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The North Eastern Railway's (NER) development of express passenger locomotives was held up by
World War 1, but resumed in 1921 with development of the very successful
Raven Atlantic C7.
Greater power and acceleration was required for the increasing loads on the East Coast Main Line, and
hence Raven developed the
C7 into a Pacific.
Authorisation to build the first two locomotives was received at Darlington on 30th March 1922.
Line drawings were published July 1922 to counter the publicity of the appearance of the
GNR's first Pacific.
Both were recorded as delivered by December 1922 in time for Grouping (1923), although No. 2400 was still
in shop grey and No. 2401 did not run until January 1923.
Raven created the A2 by stretching his
C7 design. The cylinder size was
increased, as was the boiler diameter. A wide firebox was fitted which spread out over the
trailing wheels. Although the grate was slightly shortened, this wider firebox had to be behind the
driving wheels resulting in a further increase in length.
The length of the resulting engine proved to be a significant restriction on route availability.
To counteract the adverse effects of longer boiler tubes, some of the tubes were increased in diameter.
Boiler pressure was increased from 175psi to 200psi.
The three cylinder drive on the front coupled axle was kept from the
C7 design.
The valve arrangements and steam passages were unchanged, and were a handicap to the required handling
of large volumes of steam.
The choice of three sets of independent Stephenson valve gear (contrast to
the A1's conjugated gear) had to be fitted in a confined space which
resulted in limited bearing sizes. This was satisfactory for the
C7, but the bearings on the A2 valve gear
were expected to handle significantly more power.
As can be seen, the A2s had a number of problems. The A2 was the biggest engine the NER built, but
few could claim it was their best. Opinion at the time of development was that the
A2s were rushed so that they would be completed before the NER ceased to exist.
It should also be remembered that Raven
had different thoughts as to how the East Coast Main Line passenger expresses should be developed.
Concurrent to the A2 development, Raven
was also trying to build a 4-6-4 electric locomotive to haul 450 ton passenger trains at up to 90mph.
One of the big "What ifs" of British railway history, is how things would have developed if
Raven's electrification plans for the
East Coast Main Line had come to fruition in the 1920s rather than the 1980s.
Results with the first two engines were satisfactory, and three more were ordered in
February 1923 - possibly in reaction to
Gresley's appointment as CME of the
newly formed LNER. However, before construction had begun,
Gresley modified the rear bogie
to have A1-style rear bogies.
There was little difference between the A1 and A2 when they were tested
in 1923. The main difference was that the A2 did a better job of maintaining full boiler pressure.
However, in October 1923 the future of the A2 was pretty much decided when 40
A1s were ordered.
The A1s were considered a better design and had more scope for further
development (eg. with better valve settings).
The A2s were originally allocated to Gateshead before being moved to York in 1934. With the
exception of a few visits to Kings Cross in 1923 for the trials, they spent their first 10 years between
Grantham and Edinburgh, with some runs to Leeds. Whilst at York, they mainly ran heavy secondary
express passenger trains on the East Coast Main Line, although they also occasionally ran freight
and mail trains.
From September 1929, No. 2404 was fitted with a modified A1 boiler.
Due to the small size of the class, it was not practical to build a new set of boilers, so a
spare A1
boiler was used instead. Both the new boiler and the locomotive had to be modified. The firebox was
extended to fit the longer A2, and the A2 footplate had to be made wider.
In 1933, the use of A3 boilers was considered, although none of the A2s
were eventually fitted with them. Also in 1933, standard
Gresley 8-wheeled non-corridor tenders
were ordered. These replaced the original NER 6-wheeled tenders in late 1934. The NER tenders were
fitted to newly built J39s.
A combination of their limited route availability, and the need for new boilers at about
the time the first
V2s appeared, led to withdrawal between
1936 and 1937.
Four of the original boilers were scrapped at the same time, but the fifth (from No. 2400) was
converted into a stationary boiler in 1937 for use at Darlington. It was scrapped in May 1939.
No. 2402 was the first locomotive built by the LNER to be scrapped.
The following technical details are for the A2s at Grouping in 1923.
None of the A2s survived into preservation.
In the past, DJH have produced a kit of the Raven A2 for OO gauge (4mm scale) but this is no longer available.
ACE Products sell a kit of the A2 for O gauge (7mm scale).
Thank you to Morgan Gilbert for the photograph of his finished model of No. 2400.
Thank you to Malcolm Peirson for the photographs of No. 2402 City of York and
No. 2404 City of Ripon.


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Wednesday, September 11, 2013
On Thursday, Sept. 12, Charleston Animal Society will host its 2014 Firefighter Calendar Debut Party at Woolfe St. Playhouse, 34 Woolfe St. in downtown Charleston. Sponsored by Little Black Dress and Icebox, the festivities begin with a VIP reception at 6 p.m. Doors open for General Admission at 7 p.m., with the party slated to kick off at 8 p.m. Comedian Travis Howze will serve as emcee. The "Celebrity Judging Event" party to select models for the calendar was held in May at the Music Farm and was the talk of the town. This party is sure to be at just as "hot."
Calendars will be available at a 10 percent discount that evening for ticket holders. All proceeds from the party and subsequent calendar sales will go to "Toby's Fund," Charleston Animal Society's Medical Fund, which treats the community's injured, abused and abandoned animals.
Autographed calendars signed by all the models will be available at the party for a discounted price of $30. All autographed calendars purchased after the event will be $50. There will also be an auction for dates with some of the models at local venues such as FISH Restaurant, Hall's Chophouse and Middleton Place.
Tickets for the event are already selling like "wildfire". VIP runway access packages sold out almost immediately. Still available are $85 VIP packages for one, which include one 2014 Calendar ($20 value), access to the VIP Cocktail Reception from 6-7:30 p.m. as well as access to the VIP area during the course of the event, "meet and great" time with calendar models, including photo and autograph opportunities and one complimentary drink. Some $30 standing room only General Admission tickets also remain. General admission ticket holders get half off their first calendar purchased and 10 percent off any additional calendar purchases at the party.
More information on and ticketing information for the event can be found at: http://firefightercalendar.eventbrite.com.
Tickets are also be available at Little Black Dress, 93 Society St. Only cash or checks accepted at Little Black Dress.
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subset: commoncrawl


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Gear Review
Mark Travers
Sweet shirt, runs true to size
Love this shirt! Unlike some of the other reviews, I found that this runs true to size. I usually wear a large (6'1" 210#), and this shirt in a large is spot-on. The fabric is a little bit heavier than some of the other synthetic shirts out there - I found that the weight and feel of this shirt is more like cotton, but still breathes like synthetic. Great combo!
View More Details on Backcountry Beater T-Shirt - Short-Sleeve - Men's


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Obituaries, July 1, 2014
Thomas Riley Gilmer
DURHAM — Mr. Thomas Riley Gilmer, Sr, 85, of 205 W. Weaver St., died Saturday, June 28, 2014 at his home. His service will be conducted at 12pm on Thursday, July 3, 2014 at First Calvary Baptist Church, 1311 Morehead Ave. Interment will follow in Beechwood Cemetery. The family will receive friends at the church on Thursday from 11am-12pm one hour prior to the service.
Mr. Gilmer is survived by his wife, Mary G. Gilmer, and his children, Thomas Gilmer, Gathea Peaks, Elaine Little, Sherron Williams and Harold Brooks.
Arrangements by Burthey Funeral Service
Marjorie Stohlman Leimbach
DURHAM — Marjorie Stohlman Leimbach of Durham passed away on Saturday, June 28, 2014 while at the Hock Family Pavillion. She was born in Cleveland, OH to the late Mr. & Mrs. Gordon C. Stohlman. She attended Cleveland Institute of Art and was an accomplished artist. She was a member of St. Paul's Lutheran Church where she was active in the Altar Guild and Katherine Circle. She was a long time member of Umstead Pines Golf Club where she enjoyed golf, tennis and the Women of Willowhaven (Garden Club). She loved being with her grandchildren and enjoyed gardening, especially raising orchids in her greenhouse. She was a Red Cross Bloodmobile volunteer for 15 years. She was preceded in death by her husband Richard Charles Leimbach after 56 years of marriage.
She is survived by her daughter Lori Leimbach Eberhardt and husband Bob of Mebane, N.C., her son Jim Leimbach and wife Linda, grandchildren Sydney and Emily Leimbach of Atlanta, GA, sister Carolyn Jacobson of Fairview Park, Ohio, sister-in-law Marguerite Bichsel of Dunedin, FL. , nieces Linda Khan, Marcia Rice and nephews Jack Bichsel and David Bichsel.
The Memorial service will be held on Sunday, July 20, 2014 at 2:00 p.m. at St. Paul's Lutheran Church. After the service the family invites everyone to a reception at the church.
In lieu of flowers donations may be made to: St. Paul's Lutheran Church, 1200 W Cornwallis Rd., Durham, NC 27705 or Duke Hospice – Hock Family Pavillion, 4321 Medical Park Drive Suite 101, Durham, NC 27704.
The family would like to express special thanks to Mary Wearins her caregiver, friends, neighbors and the staff of Duke Homecare and Hospice.
Online condolences can be made at www.hudsonfuneralhome.com
Carl Thomas Lyons
DURHAM — Mr. Carl Thomas Lyons, 70, passed on June 29, 2014 at Duke University Medical Center of Durham.
Surviving relatives include his loving mother, Nannie Pearl Daye Lyons; two sisters, Geraldine Lyons and Lynda P. Lyons Thomas.
Funeral service will take place at Mt. Level Missionary Baptist Church, 316 Hebron Road
Durham, NC 27704 beginning at 1 p.m. A visitation with family and friends will be held one hour prior to the service.
Arrangements are entrusted to Scarborough and Hargett Memorial Chapel and Gardens. Online condolences may expressed at ScarboroughandHargettFh.com.
Tracy Weaver
DURHAM — Tracy Weaver, 52, departed his life on June 26, 2014.
He leaves to cherish his memory his lovely wife Jonia Weaver and his son Isaac Weaver.
Viewing and funeral will be held at Mt. Zion Christian Church, 3519 Fayetteville St. on Thursday, July 3, 2014 , viewing 10:00 am, funeral 11:00am.
Arrangements entrusted to Holloway Memorial Funeral Home.
Lydia Marie Coleman Ferrell
ELBERTON, GA — Mrs. Lydia Marie Coleman Ferrell, 81, of Elberton, Georgia passed away Friday, June 27, 2014 at Elbert Memorial Hospital.
Mrs. Ferrell was born in Durham, North Carolina to the late Luther and Lola M. Coleman. She was the wife of Rev. Arthur Kenneth Ferrell, Sr.
In addition to her husband, Mrs. Ferrell is survived by two sons: Arthur Kenneth Ferrell, Jr., and Reid Ferrell and his wife, Tina. She also leaves behind four grandchildren and four great-grandchildren.
She was preceded in death by a brother, Luther Coleman and his wife, Bobbie.
A memorial service was held Sunday, June 29, 2014 at Falling Creek Baptist Church with Rev. Dwight Morgan and Rev. Jay Akin officiating. Interment was at Forest Hills Memorial Park.
Memorials may be made to T.J. & Friends Cancer Foundation, P.O. Box 6161, Elberton, GA 30635.
Online condolences may be expressed to the family at www.hicksfuneral.com.
Hicks Funeral Home of Elberton is caring for the family of Mrs. Lydia Marie Coleman Ferrell.


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Drupal: Fix for Disappearing Taxonomy Term in Views Filter
We've been using Drupal more and more here at Forum One. One of its nicer aspects is a web interface to some functionality we had for programmers in SyntaxCMS: the ability to set up a "view" of a content type, so that you only got, say, all blog posts tagged with "Environment." This is pretty powerful; it lets a savvy Internet user with no specific programming skills to set up lots of functionality by clicking a few widgets. One such user asked me what I thought was an odd question: "Any idea why one of my terms for the issue taxonomy isn't showing up in filter for a view?"
I checked, and sure enough, the first term was missing. It happened to be the longest term, and I immediately hypothesized some sort of character limit. So I tested it by shortening the term. No joy. I then wondered if there were any illegal words, so I tried eliminating "and" from the term. Still nothing. I began to suspect it something about the position or some permission around the term. I deleted and re-added the term. Still nothing.
At this point I tried adding a term that came alphabetically before the missing term. Success! All but the new term showed up. But now I had an extraneous term hanging around. Having eliminated configuration errors as a cause, I went to the Views module page, and made sure I had the latest released version for the version of Drupal I was using (5.7) (We make it a policy never to rely on beta or dev versions). I did (1.6 as of this writing). So I started looking at the bug list to find out if anyone had reported this issue–it certainly seemed big enough that someone ought to have noticed.
Lo and behold, I found this bug. Turns out it had been discovered in beta testing and a patch applied. But I was using the latest release version…reading further, I found out a change in the taxonomy module itself had obsoleted the fix and a new patch issued, tested, and committed to CVS. It is in the 1.7 pre-release versions, but as I mentioned before, we don't use pre-release software if any alternative is available. However, knowing the patch is applied in the next version siginificantly reduces the effects of applying the patch now. So now I have the patch applied and committed to my local repository for the site.
So if you're having problems with disappearing taxonomy terms when building out a view, the patch above should fix it for you for Drupal 5.7 and Views 1.6.


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Forecaster Forum: Who's drinking the school consolidation Kool-Aid?
According to Edgar Allen Beem ("The Universal Notebook," June 8), school leadership at School Administrative District 51 "could have (like the Yarmouth schools) sought high-performing exemptions" from school consolidation "had their educational leaders not drunk the consolidation Kool-Aid."
Frankly, as the SAD 51 board chairwoman during this period, I am not sure who is drinking the Kool-Aid.
Beem may be correct that the consolidation legislation is doomed, but not for the reasons he gives: "no educational benefits" and "no financial savings." If consolidation is repealed this November, it will be the result of a number of factors, including the adoption by the Legislature of a nonsensical and misunderstood school budget approval process. The unrelated and mandated change to the budget process, together with Gov. Baldacci's false promotion of immediate savings, may have doomed the effort from the start.
The final blow, however, is most likely the combination of the governor's unwillingness to bend on the time-line and the Legislature's inability to withstand political pressure from influential legislators seeking piecemeal exemptions for towns and islands like Yarmouth, Cape Elizabeth, Vinalhaven, York and Vassalboro.
Let's take a look at the facts:
Well before the governor floated his consolidation proposal, the leadership of SAD 51, Yarmouth, Falmouth, Freeport and Pownal were already meeting to consider the educational and financial benefits of consolidation. This group commissioned a study in 2006 by the New England School Development Council that showed the potential for more than $1 million in annual savings.
So really, who is drinking the Kool-Aid?
Immediately before passage and without consultation beyond a small group of legislators (which included the Gorham representative, a Yarmouth High School teacher), the consolidation law was modified to exempt Yarmouth, under the so-called exemption for "efficient, high-performing" schools. This exemption was not taken from any pre-existing definition, but was one that was designed by those who sought a way out of consolidation.
"Efficient, high-performing schools" are determined by applying two factors: having at least three schools identified as "higher performing" in the 2007 report of the Maine Education Research Policy Institute and reporting by individual districts that their 2005-2006 per-pupil expenditures for system administration represent less than 4 percent of all per-pupil expenditures.
Greely High School and Greely Middle School are classified as higher performing schools under the MERPI report. By all measures SAD 51 is an excellent school system but elementary school buildings that do not serve students in both 3rd and 4th grades, which those in SAD 51 do not, were excluded from MERPI report and so did not receive any rating at all. Without a third school rated as higher performing under the MERPI report, SAD 51 was not eligible for this exemption regardless of how high its quality of education was.
Beem also highlights that Yarmouth was exempt because its "administrative costs are low." In adopting the second part of the Legislature's design-based exemption, the Legislature failed to take into account overall expenditures. For example, Yarmouth spent $11,840 per pupil in 2008-2009 while SAD 51 spent $10,339 – a difference of $1,501 per student. If SAD 51 were to spend what Yarmouth did, the SAD 51 school budget would have been $3.3 million higher.
No one disputes Yarmouth's educational excellence. Yarmouth's student enrollment, however, is not expected to grow. Hence, contract commitments alone (without the elimination of program offerings) will result in higher per-pupil spending.
As we move forward, I hope that Yarmouth continues to attract community support for its schools and that Beem remains grateful to his school leadership for warning against consolidation. But please don't criticize those of your neighbors who were looking for a long-term solution that would slow inevitable cost increases and protect quality education.
Betts Gorsky, former chairwoman of the SAD 51 Board of Directors, lives in Cumberland Foreside.


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Description And Comments
The United States Pavilion, generally referred to as The American Adventure consists (not surprisingly) of a fast-food restaurant and a patriotic show.
The American Adventure production is a composite of everything Disney does best. Housed in an imposing brick structure reminiscent of colonial Philadelphia, the 29-minute show is a stirring, but sanitized, rendition of American history narrated by an audioanimatronic Mark Twain (who carries a smoking cigar) and Ben Franklin (who climbs a set of stairs to visit Thomas Jefferson). Behind a stage (almost half the size of a football field) is a 28 x 155-foot rear-projection screen (the largest ever used) on which motion picture images are interwoven with action on stage.
Though the production elicits patriotic emotion in some viewers, others find it overstated and boring. A man from Fort Lauderdale, Florida, writes:
I've always disagreed with you about The American Adventure. I saw it about ten years ago and snoozed through it. We tried it again since you said it was updated. It was still ponderous. Casey used the time for a nap, and I was checking my watch, waiting for it to be over. I'll try it again in 10 years.
An Erie, Pennsylvania, couple resented Disney's squeaky-clean version of American history:
Our biggest gripe was with The American Adventure. What was that supposed to be? My husband and I were actually embarrassed by that show. They glossed over the dark points of American history and neatly cut out the audio about who bombed Pearl Harbor (after all, Japan is right next door and everyone is happy at WDW). Why do they not focus on the natural beauty of America, the ethnic diversity, immigration, contributions to the world society? No, it's a condensed and Disneyfied history lesson that made us want to pretend to be Canadians after seeing it.
But an Iowa City, Iowa, father of three thinks a lot of people are missing the point:
Cramming all of American history into a 20-minute flick is no easy task, and face it, a theme park is hardly the place for a wholly objective, serious critique of the United States. I think that it's perfectly appropriate for the film, as an attraction in Epcot, to emphasize what's good about the United States.
Touring Tips
- Open for Evening Extra Magic Hours.
Architecturally, the U.S. Pavilion isn't as interesting as most others in World Showcase. But the presentation, our researchers believe, is the very best patriotic attraction in the Disney repertoire. It usually plays to capacity audiences from around 1:30 to 3:30 p.m., but it isn't hard to get into. Because of the theater's large capacity, the wait during busy times of day seldom approaches an hour, and averages 25 to 40 minutes. Because of its theme, the presentation is decidedly less compelling to non-Americans.
The adjacent Liberty Inn serves a quick, nonethnic, fast-food meal.
Attraction Photos
Special Comments
- We rate this attraction as Not To Be Missed.
Special Needs
Disney Dish with Jim Hill
One of the early ideas for The American Adventure was a flume ride, which had a section where guests floated past folk art representations of great Americans such as Eli Whitney and Harriet Beecher Stowe. But these animatronics were going to sing a 1970's pop version of Woody Guthrie's "This Land is Your Land," over and over. What's that? You're perfectly happy with the current version of The American Adventure? OK, then.
Other Attractions in World Showcase
- Agent P's World Showcase Adventure
- American Music Machine
- B'net Al Houwariyate
- British Revolution
- Canadian Lumberjacks
- Gran Fiesta Tour Starring The Three Caballeros
- IllumiNations: Reflections of Earth
- Impressions de France
- Mariachi Cobre
- Matsuriza
- O Canada!
- Paul McKenna Band
- Reflections of China
- Rose & Crown Pub Musician
- Sbandieratori Di Sansepolcro
- Sergio
- Serveur Amusant
- The Jeweled Dragon Acrobats
- Voices of Liberty
Touring Plans with The American Adventure
- Epcot One-Day Touring Plan for Active Seniors
- Epcot One-Day Touring Plan for Tweens and Their Parents
- Epcot One-Day Holiday Touring Plan for Parents with Small Children
- Epcot Late Arrival One-Day Touring Plan for Parents with Small Children
- Epcot Two-Day Touring Plan for Active Seniors - Day 2
- Epcot Late Arrival Selective One-Day Touring Plan for Adults
- Epcot Two-Day Late Arrival Touring Plan for Adults - Day 1
- Epcot One-Day Holiday Touring Plan for Adults
- Epcot World Showcase Afternoon Touring Plan
- Epcot Disney Cruise One-Day Highlights Touring Plan
- Ultimate Touring Plan: Epcot
- Epcot One-Day Touring Plan for Adults
- Epcot Unofficial Guide Two-Day Sunrise/Starlight Touring Plan - Sunrise
- Epcot One-Day Touring Plan for Parents with Small Children
- Epcot Early Riser Two-Day Touring Plan - Day 2
- Parent's Epcot Plan:1 Afternoon and 1 Full Day (Full Day)
- See all Touring Plans...
|Patriotic mixed-media and audio-animatronic theater presentation on U.S. history
|Our Rating
|Park is closed
|Grade School
|Young Adults
|Over 30
|World Showcase
|Opening Date
|October 1, 1982
|29 minutes
|Scope and scale
|Closures / Refurbishments
Inaccurate/stale data?
Let us know.


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Salmon with Smoked Bacon and Lentil Salad
- Total Time:
- 50 min
- Prep:
- 5 min
- Cook:
- 45 min
- Yield:
- 6 servings
- 1 1/2 pounds lentils
- 1/2 pound smoked bacon
- 4 tablespoons chopped scallions
- 1 tablespoon chopped garlic
- 2 ounces whole grain mustard
- 4 ounces lemon juice
- 6 ounces olive oil, plus 2 ounces
- 4 tablespoons flat-leaf parsley
- Salt and pepper, to taste
- 6 (8-ounce) pieces of salmon
Boil lentils for 25 minutes in salted water until tender cool and set aside. Saute bacon until crisp. Add scallions and garlic and saute until tender. Let cool then whisk in mustard, lemon juice and 6 ounces of olive oil. Fold dressing into lentils then add parsley, salt and pepper. Saute salmon in 2 ounces of olive until golden on each side and place in a preheated 400 degree oven until desired doneness about 8 to 10 minutes. Serve salmon on top of lentils.
Copyright 2000, Michael Symon, All Rights Reserved
Cooking Tips
More Recipes and Ideas
Salmon with Olive Vinaigrette
Recipe courtesy of Bobby Flay
Salmon with Lemon, Capers and Rosemary
Recipe courtesy of Giada De Laurentiis
Salmon With Warm Tomato-Olive Salad
Recipe courtesy of Food Network Kitchen


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The Observation Deck
A blog of opinions on local, state and national issues
Posts filtered on Tag
A smarter course for Shen
Our opinion: Public schools need to be a culture war-free zone. Young people need straightforward, comprehensive sex education. The Shenendehowa school board has an opportunity to get out of a skirmish in the culture wars and get back to the serious task of responsibly educating students about sex, if it follows a committee's sound advice. […] [Read More]
Cancerous politics invade pink ribbons
Political ambition is behind the Susan G. Komen Foundation's decision to cut funding to Planned Parenthood, which provides breast cancer screenings, education and treatment for poor and uninsured women, says Jodi Jacobson in RH Reality Check. [Read More]
Bad grades at Shen
Our opinion: Shenendehowa's decision to revisit the dismissal of Planned Parenthood is encouraging, but the district's needs to realize it can't please everyone. And it's lack of transparency is disturbing. Now everyone, parents and students alike, knows what it's like not to get straight information from the Shenendehowa School District. For students, it's the straight […] [Read More]
Shenendehowa fails sex ed
Our opinion: A school district misses some key lessons of sex education: act responsibly, not rashly, and don't give in to pressure to do things you shouldn't. Here's a pop quiz for the Shenendehowa School District: You're in a stable, long-term relationship with an organization that provides sex education. Some parents, though, are pressuring you […] [Read More]
It's Sex Ed Week. Do you know where your undergrad is?
Planned Parenthood is marking National Sex Ed Week of Action with events at college campuses, including the University at Albany and RPI. According to Upper Hudson Planned Parenthood, students in its VOX chapters will "staff tables, distribute safer sex kits, talk about healthy sexuality and promote the importance of comprehensive sex education." Both campus groups […] [Read More]
Planned Parenthood under fire
Women looking for low-cost birth control in New Hampshire were suddenly out of luck this month after a state board decided not to renew a contract with Planned Parenthood, the latest salvo by conservatives in the United States against the group from conservatives who oppose it for the abortion services it provides. New Hampshire's Executive […] [Read More]


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Lewis Saab is a family owned business located between Chichester and Emsworth on the A259 at Bosham. With over 20 years experience with the Saab Brand, we are one of the longest serving Saab Dealerships in the UK today.
Offering full Saab dealer facilities covering West Sussex and Hampshire including Southampton, Fareham, Winchester, Worthing and Brighton. We also have an extensive display of Used Saabs and selected other makes for sale.
Our aim is to offer total customer satisfaction whilst still providing value for money. Any of our experienced staff will be only too happy to help with your enquiry.
Ronic House
Main Road (A259)
West Sussex PO18 8PN
01243 574141


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Buy Tickets New Directors/New Films 2013
Show From Series
Thursday, December 26
Friday, December 27
Saturday, December 28
Sunday, December 29
Monday, December 30
Tuesday, December 31
Wednesday, January 1
New Directors/New Films 2013
March 20, 2013 – March 31, 2013
In its 42-year history, New Directors/New Films has introduced or cemented the status of some of the most familiar and critically-acclaimed directors of our time. Who will be your next favorite filmmaker? Presented by Film Society and Museum of Modern Art. Read more »


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Ellen Christine Galyon
Ellen Christine Galyon, age 49 of Seymour passed away Thursday May 1, 2014.
She was a member of Hurst Chapel Baptist Church.
She was preceded in death by her father, William Robert "Bob" Ogle, son, William Scott Greene, Sister, April Ogle, uncles, Lee Roy Parrott, Ralph Parrott and wife Vera, Aunt, Angela Parrott King, grandparents, Rev. Odes Parrott, John Davis Ogle and wife Dicie Ellen Watson, first husband, Johnny Greene.
Survivors: Loving husband of 15 years: Roger Galyon; Daughter and son-in-law: Tosha and Greg Satterfield; Step son: Jimmy Galyon and wife Linh; Grandchildren: Malachi, Blaise, and Tierce Satterfield, Blake, Logun and Miley Galyon; Mother: Sherley Butler; Step mother: Janie Dotson; Grandmother: Edna Parrott; Sisters: Rebecca Ogle, Debra Ayers and husband Tim; Nieces and Nephews: Brandon Harris, Joey Williams, Felisha Harris, Whitney Reagan and husband Sam, Cody and Tiffany Brackins;
Great Nephew: Brantley Reagan; Great Niece: Emma Harris; Many extended family members and friends.
Funeral service 2PM Tuesday at Hurst Chapel Baptist Church with Rev. Daniel Sizemore and Rev. Jack Ogle officiating. Interment will follow in Hurst Chapel Cemetery at which time we will celebrate her life with a dove release. The family will receive friends from 5-8PM Monday at Atchley Funeral Home, Sevierville. Arrangements by Atchley Funeral Home, Sevierville.


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- Kopplungskonstanten (1)
- Peptide (1)
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- acyl carrier protein, polyketide synthases, Curacin cluster (1)
- Biochemie und Chemie (16)
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- Physik (1)
- Heterologous production and characterization of two distinct di-heme containing membrane integral cytochrome b561 enzymes from Arabidopsis thaliana (2007)
- Cytochrome b561 (cyt b561) proteins are members of the recently identified eukaryotic ascorbate reducible protein family named CYBASC (CYtochrome B, ASCorbate reducible). CYBASC proteins are di-heme-b-containing membrane proteins that catalyze the transmembrane electron transfer from ascorbate. The function of the CYBASC proteins has been correlated with ascorbate recycling and/or iron facilitation uptake. Therefore, investigations on this family are of great interest as ascorbate is one of the most powerful antioxidants and iron is essential for cell survival both in animals and plants. As the amino acid sequence conservation of animal and plant CYBASC proteins is relatively high, all CYBASC members are proposed to share the same structural motifs. However, no three-dimensional structure of any representative member of the CYBASC family has been determined to date. In the Arabidopsis thaliana (A. thaliana) genome, two complete putative CYBASC open reading frames (ORFs), artb561-a and artb561-b were identified. In this thesis, these two A. thaliana CYBASC ORFs, encoding for Acytb561-A and Acytb561-B proteins respectively, were investigated and obtained main results are listed. 1. A. thaliana CYBASC proteins were heterologously produced in Pichia pastoris and Escherichia coli and purified by a single-step immobilized metal affinity chromatography (IMAC). To facilitate detection and purification, the recombinant A. thaliana CYBASC proteins were produced in both expression systems with the histidine affinity tag. Pure and stable preparations of the cytochromes were obtained via a single-step IMAC in sufficient amounts to perform biochemical characterizations. 2. Detergent solubilized recombinant Acytb561-A and Acytb561-B are dimers. As previously suggested for other CYBASC proteins, analytical gel filtration experiment suggested that both detergent solubilized cytochromes are dimers. 3. Spectroscopic features of Acytb561-B differed from those of previously described bovine chromaffin granule cyt b561. A distinctive feature of the first identified CYBASC protein, the cyt b561 from bovine chromaffin vesicles of adrenal medulla (Bcytb561-CG), is that its differential visible absorbance spectra (visible-spectra) revealed an asymmetric α-band with a maximum at 562 nm and a clear shoulder at 557 nm. This feature was recently used to discriminate CYBASC proteins from not-CYBASC proteins. However, in this thesis, it is shown for the first time that not all CYBASC proteins display in their reduced-minus-oxidized visible-spectra an asymmetric α- band and therefore, this feature can not be used as a discriminating CYBASC characteristic. 4. Ascorbate dependent reduction of the A. thaliana CYBASC proteins is inhibited by diethylpyrocarbonate (DEPC). As previously reported for the Bcytb561-CG, the ascorbatedependent reduction of the A. thaliana CYBASC proteins was inhibited by DEPC treatment. In addition, the 'ascorbate protectant' effect against DEPC that was observed on the Bcytb561-CG was also observed on the Acytb561-A and Acytb561-B proteins. Furthermore, as the physiological electron donor of all CYBASC proteins is supposed to be ascorbate, ascorbate-affinity of Acytb561- A and Acytb561-B was monitored and was found to be in the same range of the one of the Bcytb561- CG. 5. A. thaliana CYBASC proteins are Fe3+-chelate reductases. Recently, the Fe3+-chelate reductase activity of various CYBASC proteins was presented. In this thesis, it is shown that also both A. thaliana CYBASC proteins reduced Fe3+-chelates such as Fe3+-EDTA and Fe3+-citrate. Consistently, heme potentiometric reductive-oxidative titration of purified Acytb561-A and Acytb561-B indicated that the midpoint potential of the two heme centres of both cytochromes was lower than the one of those Fe3+-chelates. The values of both heme centre potentials of Acytb561-A and Acytb561-B are also consistent with the observation that both cytochromes were only partially reducible by ascorbate and were fully reduced with the non-physiological reductant Na-dithionite. In summary, this work describes the heterologous production, purification and initial characterizations of two distinct CYBASC proteins from A. thaliana: Acytb561-A and Acytb561-B. Biochemical characterization of these cytochromes showed that the shape of the α-band in the differential spectra is not a discriminating factor for CYBASC proteins but it is likely the DEPC sensitivity and the Fe3+-chelate reductase activity. Establishment of a purification strategy to obtain sufficient amounts of monodispersed and stable A. thaliana CYBASC proteins has also enabled initial screening of three dimensional crystallization conditions which are a prerequisite for a deeper understanding of this new eukaryotic redox enzyme family.
- The human GPCR nicotinic acid receptor 1 : heterologous overproduction in Pichia pastoris and the reconstitution of its complex with beta-Arrestin 1 in vivo and in vitro (2007)
- Nicotinic acid has been used in the clinical treatment of elevated blood lipid levels for over 50 years. Although it has a beneficial effect on myocardial infarction and blood lipid profiles, its widespread use has been hampered by side effects such as skin rashes and a burning sensation on the upper body. Since elevated blood lipid levels, especially ones of VLDL and LDL cholesterol are a frequent indication and high risk factor for coronary and cardiac diseases, finding a compound with an enhanced pharmacological profile, still holding the desired effects, but without inconvenient side effects, is a very appealing aim to many pharmaceutical companies. These efforts have already produced two marketed drugs, Acipimox and Acifran, but they have not been able to overcome the restrictions already imposed on the treatment by nicotinic acid. Although proposed long before, in the year 2000 the gene for the nicotinic acid receptor in mouse PUMA-G was cloned, and in 2003 the discovery of the genes HM74 and HM74A followed, which comprise the homologous low and high affinity receptors for nicotinic acid in humans. The discovery of this G Protein-coupled receptor target allowed a more directed approach for the search of alternative compounds. This work is the first report of the heterologous overexpression of the high affinity GPCR gene HM74A in the methylotrophic yeast Pichia pastoris. The protein product, NAR1, was pharmacologically characterized, and displayed a binding affinity of 224.8 nM to its ligand nicotinic acid, showing a similar activity profile compared to those displayed in human tissue, which were determined to be 60 nM to 90 nM. Additionally, inhibitory constants (Ki) for Acifran and Acipimox were determined to be 4.5 µM and 50.5 µM, respectively. Furthermore, the total yield of NAR1 reached 42 pmol/mg membrane protein, which corresponds to 0.4 mg of receptor produced per liter yeast culture, opening up the perspective of large scale protein production to facilitate high throughput screening drug discovery efforts and structural studies. In addition, NAR1 could be solubilized in n-decyl-β-D-maltopyranoside and purified to homogeneity after immobilized metal affinity chromatography and a second affinity chromatography step on immobilized monomeric avidin, yielding a single peak on gel filtration, while the purified receptor was able to bind ligand, as shown in NMR Saturation Transfer Difference (STD) measurements. It could be shown that NAR1 is desensitized by β-arrestin 1 in vivo in confocal microscopy studies on HEK and BHK cells. This finding provides a native binding partner for the stabilization of the receptor upon solubilization and purification. Finally human β-arrestin 1 could be produced as a constitutively active variant, comprising residues 1-382 in Pichia pastoris and Escherichia coli. The purified protein was used for in vitro binding experiments and shown to be capable of interacting with NAR1. Although the interaction and formation of the complex was only possible to a limited extent, it leaves open the perspective of crystallizing NAR1 in its active conformation, bound to nicotinic acid and β-arrestin 1.
- Solid-state NMR investigations of the ATP binding cassette multidrug transporter LmrA (2006)
- The development of resistance to multiple drugs is a major problem in treatment of number of infectious diseases and cancer. The phenomenon of multidrug resistance (MDR) is based on the synergetic interplay of a number of mechanisms such as target inactivation, target alteration, prevention of drug influx as well as active extrusion of drugs from the cell. The latter is mediated by over-expression of multidrug efflux pumps. The first discovered and the best characterized until now the human MDR transporter is P-glycoprotein. It is a member of the ATP binding cassette (ABC) superfamily and acts as an active transporter for a variety of anticancer agents using the energy released by ATP hydrolysis. The closest structure and functional homologue of P-glycoprotein found in bacteria is LmrA from Lactococcus lactis. The major goals of this work are to establish the selective isotope labelling of LmrA in Lactococcus lactis, to optimize LmrA sample preparation for solid-state NMR, and finally to perform first solidstate NMR investigations on LmrA shedding light on its catalytic cycle and substrate binding. For a long time the solid-state NMR applications to biological science has been limited to investigation of small molecules mostly. Recently, the solid-state NMR methods have shown potential for structuraland non-perturbing, site directed functional studies of large membrane proteins as well as ligands bound to them. However, to our knowledge neither selective isotope amino acid labelling of any ABC transporter, nor NMR investigations on full-length ABC transporter have been reported to date. Solidstate NMR experiments on a membrane protein require reconstitution of purified proteins into a membrane environment at a high density and either isotopic enrichment of the protein or bound drugs or inhibitors. Therefore, the large quantities of LmrA reconstituted at a high density in lipid membranes, sufficient for advanced NMR studies have been produced and its functional state in reconstituted form has been assessed. In the next step, a procedure for cost effective selective amino acids isotope labelling of LmrA in Lactococcus lactis has been established. Using this protocol deuterium alanine labelled LmrA reconstituted into E. coli liposomes has been prepared. Deuterium NMR has been used extensively to assess the proteins dynamics in past. However, it has never been applied to ABC transporter. Here, we report 2H NMR on selective alanine isotope labelled LmrA which has been used to shed light on the dynamics changes in the protein occurred under AMP-PNP, non-hydrolysable ATP analogue, binding and in ATP/ADP-Vanadate trapped state. It has been found that the major conformation changes affecting the protein motional characteristics occur in the ATP binding domains but not in the transmembrane domains. Additionally, the binding of several substrates to LmrA has been studied by fluorescence spectroscopy as well as by 19F and 31P solid-state NMR. The binding constants for several LmrA substrates have been obtained by fitting the concentration dependant tryptophan intrinsic fluorescence quenching curves. Based on the fluorescence studies and solid-state NMR data, the conformation changes in LmrA under substrate binding have been discussed. In addition, the preferable location of nine LmrA and P-glycoprotein substrates within the model membrane has been studied via 1H-MAS-NOESY-NMR. The results have been interpreted with respect to LmrA and P-glycoprotein binding site accessibility from the membrane interface region.
- NMR, EM and functional studies on TBsmr, a small multidrug transporter from M. tuberculosis (2008)
- Antibiotic resistance of pathogenic bacteria is a major worldwide problem. Bacteria can resist antibiotics by active efflux due to multidrug efflux pumps. The focus of this study has been the mycobacterial multidrug transporter TBsmr because it belongs to the small multidrug resistance (SMR) family whose members are a paradigm to study multidrug efflux due to their small size. SMR proteins are typically 11-12 kDa in size and have a four-transmembrane helix topology. They bind cationic, lipophilic antibiotics such as ethidium bromide (EtBr) and TPP+, and transport them across the membrane in exchange for protons. To understand the molecular mechanism of multidrug resistance, we have to gain information about the structure and function of these proteins. The research described in this thesis aimed to deduce details about the topology, transport cycle and key residues of TBsmr using biophysical techniques. Solid-state NMR (ssNMR) can provide detailed insight into structural organization and dynamical properties of these systems. However, a major bottleneck is the preparation of mg amounts of isotope labeled protein. In case of proteoliposomes, the problem is compounded by the presence of lipids which have to fit into the small active volume of the ssNMR rotor. In Chapter 3, an enhanced protein preparation is described which yields large amounts of TBsmr reconstituted in a native lipid environment suitable for further functional and structual studies. The achieved high protein-to-lipid ratios made a further characterization by ssNMR feasible. The transport activity and oligomeric state of the reconstituted protein in different types of lipid was studied as shown in Chapter 4. The exact oligomeric state of native SMR proteins is still uncertain but a number of biochemical and biophysical studies in detergent suggest that the minimal functional unit capable of binding substrate is a dimer. However, binding assays are not ideal since a protein may bind substrate without completing the transport cycle which can only be shown for reconstituted protein in transport assays.By combining functional data of a TPP+ transport assay with information about theoligomeric state of reconstituted TBsmr obtained by freeze-fracture electron microscopy, it could be shown that lipids affect the function and the oligomeric state of the protein, and that the TBsmr dimer is the minimal functional unit necessary for transport. The transport cycle must involve various conformational states of the protein needed for substrate binding, translocation and release. A fluorescent substrate will therefore experience a significant change of environment while being transported, which influences its fluorescence properties. Thus the substrate itself can report intermediate states that form during the transport cycle. In Chapter 5, the existence of such a substrate-transporter complex for the TBsmr and its substrate EtBr could be shown. The pH gradient needed for antiport has been generated by co-reconstituting TBsmr with bacteriorhodopsin. The measurements have shown the formation of a pH-dependant, transient substrate-protein complex between binding and release of EtBr. This state was further characterized by determining the Kd, by inhibiting EtBr transport through titration with non-fluorescent substrate and by fluorescence anisotropy measurements. The findings support a model with a single occluded intermediate state in which the substrate is highly immobile. Liquid-state NMR is a useful tool to monitor protein-ligand interactions by chemical shift mapping and thus identify and characterize important residues in the protein which are involved in substrate binding. In agreement with previous studies (Krueger-Koplin et al., 2004), the detergent LPPG was found to be highly suitable for liquid-state NMR studies of the membrane protein TBsmr and 42% of the residues could be assigned, as reported in Chapter 6. However, no specific interactions with EtBr were found. This observation was confirmed by LILBID mass spectrometry which showed that TBsmr was predominantly in the non-functional monomeric state. Functional protein was prepared in proteoliposomes which can be investigated by solidstate NMR (Chapter 7). Besides the essential E13, the aromatic residues W63, Y40, and Y60 have been shown to be directly involved in drug binding and transport. Different isotope labeling strategies were evaluated to improve the quality of the NMR spectra to identify and characterize these key residues. In a single tryptophan mutant of reconstituted TBsmr W30A, the binding of ethidium bromide could be detected by 13C solid-state NMR. The measurements have revealed two populations of the conserved W63 residue with distinct backbone structures in the presence of substrate. There is a controversy about the parallel or anti-parallel arrangement of the protomers in the EmrE dimer (Schuldiner, 2007) but this structural asymmetry is consistent with both a parallel and anti-parallel topology.
- Characterization of proteorhodopsin 2D crystals by electron microscopy and solid state nuclear magnetic resonance (2008)
- Proteorhodopsin (PR) originally isolated from uncultivated γ-Proteobacterium as a result of biodiversity screens, is highly abundant ocean wide. PR, a Type I retinal binding protein with 26% sequence identity, is a bacterial homologue of Bacteriorhodopsin (BR). The members within this family share about 78% of sequence identity and display a 40 nm difference in the absorption spectra. This property of the PR family members provides an excellent model system for understanding the mechanism of spectral tuning. Functionally PR is a photoactive proton pump and is suggested to exhibit a pH dependent vectorality of proton transfer. This raises questions about its potential role as pH dependent regulator. The abundance of PR in huge numbers within the cell, its widespread distribution ocean wide at different depths hints towards the involvement of PR in utilization of solar energy, energy metabolism and carbon recycling in the Sea. Contrary to BR, which is known to be a natural 2D crystal, no such information is available for PR til date. Neither its functional mechanism nor its 3D structure has been resolved so far. This PhD project is an attempt to gain a deeper insight so as to understand structural and functional characterization of PR. The approach combines the potentials of 2D crystallography, Atomic Force Microscopy and Solid State NMR techniques for characterization of this protein. Wide range of crystalline conditions was obtained as a result of 2D crystallization screens. This hints towards dominant protein protein interactions. Considering the high number of PR molecules reported per cell, it is likely that driven by such interactions, the protein has a native dense packing in the environment. The projection map represented low resolution of these crystals but suggested a donut shape oligomeric arrangement of protein in a hexagonal lattice with unit cell size of 87Å*87Å. Preliminary FTIR measurements indicated that the crystalline environment does not obstruct the photocycle of PR and K as well as M intermediate states could be identified. Single molecule force spectroscopy and atomic force microscopy on these 2D crystals was used to probe further information about the oligomeric state and nature of unfolding. The data revealed that protein predominantly exists as hexamers in crystalline as well as densely reconstituted regions but a small percentage of pentamers is also observed. The unfolding mechanism was similar to the other relatively well-characterized members of rhodopsin family. A good correlation of the atomic force microscopy and the electron microscopy data was achieved. Solid State NMR of the isotopically labeled 2D crystalline preparations using uniformly and selectively labeling schemes, allowed to obtain high quality SSNMR spectra with typical 15N line width in the range of 0.6-1.2 ppm. The measured 15N chemical shift value of the Schiff base in the 2D crystalline form was observed to be similar to the Schiff base chemical shift values for the functionally active reconstituted samples. This provides an indirect evidence for the active functionality of the protein and hence the folding. The first 15N assignment has been achieved for the Tryptophan with the help of Rotational Echo Double Resonance experiments. The 2D Cross Polarization Lee Goldberg measurements reflect the dynamic state of the protein inspite of restricted mobility in the crystalline state. The behavior of lipids as measured by 31P from the lipid head group showed that the lipids are not tightly bound to the protein but behave more like the lipid bilayer. The 13C-13C homonulear correlation experiments with optimized mixing time based on build up curve analysis, suggest that it is possible to observe individual resonances as seen in case of glutamic acid. The signal to noise was good enough to record a decent spectrum in a feasible period. The selective unlabeling is an efficient method for reduction in the spectral overlap. However, more efficient labeling schemes are required for further characterization. The present spectral resolution is good for individual amino acid investigation but for uniformly labeled samples, further improvement is required.
- Produktion, Reinigung und Charakterisierung von monovalenten Kation/Proton - Antiportern (2007)
- Eine wichtige Klasse von Membranproteinen ist die der aktiven sekundären Transporter. Diese Proteine werden in allen Spezies gefunden und verwenden einen Gradienten von löslichen Substanzen, um den Transport von Substraten voran zu treiben. Dieser Transportprozess ist essentiell, um die chemische Zusammensetzung des Zytoplasmas, wie Kalium- oder Natriumkonzentration von der des umgebenden Milieus unterschiedlich zu halten. Die Konzentration von K+ und Na+ in der Zelle sind wichtig für ein konstantes Zellvolumen, für die pH-Homöostase, für die Erregbarkeit von Nervenzellen und füür die Akkumulierung von Zuckern und Aminosöuren über Kotransportsysteme. In Bakterien wie Escherichia coli wird mit der Oxidation von Substraten durch die Elektronentransportkette ein Protonengradient und gleichzeitig eine Potentialdifferenz erzeugt. Ein Beispiel für einen sekundären Transporter, der diese Potentialdifferenz ausnutzt ist der Na+/H+-Antiporter NhaA, einer der am besten untersuchten Antiporter aus E. coli (Hunte, Screpanti et al. 2005). Dieser Antiporter ist essentiell für die Fähigkeit von Bakterien im alkalischen pH-Bereich zu überleben. Auch bei Säugetieren, sind die Isoformen der humanen Natrium/Protonen-Antiporter SLC9A1-SLC9A8 (NHE1-8) unentbehrlich für eine Reihe physiologischer Prozesse. So wird über die Antiporter-Aktivität nicht nur der Säure-Base-Haushalt und das Verhältnis des Zellvolumens zur Menge an Elektrolyten reguliert, Antiporter spielen ebenso eine wichtige Rolle bei der Adhäsion, Migration und Proliferation der Zelle (Orlowski and Grinstein 2004). Anomalien in diesem Bereich sind charakteristisch für maligne Zellen. Die Rolle von NHE1 in der Entwicklung von Tumoren ist daher ein wichtiger Ansatzpunkt für die Entwicklung von Krebsmedikamenten. Im Herz ist NHE1 die dominierende Isoform und wird damit zu einem pharmakologisch wertvollen Zielprotein (Malo and Fliegel 2006). Struktur und Mechanismus der meisten Antiporter ist bis dato jedoch noch nicht bekannt. Neben den klassischen Methoden der Pharmaentwicklung wird die strukturbasierende Wirkstoffentwicklung immer wichtiger um effiziente Medikamente ohne Nebenwirkung zu herzustellen. Hierfür werden jedoch 3D-Strukturen von Proteinen, sowie genaue Kenntnisse von deren Mechanismus benötigt. Zieht man in Betracht, dass 70% aller bis jetzt entwickelten Medikamente als Ziel ein Membranprotein haben, wird die Notwendigkeit klar, eine möglichst große Anzahl von Membranproteinstrukturen verfgbar zu haben. Wie bereits erwähnt ist die Klasse der monovalenten Kation/Proton-Antiporter aufgrund ihrer vielfältigen Aufgaben, eine äußerst wichtige Zielgruppe für die strukturbasierende Wirkstoffentwicklung. Die große Anzahl an entschlüsselten Genomen eröffnet hier ein breites Forschungsfeld füür die Strukturbiologie. In dieser Arbeit wurden daher Techniken und Methoden aus Hochdurchsatz-orientierten Strukturgenomikprojekten übernommen, um eine große Anzahl von Zielproteinen in ausreichender Menge für die funktionelle Charakterisierung und für die Kristallisation zu produzieren. Als Zielorganismen wurden Salmonella typhimurium LT2, Helicobacter pylori 26695, Aquifex aeolicus VF5 und Pyrococcus furiosus ausgewählt. Die Grundlage dieser Entscheidung hierfür waren die humanpathogenen Eigenschaften der beiden zuerst genannten Organismen und die Hyperthermophilie der beiden letzteren. Dadurch konnten sowohl klinische Anwendungsmöglichkeiten, als auch die potentiell höhere Stabilität der hyperthermophilen Proteine genutzt werden. Als Proteinzielgruppe wurden die monovalenten Kation/Proton-Antiporter aus allen 4 Organismen ausgewählt. Des Weiteren wurden Antiporter zweier eukaryotischer Systeme, Saccharomyces cerevisiae und Homo sapiens in die Zielproteingruppe aufgenommen. In dieser Arbeit wurden 24 verschiedene monovalente Kation/Proton-Antiporter untersucht. Von diesen 24 Zielproteinen konnten 12 in Expressionsvektoren kloniert und produziert werden. Von diesen 12 Antiportern konnten die Zielproteine STM0039 (STNhaA), HP1552 (HPNhaA), STM1556 (NhaC) und PF2032 (NhaC) in einer für die Kristallisation ausreichenden Homogenität und Ausbeute gereinigt werden. Mit der Ausnahme von HP1552 ist bis heute in keiner Veröffentlichung über diese Zielproteine berichtet worden. Durch Komplementationsexperimente mit dem E. coli-Deletionsstamm EP432 konnten eine Reihe von Zielproteine (STM0039, HP1552, PF2032, Aq_2030, STM1806, STM1556) bezüglich ihrer Fähigkeiten zum Na+/H+-Antiport untersucht werden. Die Ziel-proteine STM0039, STM1556 und HP1552 konnten zum ersten Mal kloniert, produziert, gereinigt und anschlieáen in Liposomen rekonstitutiert werden.Weiterhin konnte durch SSM-Messung die pH-Regulation der Zielproteine STM0039 und HP1552 gezeigt werden. Im Gegensatz zu bisherigen Literaturangaben ist HP1552 im pH-Bereich von pH 6 bis 8,5 nicht konstitutiv aktiv, sondern erfährt eine ähnliche Aktivierung wie STM0039 oder ECNhaA. STM0039 lässt sich zudem durch 2-Aminoperimidin inhibieren. Für STM0039 konnten die ersten Proteinkristalle der inaktiven Konformation bei pH 4 erzeugt werden. Weiterhin wurde in dieser Arbeit ein gegen das Zielprotein STM0039 gerichtetes scFV-Antikörperfragment (F6scFv) eingehend charakterisiert. Durch die Ko-Kristallisation des Antikörperfragments F6scFv mit STM0039 konnten die ersten 3 dimensionalen Kristalle in einer aktiven Proteinkonformation bei pH 7,5 erzeugt werden. Neben den bereits verfeinerten Kristallisationsbedingungen für das Zielprotein STM0039 wurden erfolgreich erste Kristallisationsbedingungen für STM0086 und PF2032 gefunden. Es wurde eine Vielzahl von Produktions- und Reinigungsprotokollen füür die Zielproteine etabliert. Dadurch ist der Grundstein füür weitergehende Charakterisierungs- und Kristalli-sationsexperimente gelegt. Die in dieser Arbeit etablierte Kombination von Hochdurch-satzmethoden mit klassischen Vorgehensweisen zur Proteincharakterisierung lassen sich leicht auf anderen Membranproteinklassen bertragen und die Geschwindigkeit der ver-schiedenen Schritte bis zur Strukturlösung stark beschleunigen.
- Purification and characterisation of the respiratory supercomplex III/IV from Corynebacterium glutamicum and phospholipid analysis of membrane proteins (2008)
- The respiratory chain is composed of protein complexes residing in the inner mitochondrial membrane of eukaryotes or in the cytoplasmic membrane of prokaryotes. This cellular energy converter transforms a redox potential stored in low potential substrates into an electrochemical potential across the respective membrane. Typical respiratory chains contain the complexes I, II, III and IV named according to their sequence in the respiratory chain reaction. Electrons of low potential substrates enter at complex I or II and are passed via complex III to complex IV where they are transferred to oxygen. The transport of electrons between the complexes is mediated by small electron shuttles like quinol or cytochrome c. Two different models describe their exchange either by (1) random collision of freely diffusible electron shuttles and membrane protein complexes or (2) arrangement of the complexes in supercomplexes enabling direct channeling of electron shuttles. In the Gram positive bacterium Corynebacterium glutamicum, the complex III to complex IV electron shuttle cytochrome c is not diffusible but a covalently bound part of the diheme cytochrome subunit QcrC of complex III. Therefore, the complexes III and IV have to form a supercomplex for electron transduction. The aim of this thesis was to purify and characterise this obligatory supercomplex III/IV of C. glutamicum. To gain sufficient biomass of C. glutamicum as starting material for purification, a phosphate buffered minimal medium was developed that enabled yield of total 120 g wet cell mass (38 g dry mass) in 12 L (6×2 L) shaking cultures. The determined conversion factor of glucose into biomass was 0.46 g/g indicating an intact respiratory chain. The yield was increased by bioreactor cultivation to ~690 g wet cell mass (~220 g dry mass) in ~10 L culture volume. A previously described homologous expression system was applied that produces the complex IV subunit CtaD with a fused Strep-tag II to facilitate purification. Affinity purifications using the Strep-tag II affinity to Strep-Tactin resin yielded a mixture of complexes and supercomplexes. Two supercomplex III/IV versions named supercomplex A and B and free complex IV were identified in this mixture by size exclusion chromatography, redox difference spectroscopy and two dimensional polyacrylamide gel electrophoresis including blue native polyacrylamide electrophoresis. The here presented downscaled blue native polyacrylamide electrophoresis method with analysis times of ~1 h enabled efficient screening of factors influencing the stability of supercomplex III/IV. The screening resulted that the integrity of supercomplex III/IV is preserved by using neutral detergents at minimal detergent to protein ratios for solubilisation and low detergent concentrations for purification and storage slightly above the required critical micellar concentration. Furthermore, pH <=7.5 is required for stability of supercomplex III/IV. Large biomass yields enabled upscaling of supercomplex III/IV affinity purification. Application of the identified stability conditions resulted in affinity purified samples free of supercomplex B. The major component supercomplex A was efficiently separated from residual free complex IV by preparative size exclusion chromatography. Concentration of purified supercomplex A by ultracentrifugation resulted in integrity of the supercomplex for several days at 4 °C. Purified supercomplex A contains ten different previously described subunits. The heme content of supercomplex A relative to the protein mass is heme A: 6.0 μmol/g, heme B: 6.5 μmol/g, and heme C: 5.8 μmol/g determined by redox difference spectroscopy and biochemical protein quantification. This indicates an equimolar ratio of complex III and complex IV in supercomplex A. Supercomplex A has quinol oxidase activity that is inhibited by stigmatellin or sodium azide. The turnover number of transferred electrons per complex III monomer is 148 s−1 at 25° C. The homogeneity and stability of the prepared supercomplex A enabled the growth of threedimensional crystals of up to 0.1 mm in length. Their composition of supercomplex A was verified by redox difference spectroscopy of intact crystals and blue native polyacrylamide electrophoresis of dissolved crystals. The crystals diffracted X-rays corresponding to a resolution of ~10 Å. Electron microscopy of negative stained samples revealed the uniform shape of purified supercomplex A particles with dimensions of 22 × 9 nm in the view plane. Combined heme quantification, size determination, determined activity, symmetry considerations, and particle shape indicate that supercomplex A has a central dimer of complex III and two monomers of complex IV on opposite sides. This conformation is functionally reasonable because it provides each complex III monomer with one complex IV monomer as electron acceptor. Therefore, the stoichiometry of supercomplex A is most likely III2IV2. The sensitivity of supercomplex A to detergents indicated a role of phospholipids in its stability. Therefore, a method for phospholipid identification and quantification was developed that is suitable for detergent solubilised crude and purified membrane protein samples. The analysis combines separation of phospholipid classes according to their head group by normal phase high performance liquid chromatography with evaporative light scattering detection. Calibration with external standard allows quantification of phospholipid amount in the range of 0.25-12 μg. The method is verified by analysing the phospholipid content of the well characterised complex III of Saccharomyces cerevisiae. The reduction of its phospholipid content during its purification steps is monitored. The complex III sample purified to crystallisation quality contains the phospholipid content that was also observed in previously reported structures determined by X-ray crystallography. Purified stable supercomplex A from C. glutamicum revealed a large content of bound phospholipids. The main differences between intact supercomplex A and a mixture of potentially disintegrated smaller complexes is that intact supercomplex A has a doubled phosphatidic acid content and an increased phosphatidyl glycerol content. The importance of the small anionic phosphatidic acid for mediation of contacts between complexes in a supercomplex is discussed. The total phospholipid content of stable supercomplex A is sufficient for a complete belt surrounding the supercomplex in the membrane plane. This indicates that also all essential internal phospholipid binding positions are occupied and potentially stabilise supercomplex A.
- X-ray structure of the Na+-coupled Glycine-Betaine symporter BetP from Corynebacterium glutamicum (2009)
- Cellular membranes are important sites of interaction between cells and their environment. Among the multitude of macromolecular complexes embedded in these membranes, transporters play a particularly important role. These integral membrane proteins perform a number of vital functions that enable cell adaptation to changing environmental conditions. Osmotic stress is a major external stimulus for cells. Bacteria are frequently exposed to either hyperosmotic or hypoosmotic stress. Typical conditions for soil bacteria, such as Corynebacterium glutamicum, vary between dryness and sudden rainfall. Physical stimuli caused by osmotic stress have to be sensed and used to activate appropriate response mechanisms. Hypoosmotic stress causes immediate and uncontrolled influx of water. Cells counteract by instantly opening mechanosensitive channels, which act as emergency valves leading to fast efflux of small solutes out of the cell, therebydiminishing the osmotic gradient across the cell membrane. Hyperosmotic stress, on the other hand, results in water efflux. This is counterbalanced by an accumulation of small, osmotically active solutes in the cytoplasm, the so-called compatible solutes. They comprise a large variety of substances, including amino acids (proline), amino acid derivatives (betaine, ectoine), oligosaccharides (trehalose), and heterosides (glucosylglycerol). Osmoregulated transporters sense intracellular osmotic pressure and respond to hyperosmotic stress by facilitating the inward translocation of compatible solutes across the cell membrane, to restore normal hydration levels. This work presents the first X-ray structure of a member of the Betaine-Choline-Carnitine-Transporter (BCCT) family, BetP. This Na+-coupled symporter from Corynebacterium glutamicum is a highly effective osmoregulated and specific uptake system for glycine-betaine. X-ray structure determination was achieved using single wavelength anomalous dispersion (SAD) of selenium atoms. Selenium was incorporated into the protein during its expression in methione auxotrophic E. coli cells, grown in media supplemented with selenomethionine. SAD data with anomalous signal up to 5 Å led to the detection of 39 selenium sites, which were used to calculate the initial electron density map of the protein. Medium resolution and high data anisotropy made the structure determination of BetP a challenging task. A specific strategy for data anisotropy correction and a combination of various crystallographic programs were necessary to obtain an interpretable electron density map suitable for model building. The crystal structure of BetP shows a trimer with glycine-betaine bound in a three-fold cation-pi interaction built by conserved tryptophan residues. The bound substrate is occluded from both sides of the membrane and aromatic side chains line its transport pathway. Very interestingly, the structure reveals that the alpha-helical C-terminal domain, for which a chemo- and osmosensory function was elucidated by biochemical methods, interacts with cytoplasmic loops of an adjacent monomer. These unexpected monomer-monomer interactions are thought to be crucial for the activation mechanism of BetP, and a new atomic model combing biochemical results with the crystal structure is proposed. BetP is shown to have the same overall fold as three unrelated Na+-coupled symporters. While these were crystallised in either the outward- or inward-facing conformation, BetP reveals a unique intermediate state, opening new perspectives on the alternating access mechanism of transport.
- Improving methods for the study of membrane proteins by solid-state NMR (2009)
- Solid state NMR is a emerging method for the study of membrane proteins, which has received much interest in recent years. Limiting the study of many pharmacologically relevant targets, are the often long measuring times, required to obtain especially higher dimensional solid state NMR spectra of good quality. To address this problem, multiple methods where developed in this work, which can be categorized into two groups. The first set of methods aims at the quality of certain spectra, by implementing a spectral filter, which increases the fidelity of the measured data. The second set of methods, addresses the problem of long measuring times directly, by increasing the sensitivity per unit time, as could be shown, for example, on homo- and heteronuclear singlequantum-singlequantum correlation experiments. The gains in measuring time for the latter group of methods are typically in the order of 2-3, but some experiments allow multiple methods to be employed simultaneously, which can lead to a decrease in measuring time of a factor of up to 8. It is important to mention, that none of the methods introduced in this work require any equipment in addition to the conventional setup present in most sold state NMR laboratories and no changes or addition to the samples under study are required. Therefore the gains reported in this work come at no extra cost and require only minimal implementation effort on the side of the user.
- Biophysical studies on LmrA : a multidrug resistance ABC transporter / von Ute A. Hellmich (2010)
- LmrA is a member of the ATP Binding Cassette (ABC) transporter family of membrane proteins and a structural and functional homologue of P-glycoprotein1, 2. ABC-transporters share a common architecture of two transmembrane domains and two nucleotide binding domains. The NBDs are highly conserved in this transporter family whereas the TMDs are highly diverse3. The TMDs recognize the substrate and the NBDs bind and hydrolyze ATP and thus contribute the energy for substrate translocation. ABC transporters as a protein family transport a high number of substrates including peptides, nutrients, ions, bile acids, lipids and other lipophilic compounds. LmrA is a multidrug transporter that recognizes a number of hydrophobic substrates including fluorescent dyes and antibiotics1, 4-6. LmrA is a native protein of the gram-positive bacterium Lactococcus lactis. In this thesis, L. lactis was used as a homologous expression host for the preparation of LmrA for a variety of experiments. Wildtype LmrA as well as a number of cysteine mutants were successfully expressed in L. lactis, purified and subsequently characterized by a variety of biochemical assays (Chapter 4). LmrA can be expressed to very high amounts in L. lactis. The purification and reconstitution were optimized for the requirements of solid-state NMR experiments in this thesis. For the first time, an ABC transporter has been reconstituted in synthetic lipids to a ratio of up to 1:150 (mol/mol). LmrA was shown to be active under magic angle spinning conditions with these reconstitution ratios. By taking advantage of the slower ATP hydrolysis by LmrA ΔK388 (lysine deletion in the Walker A motif), a real-time 31P solid-state NMR ATPase assay was established (Chapter 5). This assay allowed, for the first time, the investigation of all phosphor nuclei during the ATP hydrolysis cycle of a membrane protein simultaneously and in real time7. This assay has been successfully adapted to investigate both ATP hydrolysis and substrate phosphorylation of diacylglycerol kinase (together with S. Wollschlag) and ATP hydrolysis at high temperatures of the thermophilic ABC transporter ABC1 from Thermos thermophilus (together with A. Zutz). In the course of this thesis, the gene for LmrA has been cloned into expression vectors suitable for Escherichia coli and the heterologous expression of LmrA was established (Chapter 4). The functionality of the heterologously expressed protein has been investigated and compared to L. lactis LmrA. In these experiments, LmrA was shown to yield a distinct multidrug resistance phenotype in its E. coli host and to show secondary active multidrug transport in the absence of ATP and presence of a proton gradient [Hellmich et al, in prep] (Chapter 4). Previously, it had been shown that LmrA acts as a seconadary active transporter when the NBDs are truncated8. The overexpression in minimal and defined medium and the purification of LmrA from E. coli have been optimized. Isotope labeling for ssNMR has been established and the first multinuclear ssNMR experiments have been carried out on a functional ABC transporter (Chapter 8). ABC transporters couple two cycles: upon ATP binding, the NBDs dimerize, hydrolyze the ATP, subsequently release Pi and ADP and finally dissociate. During this cycle, conformational changes are relayed to the TMDs which utilize the energy from ATP binding and/or hydrolysis to translocate the respective substrate. The prehydrolysis state can be trapped by beryllium fluoride, whereas the post-hydrolysis state of this cycle can be trapped by vanadate9-12. Trapping protocols for these reagents were successfully established for LmrA in this thesis (Chapter 4). This allowed for the investigation of different catalytic states by both ssNMR and EPR. A general 19F labeling protocol for membrane proteins has been established in the course of this thesis and successfully applied to proteorhodopsin (together with N. Pfleger)13 and LmrA (chapter 6). Single cysteine mutants of LmrA that line out the dimer interface have been labeled with a fluorine label for ssNMR. In the apo state, the 19F labeling indicates highly flexible transmembrane domains, a finding that is supported by 13C ssNMR and EPR measurements. The addition of drugs has a different effect on different positions within the LmrA dimer, therefore indicating that different drugs are recognized at a different position within the protein. For P-glycoprotein and LmrA it has been previously shown by biochemical methods that different drug binding sites co-exist. For a 19F label attached at position 314 (LmrA E314C), the spectra showed two distinct peaks with similar populations. This could hint towards a structural asymmetry within the LmrA dimer that might also be reflected in the alternating ATP hydrolysis at the NBDs. E314 has been specifically implicated with drug transport. Thus, structural asymmetry at this position might be functionally relevant for guiding a substrate through the transporter. Structural asymmetry within a homodimeric ABC transporter has also been shown for BtuCD, the E. coli vitamin B12 importer14. In addition, the conserved glutamates in EmrE, a small multidrug resistance protein, were shown to be asymmetric in the drug bound state15. Both, uniformly 13C/15N labeled as well as selectively amino acid type labeled LmrA has been investigated in different conformational states. Interestingly, significant dynamic changes in the b-sheet regions of LmrA (confined to the NBDs) were observed in the pre-hydrolysis (beryllium fluoride) and transition state (vanadate trapped) state. These were interpreted as the transition from a domain in fast conformational exchange in the apo state to one of intermediate exchange in the nucleotide bound state. A significant change in NBD mobility upon nucleotide binding was previously also shown with 2H ssNMR on LmrA16. By EPR it was shown that LmrA in both the vanadate and BeFx trapped states displays a significantly higher rigidity and therefore defined distances, whereas the apo state resembled a "floppy" protein with no preferred distance distribution. This concurs with data obtained from 19F ssNMR with fluorine labeled single-cysteine mutants. Here, in agreement with the EPR data, a higher label (and possibly) protein mobility was observed in the apo state displaying rather broad line widths. Upon trapping with vanadate, the line widths of the majority of fluorine-labeled mutants decreased due to an enhanced protein rigidity and a more homogenous environment of the fluorine labels. A similar observation was made when increasing the temperature that can be explained due to higher protein flexibility at increased temperatures. Solution NMR was employed to investigate the isolated soluble NBD of LmrA (Chapter 9). First 2D and 3D spectra were successfully obtained and could be utilized for a preliminary assignment of a significant fraction of residues. Additionally, binding of ATP and ADP in absence and presence of magnesium was investigated. Finally, the effects of peptides emulating the coupling helices of the full-length transporter on the soluble NBD were investigated. Strikingly, binding of one of these peptides only occurred in the presence of nucleotides (whereas the other showed no binding at all) hinting towards a tightly coupled regulation of the NBD and TMD during the substrate translocation/ATP hydrolysis cycle based on nucleotide binding.


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|50 posts found
Joined: 12/11/08
The problem with hard drives is that they're slow. In order to read anything from a hard drive, you have to wait for the platter and head to physically move to the right spot before it can start reading. The amount of time this takes varies, but it's typically on the order of 10 ms.
But wait, 10 ms sounds fast, you say? Well, what happens if you have to load a thousand things at once? Now you're looking at 10 seconds. And a loading screen, which is boring. Hard drives may cite speeds on the order of 100 MB/s, which is plenty fast, but that's only for sequential transfers, such as when you need to load one really big file that is not fragmented at all. If you're loading small files, it's more like, read a little bit, wait 10 ms, read a little bit, wait 10 ms, and so forth. In some cases, you spend over 99% of the time waiting for the platter and drive head to physically move to the next spot before continuing.
In fact, when your computer makes you wait for something, if it's not downloading things from the Internet, it's probably making you wait while it loads stuff from a hard drive.
Wouldn't it be great if you could skip the 10 ms penalty for switching to a different spot that hard drives bring? Well, solid state drives can do exactly that. They won't eliminate loading times entirely, but they will mean that you're usually not waiting on an SSD. Many things will load in 1/2 or 1/3 of the time that they would have with even a relatively fast hard drive. Some things will see a much bigger jump in performance than that, even.
The upshot is that when you tell your computer to do something, it just does it, rather than make you sit there and wait and wait and wait and then eventually getting around to it. Big games will still take a while to load, but many programs will be loaded and ready to go faster than you are.
That only happens for loading things off of a solid state drive, though. What a lot of people do is to get both a solid state drive and a hard drive, and install the OS and programs on the SSD, while putting bulk data (e.g., videos, music, or pictures) on the hard drive.
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Originally posted by Quizzical
Ok that helps a lot. Now i'm wondering if i should skip the hard drive and get one of them bigger 500 someone GB SSD but at same time I don't have an SSD on my PC right now and not a prob far as i can tell. Hmm choices lol.
My thought on the 3T hard drive was that games are getting bigger every year not that I in fact need one and only have a 400gb one now I still don't fill.
Joined: 12/11/08
My standard guideline on storage space is to look at how much you're using now, double it, and figure that's now much you'll need on your new computer. That's not just how much you have now, but how much you're actually using.
If a few years from now, you discover that you need more space than you thought, then it's easy to buy another hard drive and add it later. It will probably be cheaper a few years from now than today, too.
The downside of SSDs is that they're very expensive in $/GB. SSDs with about 500 GB of capacity start around $300, and you should expect to pay closer to $400 for the better ones. On a $3000 budget, you can go ahead and pay that and not worry about it, but on your budget, I wouldn't.
You can get both an SSD and a hard drive. Some things don't benefit from the speed of an SSD; it's really only when you want to read or write a bunch of files at once that the SSD offers a big benefit. Programs tend to load a lot of files at once. Browsers will both load and save a lot of files at once. Games will load huge numbers of files at once at nearly any loading screen.
But videos, pictures, and music tend to be loaded one file at a time, so the speed of an SSD doesn't matter. If a large fraction of your used storage space is data files like these, then get a hard drive for them so as not to waste space on an SSD.
Originally posted by Quizzical
How much will an SSD help with gaming? I will pretty much only have games on this pc which will take up an SSD space pretty fast i'm thinking so not sure of the worth of it for just games. So a friend told me today about power use of all the parts which i forgot all about. Add it all up and add 20% of that to it but not all the parts list the power use that i can see anyways.
Elite Member
Joined: 12/19/10
SSD won't help with FPS for the most part, but it will help a lot with starting games, and anytime you see a loading screen (infrequent in some games, very frequent in others). Some games also stream textures from the disk, and SSDs can help a lot with the visual quality in these games.
So do SSD's help with gaming? It really depends. You can definitely notice in a group when someone doesn't have one - they will zone/load/instance over later than you (by a lot). But will it give you more FPS or crank up the graphics options (which is all some people care about)? No, it won't. But it makes using the computer a whole lot more enjoyable when it responds much better to you. SSD will help everything on the computer, from startup times, to even just little hitches like when you click on the Start menu and your computer has to "think" for a few seconds, to those times when your computer has to "wake up" when you right click on a file folder to load and display all the contextual menus... and your games all load a lot faster.
Personally, I recommend them to everyone, gaming or not, to the point where I will sacrifice video card budget in order to fit an SSD into a computer budget. But I don't play a lot of FPS games where FPS is all that matters.
To most people who are concerned with SSD size, I just recommend they keep the few games/programs they primarily play on the SSD, and everything else on a traditional drive. If they start to play another game more often (or get a new one), just shuffle them around (most games you can just copy/paste and update the shortcut and be fine).
As far as power draw:
Find the TDP of your video card (just Google "TDP" and your video card model) - most are between 100-200W. That's the maximum design power of your video card of choice - it very very rarely will ever hit that, so it's a good safe number to use. Double that if you plan on overclocking.
Almost every CPU uses right around 100W (again - max case, so it's a safe number to use). Double that if you plan on overclocking.
Figure in 100W for everything else - hard drives, motherboard, lights, fans, etc. It takes a lot of fans/hard drives/water pumps/whatever to go over 100W, and for the most part this number won't change if you overclock or not.
Add all that up - that's about what size you should get. I find that ~600W is a pretty good size for nearly all single video card builds - it's big enough to handle nearly any hardware combination you throw at it, but not so big as to be extremely expensive or overly wasteful.
Elite Member
Joined: 12/19/10
For games to get much bigger, they would need to start shipping on several DVDs (which some do, but it really starts to get ridiculous and costly much past 2 or 3) or Blu-Ray/Flash/similar devices (which just haven't really caught on, or the media would cost substantially more) - and for the most part software is trying to get away from relying just on physical distribution, with digitally distribution a requirement for many. Not everyone has a really fast connection and a lot of people sit on ISPs with data caps, so the install size is still a very important consideration.
Size has been constrained somewhat by some artificial means, and hasn't really changed much over the past few years. A typical AAA MMO game is around 15G, F2P tend to be a bit smaller (in at around 5G), and a few are very large (over 20G, generally those with lots of expansions under their belts, or very high resolution textures and voiceover assets).
AoC is still the largest game I have on my computer (at just over 30G), with WoW right behind it (through Cata, no MoP, at 28G). SWTOR with all it's voice acting is around 20G. LOTRO is 15G (I don't have RoR). EQ1 is around 8G (missing the last 2 expansions). RIFT is just under 10G (missing the lastest expansion).
Some newer games: PS2 is just under 10G. The last Firefall beta is 6.5G. TERA was 25G the last time I patched it (shortly after release). GW2 is at 16.5G.
I have used a 120G SSD as my primary drive for the past 3ish years. I find it's big enough for Windows (allow around 40G for this, to include cache files and temporary file space, and the swap file). And then I've usually got room for 3-4ish games on top of that.
Now, what I do use a lot of space for are multimedia - my legitimate (as hard as that may be to believe, it's true) multimedia collection has exploded, particularly once I started getting movies digitally in HD (720 and 1080). I'm just over 3T in multimedia alone -- thank goodness for NAS'es. So while games have been more or less stagnant, not everything has. It really depends on what you do with your computer. I find I can just shuffle all those off onto a network device, and I don't even need to consider them when planning a new computer - since they are on the network they are available to every computer, and my network is fast enough to stream it decently. But if I were to try to play a game over the network from the NAS, it would work, and it would be extremely painful.
I would agree, and all my current systems currently run with, a 120/128GB SSD plus 1TB+ HDD is a good standard for a modern system over $1k. There are certainly cases where a larger SSD might be worth considering but I've never personally run into them on any of my system which break down into the following:
1. My Main System (Primarily a gaming PC which I only keep my main games running off the SSD)
2. My Wife's CS6 System (doubles as a gaming PC but it was built with Photoshop in mind)
3. Our HTPC (the 128Gb SSD in this is prebably overkill as the only thing that really benefits is Windows 7)
If you in fact enjoy playing many large games (as in storage size) at the same time and don't want to hassle with moving them from HDD to SSD then you could certainly fall into the rare case where a larger SSD might be worth consideration, especially if you are looking at spending $2k or more.
Thanks for all the info :).
Ok so here is what i'm going for so if anyone wants to point out any red flags before i buy if any.
CPU and MB
Case and power
Hard drive
Optical drive
Windows 7
I don't see any red flags but based on reviews and personal experience I'm going to recommend you swap out your PSU for a Seasonic X 750.
In most head to head comparisons is generally wins out in both overall performance/reliability and is very quiet. Yes it's a bit more expensive and you'll probably lose out on the combo deal so that is why it's just a suggestion. That said you actually don't need a 750 watt power supply so you could also consider a Seasonic X 650 for the same price as the Corsair. The only time you would even need to think of getting a 750+ watt psu these days is generally for a multiple GPU setup.
A thread talking about the breakdown of the various product lines for Corsair PSU's that might interest you.
And finally a break down of OEM's for the various PSU's on the market:
Originally posted by miguksaram
Haha the PSU i was wanting is out of stock now too >.<.
Guess last thing i should cover just to be safe is place i build it at. My place is all carpet so would i want a rubber mat to stand on besides the static bracelet and do i only need one or two of them? Oh also it be on a wood table that would be ok right?
Originally posted by DM19
I never used the static braclet but I've also built all my computers on hardwood tables along with hardwood floors so it really wasn't ever an issue. I would suggest if you can help it do not build it while near carpet but if you can't just be sure to have something metal nearby to "discharge" the build up static on before you touch the electronic parts of the build.
Novice Member
Joined: 11/14/11
NO SSD drive(s)????
you can put 200GIG of ram in it but your harddisk will still be your bottleneck when loading. (i recommend the revo 3 pci-e card as a harddrive to load your games from)
16GB of RAM will do just fine....32 is overkill.
Elite Member
Joined: 10/29/07
Originally posted by miguksaram
Chanses are pretty low that something happens but not zero (I trashed a network card once, saw he spark). And yeah, I used a hardwood table as well. I think 'I built 20 or so computers without the bracelet, twice that number with.
ESD bracelets are a few bucks each, getting one is worth it even if chanses you get a spark is small if you are careful. Turning a new GFX card into a doorstop is not a good idea to save 5 bucks. And you might be able to borrow one from a friend for free.
Originally posted by miguksaram
Thanks for all the help :) Now just to get the parts and see if it ends up a bomb somehow lol
Novice Member
Joined: 10/18/12
Why such a top-notch CPU paired with such an average graphics card?
Spend a little extra and get the GTX 670 instead. ;)
Originally posted by DancingQueen
The new build is on page 3 and thats what i got :)
Novice Member
Joined: 10/18/12
Originally posted by DM19
There are only 2 pages.
Apprentice Member
Joined: 8/02/05
Haggis Humper
Originally posted by miguksaram
Tuoching the copper pipe on a radiator before you handle each part is almost as good as a bracelet if you know you're not going to build it up quickly. The only time i've ever used the bracelets was on one of my first jobs, minimum wage soldering circuit boards in a factory :)
Originally posted by DancingQueen
Hmm odd your reply is on page 4 now :) Are you using a phone?
Elite Member
Joined: 4/06/04
Talk low, talk slow, and don't say too much. - John Wayne
Originally posted by DM19
I only see one page of discussion. Look in your profile settings. Experiment.


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In their own words, Ed
The 0950 from Yarmouth has been cancelled this morning after an alternator failure on Wight Light at Yarmouth. Customers are asked to turn up at their normal check-in time.
Wightlink would like to apologise for any inconvenience that this may cause. Passengers concerned about their sailing(s) should call Wightlink reservations on 0871 376 1000.
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Monday, 18th February, 2013 8:49am
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Blackberry qwerty keyboard is proven asset by Jean Louis Gassee
Jean pointed out a reason why iPad sales are not as near projected as it should be because many tasks need require keyboards where people resort to their macbook and the sweet spot is where users have iPad & MacBook to do their work. With that said, John Chen is taking the right path listening to the consumers on a need of qwerty Keyboard by bringing back the Blackberry Curve and also adding a new innovative Passport which will fill the niche for the consumers who needs something mobile enough to still do their spreadsheet and business task.
Blackberry at a glance
Blackberry stocks were set back recently with the news of Apple & IBM paring up for the enterprise-sector, however was recently approved to manage android/iphone and other mobile platforms.
Blackberry patents-portfolio are undervalue as they continue to beef up their security with acquisition of SecuSmart
Blackberry CEO John Chen will raise value for years to come as strong signs of success on turnaround plans
Evaluating John Chen will need judgement of pass performance, unlike stocks where this is not recommended. When looking at a person chances of succeeding in the business world, you must look at it in terms of credit-score if they a person have a great credit score, if it's great the likelihood they will deliver payments on time are higher. John Chen will do just that as many company are focus less on security and more on designs. It may be better on a consumer market for likes of Android/iPhone to succeed in sales, but big and small business runs the economy and the ability to run so under a secure enterprise platform to lower chances of losses due to downtown is invaluable.
Conclusion: Release financial report will be on sept 26, 2014 and likely to outperform analysis expected results.
*** These are no financial advice***