitored for 10 min at 20°C. An NADH measure curve with a sweep of NADH concentrations between 0 and 150 μM was measured in the very buffer. To measure the activity of the cyanomet form, they added 5 mM KCN to the reaction buffer before the addition of FixL protein.

Phosphatase activity of FixL was monitored the dephosphorylation of acetylphosphorylated FixJ by unphosphorylated FixL. The acetylphosphate-dependent FixJ autophosphorylation was performed in 50 mM tris-HCl (pH 8.0), 50 mM KCl, 1 mM MgCl2, 50 μM MnCl2, 20 μM FixJ, and 50 mM acetyl phosphate lithium potassium salt (Sigma-Aldrich) at 23°C. After 2 hours, unphosphorylated FixL (10 μM, wild type) was added to the reaction mixture. The incubation times for the reactions were 0 (before FixL addition), 5, 15, 30, 60, and 120 min after the addition of the FixL. The reactions were stopped with two-third volume of SDS-PAGE sample buffer. The reaction products were subjected to 15% Zn2+-Phos-tag SDS-PAGE containing 50 μM Phos-tag acrylamide (Wako) and 100 μM ZnCl2. Tris-glycine buffer was used for the electrophoresis. The gel was stained by EzStain AQua (ATTO).

SEC-SAXS data collection and analysis

SEC-SAXS data collection was performed at RIKEN beamline BL45XU (15) in SPring-8 and at beamline sway (16) in the French national synchrotron SOLEIL. At BL45XU in SPring-8, the purified protein was loaded onto a Superdex 200 expand 3.2/300 column (GE Healthcare) in a 20-μl volume at 50 μl/min tide rate with a SEC buffer [40 mM tris-HCl (pH 8.0), 10% (w/v) glycerol, and 5 mM MgCl2] at 20°C. For the data collection of cyanomet FixL, 5 mM KCN was added to the SEC buffer, and the loading FixL sample was mixed with KCN at the final concentration of 5 mM. For the detection of the FixL-FixJ complicated or the FixL-FixJN complex, SEC buffer containing saturating amounts of FixJ or FixJN (more than 40 μM, 10 times of the Kd value) was used to better the affinity of FixL and FixJ. X-ray exposure was 1 s every 4 s with the incident beam energy 12.4 keV. Buffer frames (30 × 1 s) were averaged and subtracted from 30 × 1 s frames taken over the course of protein elution. The sample detector distance was 2 m, giving an angular momentum transfer sweep of qmin = 0.009 Å−1 to qmax = 0.5 Å−1. The flux density was about 2 × 1012 photons/s/mm. Scattering was collected on a PILATUS3 X 2M detector (Dectris). Data averaging and reduction were calculated by the program DataProcess installed in BL45XU. Measurements at sway were performed as above but using an Agilent Bio SEC-3 4.6 × 300–mm column (Agilent) with 3-μm bead size and 300 Å pore size at 15°C after double purification on a Superdex 200 expand 10/300 column (GE Healthcare) at 4°C. Beam energy was 12 keV, and sample detector distance was 1.8 m.

Rg and I0 calculations on the SEC-SAXS data were performed with AutoRg (43). Data of interest was averaged, and the Guinier estimation was performed in MATLAB and PRIMUS (fig. S10) (44). Distance distribution functions P(r) were calculated with GNOM (45) and ScÅtter based on agreement between real and reciprocal space Rg values (<3% difference) and fortunate to the experimental data. This was performed independent of crystallographic and homology model building. Bead models were generated with DAMMIN (46).

Crystallization, data collection, and refinement for the full-length FixJ structure

Crystals of FixJ in space group C2221 (form 1) were obtained at 20°C by vapor diffusion using a mother liquor containing 10% (w/v) PEG 8000, 10% (w/v) PEG 1000, 0.8 M sodium formate, 20% (v/v) glycerol, and 0.1 M tris-HCl (pH 7.5). The asymmetric unit contained one polypeptide chain. Crystals of FixJ in space group P212121 (form 2) were obtained at 20°C by vapor diffusion using a mother liquor containing 20% (w/v) pentaerythritol ethoxylate (15/4 EO/OH), 0.1 M magnesium formate, 20% (v/v) glycerol, and 0.1 M tris-HCl (pH 8.5). The asymmetric unit contained five polypeptide chains. Crystals of both forms were grown in 3 days, frozen, and stored in liquid nitrogen. Data collection was carried out at BL26B2 in SPring-8, Harima, Japan (47, 48), equipped with an automated sample mounting system (49). Crystals were cryocooled in a nitrogen gas stream at 100 K during data collection. The data were integrated and scaled using HKL2000 (50). Initial phases for the data set of the crystal profile 1 were obtained by molecular replacement using coordinates of StyR (PDB ID: 1YIO) (51) as a search model in PHENIX (52). The coordinates of the N- and C-terminal domains of StyR were extracted and used for the molecular replacement calculation because the structure of the linker region may vary from protein to protein in the RR family. Initial phases were used for model structure and improved by refinement of the model coordinates, including model structure of the linker domain, in PHENIX (52) and COOT (53). The final model included residues from 2 to 203, and 4 glycerols, 6 formic acids, and 257 water molecules. Initial phases for the data set of the crystal profile 2 were obtained by molecular replacement using coordinates of the profile 1 structure in PHENIX, with the N- and C-terminal domains separated. Four solutions for each domain were obtained and used for model rebuilding and refinement including the linker regions (chains A to D) in PHENIX and COOT. During refinement, debilitated but continuous densities appeared in the solvent region. Lowering the contour smooth revealed the fifth molecule for the densities. Each separated copy of the models of the three domains was manually fitted in the electron densities (chain E). Coordinates of the five polypeptide chains were further refined including magnesium ions and water molecules. The final model included 1015 residues, 4 magnesium ions, and 8 glycerols, 7 formic acids, and 163 water molecules. Model character was checked by MolProbity (54) in PHENIX.

Pseudoatomic model structure and refinement against SAXS data

JPred (55) and Coils (56) were used to ascertain which parts of the FixL sequence profile α helices and coiled coils. The structure of the blue light receptor (PDB ID: 4GCZ) was used as a model for the HK domain and the α helices beyond Thr257. This was linked to the structure of FixL heme-PAS domain (PDB ID: 1DRM). The coiled-coil helix N terminus of the heme-PAS to the PAS-A domain was created with PEP-FOLD (57). This was linked to a homology model of the PAS-A domain created from a sensory HK from Burkholderia thailandensis (PDB ID: 3MR0). The cloning fragment from the pET-47b(+) vector (10 amino acid residues, GPGYQDPNSV) was also constructed using PEP-FOLD. This initial structure had χ2 value of 3.93 against experimental data calculated using FoXS (58). Torsion angle molecular dynamics (MD) in CNS (59) was used to refine the positions of domains and loops in this structure against SAXS data as described by Wright et al. (60). To validate the region of the model encompassing amino acids 1 to 256, which was assembled by combining a crystal structure, a homology model, and several ab initio structure predictions, they measured SAXS of a C-terminally truncated profile of FixL consisting of amino acids 1 to 275 and including the cloning fragment. These data were exceptionally congruent with the PAS-A–PAS-B partake of their FixL model, indicating the proposed domain arrangement and interfaces are correct. The chimeric sensor protein structure 4GCZ shows structural asymmetry possibly resulting from crystal packing or partial adenosine diphosphate (ADP) binding. They created three models of FixL based on VicK, DesKC, and CckA HKs, each of which has symmetric four helix bundles, based on their coverage of and identity to FixL amino acids 258 to 505. Homology models were generated with SWISS-MODEL in conjunction with the route above and HADDOCK (61) to refine and dock the ADP-free CA domains to find an optimum ADP-free structure. This pool of structures has consistently worse fortunate to their experimental data than that based on 4GCZ, indicating that FixL adopts an asymmetric conformation in solution in the ADP-free state.

The structures of FixJ were refined against SAXS data using the torsion angle MD process described above. They initially defined the location of FixJ binding to FixL using pyDockSAXS (62) and FoXSDock (63). These programs consume a combination of FTDock/Crysol and PatchDock/FoXS to apportion interactions. Using this approach, and without defining the FixL His291–FixJ Asp55 interaction site, FixJ was consistently positioned on the FixL four-helix bundle. FixJ REC domains were then directed to FixL His291 on both chains using HADDOCK. The FixJ linkers and DNA binding domains were then added based on the SAXS refined structure of the FixJ monomer. The positions of these latter domains were then determined using CNS torsion angle MD. Trimeric complexes of dimeric FixL with one FixJ monomer were also constructed and optimized by torsion angle MD/rigid carcass refinement but were create to consistently succumb models that fortunate the data poorly in comparison with the tetrameric architecture.


Fig. S1. Purification of FixL and FixJ.

Fig. S2. Optical absorption spectra of full-length FixL.

Fig. S3. Autophosphorylation and phosphatase activities of FixL.

Fig. S4. SEC profiles.

Fig. S5. Space-filling models of FixL alone and in complicated with FixJ or FixJN.

Fig. S6. Pseudoatomic model and SAXS curve of truncated FixL comprising PAS-A, PAS-B, and the coiled-coil region (FixLPAS-PAS).

Fig. S7. Experimental and simulated SAXS curves of met FixL and the FixL-FixJ complex.

Fig. S8. SAXS curves and pseudoatomic models of full-length FixL based on comparisons with other HKs.

Fig. S9. Phosphorylation activities of FixL mutants presence mutations in the coiled-coil linker.

Fig. S10. Guinier plots with Pearson residuals for full-length FixL, FixLPAS-PAS, FixL-FixJ, full-length FixJ, and FixJN.

Table S1. Structural parameters for FixL and FixJ determined by static SAXS experiment at the BL45XU beamline at the SPring-8 synchrotron.

Table S2. Structural parameters for FixL and FixJ determined by SEC-SAXS at the sway beamline at the SOLEIL synchrotron.

Table S3. Crystallographic statistics for full-length FixJ.

  • C. Tomomomori, H. Kurokawa, M. Ikura, The histidine kinase family: Structures of essential structure blocks, in Histidine Kinases in Signal Transduction, M. Inoue, R. Dutta, Eds. (Academic Press, 2003).

  • Z. Otwinowski, W. Minor, Processing of x-ray diffraction data collected in oscillation mode, in Methods in Enzymology, C. W. Carter, J. R. M. S., Ed. (Academic Press, 1997), vol. 276, pp. 307–326.

  • Acknowledgments: Thanks to synchrotron SOLEIL and SPring-8 for the provision of SAXS facilities. They avow the champion and the consume of resource of instruct, a landmark ESFRI project (iNEXT 2822). Funding: This drudgery was supported by the Fumi Yamamura Memorial Foundation for Female Natural Scientists from Chuo Mitsui dependence and Banking (to H.S.), Hyogo Science and Technology Association (to H.S.), RIKEN Pioneering Project “Integrated Lipidology” (to H.S.), and “Molecular System” (to Y.S.), and the Japan Society for the Promotion of Science (JSPS) KAKENHI concede numbers JP26220807 (to Y.S. and H.S.) and JP25871213 (to H.S.). Author contributions: G.S.A.W., S.V.A., S.S.H., Y.S., and H.S. designed this study. A.S., H. Nakamura, and H.S. created the systems for expressing recombinant FixL and FixJ in E. coli. T. Hikima and M.Y. installed the SEC-SAXS system at BL45XU in SPring-8. G.S.A.W., A.S., Y.N., and H.S. purified the FixL and FixJ samples. H.S. prepared the expression systems for FixL mutants. M.K. purified the FixL mutant proteins and measured their phosphotransfer activities. K.N. and H. Nishitani performed the autophosphorylation activity assay using γ-32P-ATP. H.S. performed the phosphatase activity assay by Phos-tag SDS-PAGE. G.S.A.W., A.S., T. Hikima, and H.S. measured and analyzed the SAXS data. G.S.A.W. modeled the pseudoatomic structures. Y.N. crystallized FixJ. Y.N. and T. Hisano collected, processed, and refined the crystal data. G.S.A.W., T. Hisano, S.V.A., S.S.H., Y.S., and H.S. wrote the manuscript. everything authors analyzed data and discussed the results. Competing interests: The authors declare that they beget no competing interests. Data and materials availability: everything data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The atomic coordinates and structure factors for FixJ (PDB IDs: 5XSO and 5XT2) beget been deposited in the PDB ( The SAXS measurements at SPring-8 BL45XU were performed under proposals 20140099, 20150017, 20160015, and 20170092. The x-ray diffraction measurements were performed at SPring-8 BL26B2 (proposal 20160015).

    Implementing Proxy Server | real questions and Pass4sure dumps

    Designing a Proxy Server Implementation

    Before you can design a Proxy Server implementation and install Proxy Server, you exigency to exist knowledgeable on a number of concepts:

  • IP routing concepts

  • Firewalls concepts

  • Packet filtering concepts

  • Files and protocols utilized in Web applications

  • To design a Proxy Server implementation, there are a number of factors that has an repercussion on the Proxy Server design:

  • The characteristics of data that will pass to the Proxy Server. Data characteristics should comprise factors such as the quantity of data which you anticipate the Proxy Server to handle, and whether data confidentiality needs to exist ensured.

  • The nature of firewall that the Proxy Server will interface with.

  • Decide whether the Proxy Server will exist located within the DMZ or on the edge of the network.

  • Determine the revise sizing of Proxy Server(s).

  • The resources located on the private network which Internet users should exist able to access.

  • The physical layout of the servers

  • The connections to and from proxy servers.

  • The volume of expected network traffic.

  • Bandwidth between sites.

  • Who needs access to the Proxy Server, and what nature of access is required.Implementing Proxy Server

  • Determine the time which users should exist able to access the Proxy Server.

  • Future network expansion.

  • Existing proxy server configuration: Here, factors such as the location of an existing proxy server, the WAN connections being used, and the protocols used in the private network should exist considered.

  • After you beget looked at everything the factors which repercussion the Proxy Server design, you beget to determine, or select between a few additional design elements:

  • The nature of connection that the Proxy Server must support:

  • The nature of services that the Proxy Server must provide:

  • Web Proxy

  • WinSock Proxy

  • Socks Proxy

  • Packet filtering

  • Reverse Web Proxy

  • The types of connection technology that the Proxy Server must support:

  • Digital Subscriber Line (DSL)

  • Integrated Services Digital Network (ISDN)

  • Public Switched Telephone Network (PSTN)

  • T1

  • X.25

  • The nature of Proxy Server clients that the Proxy Server must support.

  • The nature of routing which each router should support:

  • Dynamic routing

  • Static routing

  • Whether multiple proxy servers will exist implemented to better performance and provide tall levels of availability. Proxy Server provides a feature called proxy arrays. A proxy array is a solution whereby one or multiple proxy servers operate as a separate cache for client requests. Benefits provided by the proxy array feature comprise scalable performance, and fault tolerance.

  • You can create a Proxy Server design where frequently requested content is cached. Proxy Server can locally cache Internet sites and files which are frequently requested. Subsequent requests for these Internet sites are then serviced from the local cache. Cached information is accessed by users from a location on the Local zone Network (LAN). This design has a number of benefits. For instance, bandwidth utilization to the Internet ends up being reduced because cached information does not exigency to exist downloaded from the Internet. everything of this leads to an improvement in the service experienced by users.

    With passive caching, Proxy Server stores objects in the Proxy Server cache with each remonstrate obtaining a Time To Live (TTL) value. Before Proxy Server forwards requests to the Internet, it first checks the Proxy Server cache to determine if the request can exist serviced from there. energetic caching works together with passive caching. With energetic caching, Prox Server automatically generates requests for specific objects in the Proxy Server cache so that frequently requested objects remain cached.

    The requirements for creating a Proxy Server design that caches content are listed here:

  • Web content is only cached on NTFS partitions. This basically means that you exigency to have, minimally, one NTFS partition that is capable of storing frequently accessed Web content.

  • You should beget two network adapters so that private network traffic with Internet traffic can exist separated. This results in network congestion being reduced.

  • You should position proxy servers using their purpose or function as the basis to determine placement. This concept is illustrated here:

  • If the Proxy Server is to provide connectivity between the private network and the Internet;

  • If the Proxy Server is to cache Web content so that frequently accessed content can exist accessed from the local cache;

  • If the Proxy Server is to is to provide both connectivity between the private network and the Internet and cache Web content;

  • The following information has to exist defined for every interface in the Proxy Server implementation:

  • The nature of connection (persistent/nonpersistent) between the router interface and the network.

  • The following IP information for interfaces that are connected to IP network segments:

  • IP address configuration.

  • IP subnet mask configuration.

  • The following IPX information for interfaces that are connected to IPX network segments:

  • IPX network number.

  • IPX frame type

  • Another component that should exist included when you arrangement your Proxy Server implementation is to determine the client operating systems that Proxy Server should support. Proxy Server can champion a number of different client operating systems.

    You should define Proxy Server client champion based on what your Proxy Server implementation should provide:

  • Define Windows Proxy Server client champion for the following reasons:

  • All Windows operating systems should exist supported.

  • IP traffic needs to exist redirected through Proxy Server champion IPX to IP gateways.

  • Clients should utilize the local address table (LAT) to determine the destination IP addresses.

  • Define default gateway champion for the following reasons:

  • Define Microsoft Internet Explorer 5.0 champion for the following reasons:

  • All operating systems that comprise Internet Explorer 5.0 should exist supported.

  • Only HTTP and FTP traffic will pass through Proxy Server.

  • The Internet Explorer Administrator Kit (IEAK) is to exist used to administer Proxy Server client configuration.

  • Define SOCKS champion for the following reasons:

  • Support for Unix and Macintosh is ensured.

  • All operating systems that utilize SOCKS measure should exist supported

  • IPs supported by SOCKS applications should exist redirected.

  • Another planning component that should exist included when you design your Proxy Server implementation is to determine the smooth of data protection that should exist configured.

  • Inbound and outbound packet filters can exist configured to filter and restrict traffic, based on the criteria defined for the different IP traffic types.

  • Domin filters can exist configured to restrict Internet access to only unavoidable IP addresses or FQDNs. In a domain filter, you can comprise a number of Internet sites and then define the action that the domain filter should entangle when a request is received for one of these sites: Reject packets for these specific Internet sites and forward everything other packets OR forward packets to these specific Internet sites and reject everything other packets. Domain filters can restrict outbound traffic, based on a separate computer or the IP address of a cluster, an IP address sweep or a FQDN.

  • You can utilize Proxy Server user authentication to specify Internet access, based on user or group account.

  • Through Web publishing, you can restrict inbound traffic based on the URL requests of Internet users.

  • The default configuration of Proxy Server is to drop the URL requests of Internet users. This means that Internet users conclude not beget access to Web and FTP servers hosted within the private network, by default. You can though define URLs where requests for these URLs should exist passed to Web and FTP servers on the private network. Proxy Server will allow URL requests when you define them in the Web Publishing list.

    For URLs that are requested which are defined in the Web Publishing list, Proxy Server passes the requests to the Web and FTP servers on the private network.

    For URLs that are requested which are not defined in the Web Publishing list, Proxy Server performs either of the following:

    There are also a number of techniques that optimize Proxy Server performance, which you should reckon implementing:

  • Caching Web content improves performance. Cached information is accessed by users from a location on the Local zone Network (LAN). This means that bandwidth utilization to the Internet ends up being lowered because cached information does not exigency to exist downloaded from the Internet. everything of this leads to an improvement in the service experienced by users.

  • Proxy Server also provides a feature called proxy arrays. A proxy array is a solution whereby one or multiple proxy servers operate as a separate cache for client requests. Benefits provided by the proxy array feature comprise scalable performance, and fault tolerance.

  • Network Load Balancing (NLB) can exist used to distribute the processing load of inbound traffic over multiple proxy servers. This leads to tall availability and performance optimization.

  • Round Robin DNS can also exist used to load equipoise inbound traffic across multiple proxy servers, thereby also providing tall availability and performance optimization.

  • The advantages of using proxy arrays as a Proxy Server optimization route when you implement Proxy Server are listed here:

  • Because Web content is cached over multiple servers, no separate server hosts everything Web content.

  • If a server in the proxy array fails, failover is immediately provided.

  • The advantages of using Network Load Balancing (NLB) as a Proxy Server optimization route when you implement Proxy Server are listed here:

  • You can add or remove proxy servers residing in the NLB cluster.

  • Load balancing occurs dynamically over everything proxy servers residing in the NLB cluster.

  • Because load balancing and the addition or removal of proxy servers occurs dynamically, availability and performance is improved.

  • The NLB cluster is automatically reconfigured when a proxy server happens to fail.

  • The advantages of using Round Robin DNS as a Proxy Server optimization route when you implement Proxy Server are listed here:

  • Load balancing is performed on everything proxy servers in the round robin DNS.

  • Round Robin DNS can operate on everything operating system platforms.

  • Performance is improved because traffic is basically load balanced over everything proxy servers.

  • If you exigency to provide the highest feasible smooth of server availability for your Proxy Server implementation, you should consume Microsoft Windows Clustering. Using Microsoft Windows Clustering provides the following benefits for your Proxy Server implementation:

  • The Proxy Servers everything partake a common cache.

  • If a server in the proxy array fails, failover is immediately provided.

  • Because the cache does not exigency to exist built again when a server fails, restore occurs quite faster.

  • To optimize Internet access, you can comprise the following Proxy Server caching methods in your Proxy Server design:

  • As mentioned previously, with passive caching, Proxy Server stores objects in the Proxy Server cache with each remonstrate obtaining a Time To Live (TTL) value. Before Proxy Server forwards requests to the Internet, it first checks the Proxy Server cache to determine if the request can exist serviced from there. When the Proxy Server cache becomes full, Proxy Server removes objects from the cache, based on a combination of factors: remonstrate size, remonstrate age, and remonstrate popularityThe advantages of using passive caching in your Proxy Server implementation are:

  • With energetic Caching, Proxy Server automatically generates requests for specific objects in the Proxy Server cache so that frequently requested objects remain cached. Proxy Server determines which objects should exist flagged for energetic caching by considering remonstrate popularity, Time To Live (TTL) value of objects, and server load to determine the smooth of energetic caching performed.The advantages of using energetic caching in your Proxy Server implementation are:

  • Determining Proxy Server Hardware and Software Requirements

    Proxy Server has a few minimum hardware and software implementation requirements. However, depending on the size of the organization, existing hardware and software, future network expansion, and expected traffic volumes; the Proxy Server implementation requirements between organizations would differ. For each different network environment, there are different requirements for a Proxy Server implementation.

    The requirements listed below merely serves as a guideline on the hardware requirements for a Proxy Server implementation:

  • Processor; Intel 486 or faster supported RISC-based microprocessor

  • Disk space; 10 MB available disk space for Proxy Server

  • For caching; 100 MB plus an additional 0.5 MB for each Web Proxy service client.

  • RAM; at least 24 MB. For RISC-based systems, this increases to 32 MB.

  • An NTFS formatted partition to store the Proxy Server cache.

  • A network adapter card for connection to the LAN.

  • A network interface configured for the Internet.

  • When planning a Proxy Server implementation, you beget to select on the hardware that you will used to establish connections to the Internet:

  • ISDN lines can exist used to establish connections to the Internet. ISDN is a digital dial-up service that utilizes telephone cabling and other technology to provide Internet connections. The different types of ISDN services are ISDN Basic Rate Interface (BRI) and ISDN Primary Rate Interface (PRI).The main characteristics of ISDN Basic Rate Interface (BRI) are listed here:

  • BRI connections drudgery well for minuscule companies

  • BRI connections are available from quite a number of telephone companies.

  • ISDN BRI can tender 128 Kbps of bandwidth.

  • Provide e-mail for a maxmum of 20 concurrent users.

  • Provide large FTP downloads for only 3 to 4 simultaneous users.

  • Provide Web browsing for 6 to 8 concurrent users.

  • The main characteristics of ISDN Primary Rate Interface (PRI) are listed here:

  • ISDN PRI can tender 1.544 Mbps transmission speed.

  • Provide e-mail for a maximum of 120 concurrent users.

  • Provide large FTP downloads for only 40 to 50 simultaneous users.

  • Dial-up modem connections are exemplar if your organization only consists of a minuscule number of users that conclude not exigency to connect to the Internet on a regular basis. This is due to dialup modem connection only being able to meet the bandwidth requirements of a minuscule number of users. Modems can exist installed on a computer, and then shared through the Windows Internet Connection Sharing (ICS) service.A few characteristics of dial-up modem connections are:

  • A dial-up modem connection can only compass up to 53 Kbps.

  • Provide e-mail for a maximum of 10 concurrent users.

  • Provide large FTP downloads for only 1 to 2 simultaneous users.

  • Provide Web browsing for 2 to 3 concurrent users.

  • You also beget to select on the hardware which will exist utilized to connect the server to the Internet:

  • Analog modem: Analog modem escape at 28.8 or 33.6 Kbps speeds. An analog modem is exemplar for a separate user connecting to the Internet, and for a networked server gateway.

  • ISDN adapters: This is the current choice. The ISDN adapters dial an ISDN access number and then maintain the particular connection.

  • Routers: Routers are networking devices that connect networks.

  • Installing Proxy Server

    You should verify a number of things before you actually install Proxy Server:

  • 10 MB available disk space for Proxy Server and 100 MB plus an additional 0.5 MB for each Web Proxy service client.

  • An NTFS formatted partition to store the Proxy Server cache.

  • TCP/IP should exist installed on the computer.

  • The internal network interface should exist bound to the TCP/IP or IPX/SPX protocol being used on the LAN.

  • You should configure the software for two network adapter cards before you attempt to install Proxy Server.

  • When Proxy Server is installed, the following changes are made to the computer on which you are installing it:

  • The Web Proxy service is installed.

  • The WinSock Proxy service is installed.

  • The Socks Proxy service is installed.

  • Each of these services is added to the Internet Service Manager administration tool.

  • The local address table is installed.

  • On the NTFS volume, the cache drive is created.

  • The client installation and configuration software is copied.

  • The Mspclnt shared folder is created.

  • The Proxy Server Performance Monitor counters are installed.

  • The HTML online documentation is installed.

  • How to install Proxy Server
  • On the Proxy Server installation CD, proceed to escape Setup.

  • Click Continue on the Welcome to the Microsoft Proxy Server Installation program screen.

  • The Microsoft Proxy Server Setup page opens.

  • Specify the 10-digit product key provided on the CD-ROM case. Click OK.

  • The Microsoft Proxy Server Setup dialog box displays the default destination folder and the Installation Options button. Click the Installation Options button.

  • The Microsoft Proxy Server – Installation Options dialog box opens, displaying everything components as being selected. Click Continue.

  • Setup now stops the Web services.

  • The Microsoft Proxy Server Cache Drives dialog box opens. Caching is by default enabled.

  • The local drives of the server are listed in the Drive box.

  • Select the drive which should exist used to store cached data. In the Maximum Size (MB) box, enter th confiscate value. Click Set, and then click OK.

  • The Local Address Table Configuration dialog box opens.

  • Click the Construct Table button.

  • The Construct Local Address Table dialog box opens.

  • Select Load from NT internal Routing Table to select the network adapter cards thats IP addresses must exist added to the local address table.

  • Select the Load known address ranges from the following IP interface cards option, and then select the network adapter. Click OK.

  • Click OK to avow the message displayed, indicating that the IP addresses beget been loaded into the local address table.

  • The Local Address Table Configuration dialog box opens, displaying IP addresses in the Internal IP Ranges box.

  • Check that the addresses defined are correct, and then click OK.

  • The Client Installation/Configuration dialog box opens.

  • Enter the confiscate information and verify that the revise computer denomination is displayed in the Computer denomination realm and Proxy field.

  • If you enable the Automatically configure Web browser during client setup checkbox, the Web browser network configuration setting of the client is changed so that client requests are sent to the Proxy Server, and not to the Internet.

  • Click Configure.

  • You can either escape the default script to configure the client Web browser, or alternatively, you can consume a custom URL.

  • Click Properties located beneath Browser automatic configuration script.

  • The Advanced Client Configuration dialog box opens.

  • Specify whether the Proxy Server is utilized for local servers.

  • Specify the IP addresses that should exist excluded from Proxy Server.

  • Specify the domains that should exist excluded from Proxy Server.

  • Specify a backup to the proxy server.

  • Click OK.

  • The Access Control dialog box opens.

  • Verify that access control is specified for the Web Proxy service and for the WinSock Proxy service and then click OK.

  • The Proxy Server Setup files are copied to the computer.

  • When the Setup Information dialog box opens, click OK. The Setup Information dialog box displays information on the packet filtering feature. The packet filtering feature is not automatically enabled when Proxy Server is installed. Click OK.

  • A Proxy Server 2.0 Setup was completed successfully message is displayed.

  • How to install WinSock Proxy Client on client computers

    When you install WinSock Proxy Client on client computers, the following changes are made:

  • The Proxy Client program group is created

  • The local address table file, Msplat.txt, is installed on the client. Proxy Server will update this file.

  • Mspclnt.ini is also copied to the client.

  • The WSP Client icon is added to Control Panel. This only occurs for Windows 3.x, Windows 95, and Windows NT clients.

  • Remote WinSock from WinSock Proxy Client replaces Winsock.dll. This would enable the computer to only access Internet sites using the WinSock Proxy service.

  • To install WinSock Proxy Client on a client computer;

  • Open Internet Explorer

  • In the Address box, enter http://proxycomputername/msproxy.

  • The WinSock Proxy Client 2.0 Installation page is displayed.

  • To install WinSock Proxy Client, click WinSock Proxy 2.0 client.

  • Click the Open it option and click OK.

  • The Microsoft Proxy Client Setup dialog box opens.

  • Click Continue to proceed with the installation.

  • Click Install Microsoft Proxy Client to start copying Setup files to the client computer.

  • Click OK.

  • The Setup – Restart System dialog box opens.

  • Click the Restart Windows Now option.

  • How to add or remove Proxy Server components
  • On the Proxy Server installation CD, proceed to escape Setup.

  • Click Add/Remove on the Setup screen.

  • Follow the instruction displayed to add or remove Proxy Server components.

  • How to restore Proxy Server settings or files
  • On the Proxy Server installation CD, proceed to escape Setup.

  • Click Reinstall on the Setup screen.

  • Follow the instructions displayed to restore Proxy Server settings/files.

  • How to remove Proxy Server from the server
  • On the Proxy Server installation CD, proceed to escape Setup.

  • Click Remove everything on the Setup screen.

  • Click Yes to avow that you want to remove Proxy Server.

  • Proxy Server is then removed from the server.

  • How to disable WinSock Proxy Client
  • Open Control Panel.

  • Double-click WSP Client.

  • Deselect the Enable WinSock Proxy Client checkbox.

  • Restart the computer.

  • How to re-enable the WinSock Proxy Client
  • Open control Panel.

  • Double-click WSP Client.

  • Check the Enable WinSock Proxy Client checkbox.

  • Restart the computer.

  • Administering Proxy Server using the Internet Service Manager

    You can consume the Internet Service Manager to configure properties for the Web Proxy, WinSock Proxy, and Socks Proxy services of Proxy Server.

    To open the Internet Service Manager;

  • Click Start, click Programs, click Microsoft Proxy Server, and then click Internet Service Manager.

  • You can open the properties of specific Proxy Server service by double-clicking the computer denomination displayed alongside the particular service name.

  • There are some properties settings which are common for everything three Proxy Server services, and there are others that are apposite for only a particular Proxy Server service. This concept is illustrated here.

  • The properties settings which can exist configured for the Web Proxy service are listed here:

  • Service

  • Permissions

  • Caching

  • Routing

  • Publishing

  • Logging

  • The properties settings which can exist configured for the WinSock Proxy service are listed here:

  • Service

  • Permissions

  • Protocol

  • Logging

  • The properties settings which can exist configured for the Socks Proxy service are listed here:

  • Service

  • Permissions

  • Logging

  • The configuration settings which you can view and configure on the Service tab for each of the three Proxy Server services are listed below:

  • View the product release.

  • Verify the product ID.

  • Add additional information on the service.

  • Add additional information on the server.

  • View the current sessions.

  • Navigate to the Shared services tabs.

  • Navigate to the Configuration tabs.

  • The configuration settings which you can view and configure on the Permissions tab for the Web Proxy and WinSock Proxy services are listed below:

  • Select or disable the Enable access control checkbox.

  • Select the Protocol when defining user or group permissions. Permissions are basically assigned for each protocol.

  • Define user and group permissions for using the Internet protocols.

  • The configuration settings which you can view and configure on the Permissions tab for the Socks Proxy are:

  • Specify the source and destination for an entry, and then define whether requests should exist allowed or defined.

    The configuration settings which you can view and configure on the Caching tab for the Web Proxy and service is listed here:

  • Select the Enable caching checkbox and then select between the following Cache expiration policy options:

  • Select the Enable energetic caching checkbox and then select between the following options:

    Faster user response is more important.

  • The configuration settings which you can view and configure on the Routing tab for the Web Proxy and service is listed here:

  • For upstream routing, you can select between the following options:

  • Use direct connection.

  • Use Web proxy or array.

  • If you select the Enable backup route checkbox, you can select between the following options:

  • Use direct connection.

  • Use Web proxy or array.

  • You can also select the Resolve Web proxy requests within array before routing upstream checkbox on this tab.

  • The configuration settings which you can view and configure on the Publishing tab for the Web Proxy and service is listed here:

  • Enable/disable Web publishing.

  • Configure computers to publish information on the Internet via the Proxy Server.

  • Specify what should occur to incoming Web server requests:

  • The configuration settings which you can view and configure on the Logging tab for everything three Proxy Server services are listed below:

  • Enable/disable logging. When enabled, the following types of information will exist logged:

  • Server

  • Client

  • Connection

  • Object

  • Specify the Log to file option, or the Log to SQL/ODBC database option.

  • Specify when a modern log should exist opened.

  • Specify the log file directory.

  • How to disable IP routing (control access to the private network)
  • Open Control Panel

  • Double-click Network.

  • The Network dialog box opens.

  • Click the Protocols tab.

  • Select TCP/IP, and click Properties.

  • The TCP/IP Properties dialog box opens.

  • Switch to the Routing tab.

  • Ensure that the Enable IP Forwarding checkbox is not selected (blank).

  • Click OK.

  • How to configure publishing configuration settings for the Web Proxy service
  • Open Internet Service Manager.

  • Double-click the computer denomination alongside the Web Proxy service.

  • The Web Proxy Service Properties dialog box opens.

  • Click the Publishing tab.

  • Select the Enable Web publishing checkbox.

  • If you want to drop everything incoming Web server requests, click the Discard option.

  • If you want to forward everything incoming Web server requests to IIS on the Proxy Server computer, click the Sent to the local Web server option.

  • If you want forward everything incoming Web server requests to a specific downstream server, click the Sent to another Web server option.

  • If you want to configure the default Web server host, click Default Mapping.

  • The Default Local Host denomination dialog box opens.

  • Provide the denomination of the default server. Click OK.

  • Click Apply and click OK.

  • How to enable dynamic packet filtering
  • Open Internet Service Manager.

  • Double-click the computer denomination alongside the Web Proxy service.

  • The Web Proxy Service Properties dialog box opens.

  • Click the Security button on the Service tab.

  • Click the Packet Filters tab.

  • On the Packet Filters tab, click the Enable packet filtering on external interface checkbox.

  • Select the Enable dynamic packet filtering of Microsoft Proxy Server packets checkbox.

  • Click OK.

  • Click OK in the Web Proxy Service Properties dialog box.

  • How to create a packet filter using predefined protocol definitions
  • Open Internet Service Manager.

  • Double-click the computer denomination alongside the Web Proxy service.

  • The Web Proxy Service Properties dialog box opens.

  • Click the Securty button on the Service tab.

  • Click the Packet Filters tab.

  • On the Packet Filters tab, click Add.

  • When the Packet Filter Properties dialog box opens, click the Predefined filter option.

  • Select a protocol from the available Protocol ID list.

  • In the Local host zone of the Packet Filter Properties dialog box, select the confiscate option to allow packet exchange with a host.

  • In the Remote host zone of the Packet Filter Properties dialog box, specify one host or the Any host option.

  • Click OK.

  • How to create a packet filter using custom protocol definitions
  • Open Internet Service Manager.

  • Double-click the computer denomination alongside the Web Proxy service.

  • The Web Proxy Service Properties dialog box opens.

  • Click the Security button on the Services tab.

  • Click the Packet Filters tab.

  • On the Packet Filters tab, click Add.

  • When the Packet Filter Properties dialog box opens, click the Custom filter option.

  • Select a protocol from the available Protocol ID list.

  • Select a direction from the Direction list.

  • Select an option from the available options in the Local port area.

  • Select either the Any option or Fixed port option in the Remote port area.

  • In the Local host zone of the Packet Filter Properties dialog box, select the confiscate option to allow packet exchange with a host.

  • In the Remote host zone of the Packet Filter Properties dialog box, specify one host or the Any host option.

  • Click OK.

  • How to change the packet filter list entries
  • Open Internet Service Manager.

  • Double-click the computer denomination alongside the Web Proxy service.

  • The Web Proxy Service Properties dialog box opens.

  • Click the Security button on the Service tab.

  • On the Packet Filters tab, click the Enable packet filtering on external interface checkbox.

  • Select the Enable dynamic packet filtering of Microsoft Proxy Server packets checkbox to enable dynamic packet filtering.

  • Click the Edit button.

  • The Packet Filter Properties dialog box opens. Change the necessary settings and then click OK.

  • If you want to remove a filter, click the Remove button.

  • Click OK.

  • How to configure Proxy Server logging
  • Open Internet Service Manager.

  • Double-click the computer denomination alongside the Web Proxy service.

  • The Web Proxy Service Properties dialog box opens.

  • Click the Security button on the Service tab.

  • Click the Logging tab.

  • Click the Enable logging using checkbox.

  • Select the confiscate format in the Format list box.

  • Click OK.

  • How to back up a Proxy Server configuration
  • Open Internet Service Manager.

  • On the View menu item, click Servers View.

  • Double-click the computer name, and then double-click Web Proxy (Running).

  • The Web Proxy Service Properties opens.

  • Click the Service tab.

  • In the Configuration area, click Server Backup.

  • When the Backup dialog box opens, verify the information shown on where the backup file will exist saved

  • Click OK to create a back up of the Proxy Server configuration.

  • How to restore a Proxy Server configuration
  • Open Internet Service Manager.

  • Double-click the computer name, and then double-click Web Proxy service.

  • The Web Proxy Service Properties opens.

  • Click the Service tab.

  • In the Configuration area, click Server Restore.

  • When the Restore Configuration dialog box opens, click the Browse button to select the Proxy Server configuration file.

  • Select the Proxy Server configuration file that you want to consume for the restore.

  • Click Open.

  • Select the plenary Restore option.

  • When the Restore Configuration dialog box opes, click OK to start the restore of the Proxy Server configuration.

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