text:
Dear Uncle Tusk,
So anyways, here I am playing a little Goldeneye multiplayer with a couple of my buddies here at college. (I was winning, of course) One of the guys kills another guy. After the death-throes and all, he continues to plug the dead guy with the ol' RCP-90. I, of course, questioned him on this action. He replied, "Because I want the 'Most Dishonorable' award!" I responded by pumping him full of auto-shotgun shells. I thought the award was if you had the highest ratio of people hit in the back, or while they weren't carrying a gun, or something like that. It was then that I realized that none of my friends know what all the multiplayer awards mean. Some are, of course, kinda obvious, i.e. Double Kill, Where's the Ammo, and so on. Some are a little ambiguous, like Marksmanship Award, Most Honorable, etc.
So, if you're in a generous mood. Could you slap those GE designers around and have 'em cough up a list of the award meanings?
Say "hi" to Maya for me,
Scott Zdankiewicz
Uncle Tusk replies:
Not a problem.
"Most Dishonourable goes to the player with, as he surmised, the highest number of shots in other players' backs. Continuing to shoot at dead bodies gets the actually-not-in-the-game-at-all, 'Waste Of Ammo' award. Most Honourable should, in light of the above, be obvious. Marksmanship goes to the skilful spy with the highest percentage of head shots. And nobody slaps me around."
These last few words were, of course, wailed back over his shoulder as he ran off clutching his face.
Dear Uncle Tusky-Poo;
I was recently turned on about the fact of the Donkey Kong Country TV Show. I saw it on the Fox Family channel and I instantly hit the roof. Of course it hurt. But what in the heck were you thinking when you made the show dudes? Try to stay along the storyline of the game. Where is this magic coconut in DKC 1-3???
Peeved, but OK
Chris Glass
Uncle Tusk replies:
Beats me. Nothing to do with us. I suppose the writers felt they needed to broaden the span of the DKC storyline if they were going to get a whole series out of it. Me, I'm looking forward to the one where DK takes the damn coconut and shoves it up Cranky's ("museum" - Ed).
Dear Uncle Tusk,
I think that there is one peculiar thing about Banjo-Kazooie that has never been explained: Where is the second yellow pot? I have 100 jiggies, 900 notes and all that stuff, but I've never seen the second yellow pot. Is it in Banjo-Tooie, functioning as the secret passageway between the two games, perhaps? Or is it placed in a really obvious place in BK, making me look stupid for not finding it? Please answer, or I will call upon my viking neighbour, Steinar, who will pay a fortune in money for the airline-ticket, take a week off his work, and travel all the way to the jungle outside Twycross just to... er... "teach you a lesson".
Thomas Ertresvg
Uncle Tusk replies:
It appears that stupidity may indeed be your problem. According to the game's designer, you should be able to find one of the pots behind the starting point of Grunty's Furnace Fun, and the other in the room at the top of the tower with the 810 note door. Now stop being thick. It's getting tedious.
Dear Uncle Tusk,
Okay, I have a lot of questions, but this is the one that has bothered me for three years now so I have to ask it: Where on earth did you get Dixie Kong's first name from?! That is my mother's first name, and since I look so much like her, my uncle calls me "Little Dixie." Then, when his grandson bought the game (DKC 3) and found out I was a million times better than his grandson, I got teased endlessly for it. I have long blondeish hair and greenish eyes, too, and that didn't help! Dixie is a good name and all, but I think you could have named her something else!
Christina Shepherd
Uncle Tusk replies:
Be grateful your nickname isn't Donkey. Anyway, here's the designer with a deeply technical explanation for this one.
"Using our Rare Random-O-Matic Name Creator, the best we could do was 'Dixie'. We thought that was OK - surely better than early suggestions of 'Diddiane' and 'Diddette'. By the way, if you can jump off tall objects and spin your hair around like a helicopter we may have a job for you..."
Dear Uncle Tusk,
This is a bit of an odd cry for help, but the other day I borrowed my friend's (albeit dust-laden) NES, and Battletoads. Getting to the point, how in the name of all things fatal does one finish the second rat race? The guy's just too quick! He always wins! Is this just really a cruel ending - the programmers gave up and made the rest of the game impossible to reach?!
Uncle Tusk replies:
Apparently, if you "give the rat's ass a good kickin' on the way down", it will serve to "slow his rather rapid progress". Didn't you try that? What, are you one of those mincing pacifist types, or something? I'll kick your face off.
Dear Uncle Tusk,
Hello, I have a ton of questions for GoldenEye but I'll give them to you one at a time. My most important question is (you'll probably need the people who made the game answer these) I found a GameShark code that gives me a weapon that I want, now what startled me was there were a new weapon and a gadget that I found. The weapon was a briefcase that you could throw, it would clamp onto anything and would blow up in 5 second. The gadget looked like a covert modem but had an antenna and a thing where the camera could see. Now what I want to know is why you hadn't put those two things in for, because the briefcase bomb is cool, and what was supposed to be the use of the camera?
Gabe Stickland
Uncle Tusk replies:
None too impressed with your GameShark corruption stupidity, the designer just muttered something about things being unlikely to make sense if you're going to fiddle about with the game's inner workings. He didn't know what you were on about as far as the 'camera' is concerned - probably just corrupted graphics - and you've just given the briefcase graphic Timed Mine properties rather than finding a brand new weapon, you dunce. Remember, kids - messing about with perfectly good code can be bad for your health when Uncle Tusk arrives on your doorstep with the Big Stick of Good Behaviour.
Yo Tusky,
What happened to Thunder from the original KI? Are you his son? Did you eat him? You live in Montana, don't you? I think Jago and Orchid should hook up. And how about that chick Dixie? Grrrrrrrow! She's hot. If only I could kill Diddy... um, no! I didn't say that! You should battle the Jinjos. I know where Winky went! Try Monkey Museum. Donkey and Candy Kong should make a... never mind. See ya Tusky.
Skull Monkey the 7th
Uncle Tusk replies:
Thunder smacked Spinal to bits then went home. That's all there is to it. Considering I was born over 2000 years before him, I'd say he's unlikely to be my long-lost Dad. Oh, and Montana doesn't exist where I'm from, and Jago and Orchid 'hooking up' would be an arrestable offence, you sick fool.
I just read a letter from M. Stephen about the "deep, dark, secret" in DKC. I didn't think anyone else noticed that! Anyway, in answer to his question, I found a warp from the beginning of the level to almost the end. It could of been a glitch, but I doubt it. Lemme tell you how I found it.
Back when I still owned the game, I had a Game Genie (I happened to use it for things other then cheating). One of the "cheats" was to be able to keep an animal to the next level. So I brought the fish into the level. I found out fish could fly, so I took some flying lessons. Above the first cart, I flew above the screen. Suddenly, I heard the sound associated with a warp barrel, and then the screen moved through the level to the last kart, where it launched me into the cart. My fish turned into a blue Diddy and then it was over.
So Mister Tusk, do you accept or deny this secret?
Larry Gajewski
Uncle Tusk replies:
Another one for the designer, then, because I'm bored of the whole stupid subject already.
"Yes, the warp barrel does exist. In fact, there are warp barrels near the start of every level of the first two worlds. And everyone knows that fishes that turn into blue chimps can fly."
Dear Uncle Tusk,
I'm going CRAZY!!!!! I have beaten DKR from top to bottom and it's getting so easy, that just today I started racing looking at the screen upside-down!! I need to know if there is anything else to do that I haven't done, so that I can stop trying to beat my Ancient Lake record of 00:52:13 in the T.T. Time Trials. I have 47 balloons in Adventure 1 and Adventure 2. I have beaten all 4 Wizpig races (2 in each Adventure). I also have the Future Fun Land trophy in both adv. (I'm tired of saying adventure already). I have Drumstick and T.T. as well.
Jason W.
P.S.: 00:52:13 is without balloons.
Uncle Tusk replies:
"There is a hidden method of controlling the characters that can be used to increase your speeds on the tracks by enormous amounts. It involves the A Button but is very difficult to do, though testers at Nintendo had used it to get times on Ancient Lake of around 34 seconds!"
Thus spake the DKR man, so get to it. Alternatively you could just buy yourself a new game, you tightarse.
Dear Uncle, (that is if I may call you uncle).
I am a pathetic single bloke desperately in need of love. I am in Leeds. Do you know if you could give me some tips to get on with sewing my wild oats.
A. M. Modha
Uncle Tusk replies:
While I understand the extra stress that your location must place on your social life, the fact that there's no mention whatsoever of Rare games in your query means there's nothing I can do for you. Unless of course you're one of those sad fools trying to scavenge a picture of Orchid in her pants, in which case I'll be more than happy to pop round and smack some sense into your stupid head with a brick.
Dear Mr. Tusk...
I bought a SNES a few years ago with DKC. Yesterday I decided to go back and revisit the wonders of a console (I use a PC now). I realised that I had PASSED the game but I hadn't found all the secrets. I had only 94% and had taken 4 hours and 4 mins.
Is it possible to get 103 & 104% like what I have in DKC2&3??
Thankyou for your help....
A guy named Mephisto in Australia. :D
Uncle Tusk replies:
As far as we can remember, the original DKC 'only' went up to 101%. Though even that was pretty wacky in those days. If that's not enough for you, and you're at least moderately dull-witted, you could always break some of your fingers to increase the challenge.
Dear TUSK,
I have a Question about BANJO-KAZOOIE.
In the tippy top portion of the lair, there is the room that Grunty has two transforming devices that are controlled by a red lever in the center. Is there any way to move the lever and open the machine doors? And also is there a way to open the wooden doors on either side of the room?
Thanx TUSK (you brute).
Drew Hamann
Uncle Tusk replies:
The official word is "No, and no." I HOPE that's Answered your Question, you STRANGE young man with Your Mad GRAMMAR.
Dear Uncle Tusk,
My problem is this: I'm playing Goldeneye, on Agent level, in the Control Center. In the first hallway, outside the elevator, there are no drone guns! Everything else is there: The guards; the computers; that little pest called Boris, but the first few drone guns are just NOT THERE! Are they supposed to be missing on Agent level, though I don't think they are? Is it a glitch?! Is my copy messed up?!! Or could it be just some weird one-time glitch, and I should just calm down and try again? Thanks, O Great and wise Uncie Tusk.
Losing control in the Control Center
Uncle Tusk replies:
Sounds to me like this is a mere surface manifestation of some far more deep-rooted paranoid anxiety problem. See a doctor, you nutcase. There were never any drone guns in that area on Agent level, mainly because Agent is the default Easy setting. Easy = less obstacles. See? Most people notice that the first time round, but then again most people aren't preoccupied with running round in circles, firing at thin air and screaming like a madman.
Dear Tuskie,
How come you never answer my letters, you good for nothing (word I'm not allowed to say)! I've sent two very good letters, also. I'm just wasting my time writing to you, aren't I? Geez, how you almost never answer your mail? AND WHEN IS THE FREAKIN' HECK IS BANJO-TOOIE COMING OUT????????????????????? You better answer my letter or uh, uh, something REALLY bad is gonna happen to you!
Uncle Tusk replies:
At the moment, Banjo-Tooie is aiming vaguely for a 4th quarter '99 release. You know we don't like release dates, so that's the most concrete answer you're going to get from us right now. We'll release it when the team's happy with it. And please don't threaten me, Mr. GamerX. I bet you're hard as nails and there's no way I could, for example, turn you inside-out with one hand.
You da man Tusky me boy, you da man!!
The way I wait for you to answer
another mailbag for me to read borders on idolatry. I have a small shrine set up in your honor in my basement where I regularly butcher one of my idiot neighbors during joyous celebrations to you oh great Tusky. Whenever I fight against you in KI I drop my controller and allow myself to be hacked to ribbons so that you are not angered with me. Okay, I go past bordering on idolatry. But if worshipping your great barbarianship is wrong then I don't wanna be right. I only have one small favor to ask of you. Would you mind taking your sword and carving your name into the back of my skull, it means a lot to me if you would. In Tusky's name I pray. Amen.
Uncle Tusk replies:
Look, stop messing about. If you want a fight, just come out and say it.
Dear Uncle Tusk,
Can you please settle a long running argument that I have with some friends.
Is it right when playing Goldeneye to look at other players' screens? I think it's perfectly okay. My friends think it's the work of the devil. What do the creators think? Cheers.
Andrew Middleditch
Uncle Tusk replies:
The creators have these statements prepared:
"We have too much innate honour to do something so despicable. We don't mind who watches what; it's up to the individual to decide. We're so amazing we watch everyone else's screen because we don't need to watch our own. We are not bothered because that means the result comes down to skill.
"Delete the replies that make you feel bad."
Dear Uncle Tusk,
Diddy Kong has been bugging me for quite some time... this character seems to have a glaring consistency problem. "What is it?" Why, quite simply...
How did Diddy Kong change from a chimpanzee to an ape?
In DKC and DKC2, Diddy clearly has a long tail (he even uses it to hang from things in DKC2), whilst in DKC3 and DKR, Diddy's tail has vanished. Which of the following is true?
1) Diddy was transmogrophied (He was what? - Tusk) when Rare realized how silly it was to give the Kong family name to a chimp...
2) During the Beta testing for DKC3, Diddy didn't quite make it through a nasty trap of some kind.
3) There are 2 Diddys - Diddy Kong (of DKR), and Dyddy Kong (appearing in DKC -- obviously a "secret character" called by the D-Y-D-D-Y code).
Please, tell me ... the world must know!
David Dayton
Uncle Tusk replies:
Several minutes of dubious umming and ahhing from the designer of DKC 1 & 2 led to the following shameful excuses:
"In DKR he sits on his tail in his vehicle to keep it warm, and in DKC3... grrr... ahh... he cut it short as this was the fashion then. Is that good enough?"
Not by anyone's standards, I'd say. So I prodded the lead artist on DKC3 for answers instead.
"Er... I don't remember modelling him without a tail. Anyway, he was only in it for a split second. Hold on, I'll find a cartridge with a saved game." (Long pause) "Right, I've just beaten the boss and Diddy's quite clearly got a tail." Just as I thought. Stupid people writing in again.
Dear Uncle Tusk,
Whilst perusing the collected outspurtings of your wisdom, I stumbled across a reference to the all-platinum end scene to Blast Corps. Yet in every other demesne of the Web, one cannot swing a dead Java script without hitting a complaint that the platinum standard, although inhumanly difficult to achieve, confers upon its proud champions no additional end scene.
Don't lie to Nephew: is there truly a platinum end scene, or was this merely another example of the sparkling wit that makes you such a sought-after guest at barbarian dinner parties?
(And as long as I'm asking dumb questions, where's the hidden George Lazenby character in Goldeneye?).
Bennett Darrow-Chicago
Uncle Tusk replies:
Depends on your definition of the word 'scene'. There's no forty-minute FMV extravaganza, no, but the rankings keep on going up as the Platinums are collected until the player is faced with a devastating exposé of his or her individual destiny. It's true.
As for George Lazenby, you must have blinked and missed him. Again.
Dear Uncle Tusk,
My brother claims (this is not a joke letter!!!) he was playing Goldeneye at a toy store when the game came out. He was on Facility and said a chef with a mustache, knife, chef hat, and an apron came after him swinging his knife. The chef, he claims, came out of the door down the stairs to the right. Is it true that this could have happened? Was the chef put in the game and then taken out? My brother is very serious about this and I don't think he is lying.
Ten K
Uncle Tusk replies:
Well, that makes one of you. This is the official word on your brother's wild claims:
"There is no truth to his assertion. Whenever he refers to this incident, pat him kindly on the back and say 'There, there. We believe you' in your most patronising tone of voice. Cut down on his sugar intake and give him crayons to draw with. You might also consider dressing as a moustachioed chef and jumping out at him in the middle of the night."
To a "Mr. Tusk",
A cat who claims to be yours was recently sent to our islands to be blown up in a nuclear bomb explosion. We regret to inform you that your so-called "cat" has digested our entire supply of armed test bombs, has horribly mutated and seems to have it in for your wife, a Mrs. Maya Tusk, for, as the cat put it, "repeated kicks to the head during kittenhood". We just thought we should warn your wife of the impending doom.
The Mayor of the Biggest Falkland Island
Uncle Tusk replies:
Aha! I wasn't sure how convincing that wig and bikini would be on me, but they seem to have done the trick. Now, as soon as Maya's out of the way, there's nothing to stop me and Sniffles from -
Oi! This is none of your business, alright? Leave us alone! We just want to be happy...
Friday, October 23, 1998
Ask Uncle Tusk: October 23, 1998
Posted by The Arscribes at 6:00 PM
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text:
I was playing around with some pictures we had on the digital camera and here's a picture of my eye.
PEEKABOO I SEE YOU! lol.
Anyway, the hubby and I often have discussions as to what color it is, especially when we're trying to decide whose eye color our children have. He says my eyes are blue. I disagree. I say they are grey with greenish tints. Sometimes they look turquoise, but not exactly blue.
We had one of those discussions this very morning, lol.
So what do you all think?

subset: commoncrawl


text:
Loosely associated statements
A loosely associated statement is a type of simple non-inferential passage wherein statements about a general subject are juxtaposed but make no inferential claim.[1] As a rhetorical device, loosely associated statements may be intended by the speaker to infer a claim or conclusion, but because they lack a coherent logical structure any such interpretation is subjective as loosely associated statements prove nothing and attempt no obvious conclusion.[2] Loosely associated statements can be said to serve no obvious purpose, such as illustration or explanation.[3]
Included statements can be premises, conclusions or both, and both true or false, but missing from the passage is a claim that any one statement supports another.
In A concise introduction to logic, Hurley demonstrates the concept with a quote by Lao-Tzu:
Not to honor men of worth will keep the people from contention; not to value goods which are hard to come by will keep them from theft; not to display what is desirable will keep them from being unsettled of mind.— Lao-Tzu
While each clause in the quote may seem related to the others, each provides no reason to believe another.
See also
- ↑ Hurley, Patrick J. (2008). A Concise Introduction to Logic 10th ed. Thompson Wadsworth. p. 17. ISBN 0-495-50383-5.
- ↑ "The logic of arguments". Retrieved April 28, 2012.
- ↑ "NONargument - Loosely associated statements". Retrieved April 28, 2012.


text:
After my post about our new dining room setup I discovered, via a comment from my brother, that I never showed you our newest furniture addition.
Back at the end of December we went to Biloxi for a family Christmas Party and while there we visited a furniture store. We had been on the hunt for a new kitchen table for almost a year but just couldn't find something we were sold on. That was, until we laid our eyes on this beauty.
It fit all the needs and wants on our list
- – Seat 6, check
- Solid wood, check
- Bench on one side so the table can be pushed against the wall, check
- Comfy on the tookus, check
- Last us the rest of our lives, [pre-determined] check
The chairs will be perfect for a booster seat when Sarah gets to that point
I'm a huge fan of the chairs. The lines are a combo of smooth, semi-curvy, & sturdy
This is the view from behind the kitchen counter
I felt like I was photographing a model with this angle… I'm such a big fan of this table
Despite the fact that the kitchen is all setup, we still have some work to do. The table needs to be stained. We got the table in the winter while I was almost 7 months pregnant so staining it indoors wasn't an option because of the fumes, and we don't have a covered outdoor space that will work. Over the past 9 months we did find a stain we both like (procrastinate much?!?). Now we just gotta figure out the when & where so the table can be completed. We should probably do that soon since this is the table we plan on having for the rest of our lives. It should definitely hold up that long and longer. Perhaps if she likes it (and her future-friends don't damage it too much) we will will it to Sarah for her family one day :)
And that's the story of our [belatedly new] kitchen table. (your welcome, Matt) ;)


text:
I Think This Sums Up Our Team
Yeahhhh. We suck.
Re: I Think This Sums Up Our Team
That alone should be reason enough to play Biyombo over Diop. Look how winded he is between shots. He needs to sit his big over-paid ass on the bench.
Re: I Think This Sums Up Our Team
It really makes no sense.
Honestly I'm not impressed at all with Silas' coaching this year. I know he has very very few options, but his rotations leave a LOT to be desired and his lack of any play calling when our team is so young is pretty incredible. When he put that as a shortcoming on Kemba's part that he couldn't just "ad lib" with the group as a PG he pretty much lost me for good.
I see no excuse for not giving Smack all the minutes he can handle...and especially over Gana.
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text:
Before dawn on Sunday, Muslim pilgrims from around the world began ascending a hill just outside Mecca where the Prophet Muhammad delivered his final sermon some 1,400 years ago.
The day spent on Mount Arafat is the pinnacle of the five-day hajj pilgrimage, which all able-bodied Muslims are required to perform at least once. Muslims spend the day there in deep prayer, many openly weeping as they repent and ask Allah for forgiveness.
Prayer on this day at Mount Arafat, about 20 kilometres east of Mecca, is believed to offer the best chance of erasing past sins and starting anew. Many Muslims who are not performing the hajj fast from dawn to dusk on this day, for similar reasons. Many of the roughly two million pilgrims taking part in this year's hajj will climb a hill called Jabal al-Rahma, or mountain of mercy, in Arafat and spend time there in supplication. It was here where the Prophet Muhammad delivered his final sermon, calling for equality and for Muslims to unite.
The white terrycloth garments worn by men throughout the five-day hajj are forbidden to contain any stitching - a restriction meant to emphasize the equality of all Muslims and prevent wealthier pilgrims from differentiating themselves with more elaborate garments.
The day on and around Arafat is the one time during the hajj when roughly all pilgrims are in the same place at the same time. The sight of people from more than 160 different countries, with all the men dressed in simple white garments, is breathtaking. Egyptian pilgrim Mahmoud Awny said the feeling of being in Arafat is "indescribable." "All Muslims on Earth wish they could have been here today. Thanks to Allah for enabling me to be here," he said. The hajj is a physically and emotionally exhausting experience, and this year temperatures soared to 42 C in Arafat. Volunteers passed out water, juice and umbrellas to shade pilgrims from the sun.
Around sunset, the pilgrims will head to an area called Muzdalifa, nine kilometres west of Arafat. Many walk the route, while others use buses. They spend the night there, most in the open air huddled near one another, and pick up pebbles along the way that will be used in a symbolic stoning of the devil in Mina, where Muslims believe the devil tried to talk the Prophet Ibrahim - named Abraham in the Bible - out of submitting to God's will.


text:
Well, there are a bunch of sites where you can find good music you can use for free in your video. Some like ccMixter are community sites with loads of composers brought together, others are run by individual composers and artist who want to get their music out there, by any means, to get some publicity.
Pretty much all these sites licence their work under the creative commons licence. There are four types of licence, which you can read about on the Creative Commons site http://creativecommons.org/about/licenses/
Mostly you'll find these composers will let you use what ever you want – however you want. All you need to do is give them a credit at the end of your video. But, do check which licence each composer is using to release their work.
So where to start?
This is all music composed and/or produced by Kevin MacLeod, who believes that: "If your art isn't experienced by people, it serves no purpose in society."
He is offering his royalty-free music for free under the creative commons license. Give him a credit and you can legally use his music in your video. He also does a nice line in different types of graph paper (including storyboards).
The best page to start is here http://incompetech.com/m/c/royalty-free/ You can browse by genre, by feel (from action to uplifting) or do a keyword search.
In the genre section there are a few classical pieces from Mozart, Bach and Saint Saens.
Once you have listened to the music and chosen something you like just click the download button to grab yourself a copy.
Music 4 Your Vids
The title is a big hint. Rick Clarke (also known as RickVanMan) is also offering free to use royalty-free music. Like Kevin (above) all he asks is you give him a credit and he would appreciate a link to his site. A small price to pay as there is some really good music here.
Searching for music can be time consuming, so I really like his idea of putting samples of his music together in short compilation videos. Within a few minutes you can get an idea of what is available and which tracks you want to hear in full.
The Vimeo Music Store
Vimeo is usually the place I recommend to upload videos. But, they obviously realised that their subscribers need music and have now opened their own music store.
It is simple to search by composer, genre or keyword. Finding the right music is time consuming so you want to be able to bookmark something interesting, so I like that I can save my favourite tracks for later.
Some of the results will be free and some will be modestly priced ($1.99) if it is for personal use. But, some are expensive if it is for commercial use
Hang on, this is Vimeo - they have a video to explain all this.
Some of the results will be free and some will be modestly priced ($1.99) if it is for personal use. But, some are expensive if it is for commercial use
"ccMixter is a community music site featuring remixes licensed under Creative Commons where you can listen to, sample, mash-up, or interact with music in whatever way you want."
The looking for…? page is a good place to start as it shows everything the site has to offer. There's loads of music, mixes, samples and acappellas to be had. You'll easily spend hours listening to it all. Just click the play icon and the music will play straight off the page. If you like it – you can download it just as simply. There is no need to register to downoad.
The Freesound Project
"Freesound focusses only on sound, not songs." http://www.freesound.org/
So, if you need the sound of someone hammering, perhaps the sound of forest birds singing in the evening or some punching and slapping for that extra dramatic effect – this is a great place to come.
The files are usually .wav files 44,100Hz, 1411kbps, 16-bit, Stereo.
Just click the play icon and the audio will play straight off the page. If you like it – you can download it just as simply. But, you must register and login to download.
Audio Farm
Audiofarm: "It´s the best and easiest way to find new and interesting music and audio recordings from around the world."
This has a wide range of audio from Punjabi jingle bells (honest) to Electronica. You can search, check to see what is the most liked and take a peek at what's new. Once you click on a link it opens on the clips page and the audio starts to play automatically and almost immediately. There is no need to register to download.
Yes, the multi award winning composer and performer is giving away his music – kinda.
If your video is part of a not-for-profit project – then Moby has very generously opened up some of his music. http://www.mobygratis.com/film-music.html
You will need to register and let him know what his music is being used for. There is some good stuff on the site (if you are a Moby fan).
Musopen calls itself a musical warehouse "our mission is to set music free". You'll be able to find music by composer, performer, instrument, genre and also by period.
Not all of the music here has the full ambience of a professional orchestra in London's Royal Albert Hall, but there a a few gems worth downloading - everything is free.
Jewelbeat sells individual WAV and MP3 tracks from 99 cents, and has more than 35,000 tracks in its library (the full library costs $999 and will be delivered to you on disk). There are also JewelSticks (themed libraries of hundreds of tracks on a USB stick) for $99.
For flexibility, like most traditional music libraries, there are multiple versions of tracks, with alternate mixes, 60-second, 30-second, 15-second and 10-second versions, and stingers.
There are also more than 1,000 free sound effects and loops available with any purchase (even a single 99 cent track).
If your project is non-commercial or educational there are some dowloads here you a can use for free.
Royalty Free Classical Music
If you can afford to pay then Royalty Free Classical Music is a great place to start. Each music download is $34.99. Compilations of 10 tracks are also available for $129.99.
The music here has high production values with a full professional orchestra - not some guy in the spare bedroom with a synth. If you know your Brahms from your Verdi - then search by composer. Alternatively you can check out their most requested top 200.
Shockwave-Sound.com offers lots of royalty-free tracks, although not FREE music, including what it claims to be the largest selection of royalty-free classical music on the web.
It does have a lot of good information about royalty-free music, and many articles about using music in your video, sound mixing, some hardware reviews, etc. Worth a look.
AudioJungle has a wealth of good cheap music. Some cost as little as $2 up to $14 for royalty free music tracks in over twenty different genres.
I also liked the Logos and Idents section for short snappy music clips between 10 and 30 seconds long.
I've been recommending Pond5 video stock footage to my clients for ages. But, over the years they have branched out into music (plus photos and illustrations). If you have footage you want to sell they do a good deal too.
For ease of use, they have to be the best.
For music click on the music tab at the top of their webpage. There you'll see around 40 different categories to choose from. But, you can also use search words too. Once you've narrowed it down I'd recommend you place put them in order... If you use the - Sort by: PRICE - starting with cheapest first - you'll see some great bargains. Some tracks start at only $2. If you are in a hurry try doing a sort by NUMBER OF SALES. that way you'll quickly see which are the most popular.
Your results will all appear on screen and listening to them is so simple. Just hover your mouse of the music waveform thumbnail and you can hear the music and see how much it cost. If you want to know more about the track - just click on it and you'll be taken to the info and download page.
OK not a download site - but, this music software is simple to use...and you'll own the copyright as you created the music.
If you are a Mac owner you already have GarageBand loaded on your machine. There is help on the Apple site to show you how simple it is to make music - even with limited musical ability.
I often recognise background music on UK TV programs that was created using GarageBand music loops.
Find Sounds is a site full of free sound effects www.findsounds.com
A couple of years ago Karen Abad left a post on a Vimeo forum of suggestion for music resources. It has been added to over the years - you'll definitely find something useful here
If you can afford to pay, go to the Performing Rights Society website
The UK Intellectual Property Office has a wealth of info on copyright.
Impress your friends with your legal knowledge... read the UK copyright act at legislation.gov.uk
If you are in the UK check out the Institute of Videographers (IOV) - they have simplified buying a music licence.
Public Domain 4 U hosts music, old and new, which is in the public domain. You can download to enjoy - but you would have to check with the artists if it is OK to use their music in your video.
Some of the old 78 stuff is very evocative - complete with scratches and fluff on the needle.


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Abu Dhabi ISAF Sailing World Cup 2014
Open Kiteboarding
Riccardo Leccese (ITA) came out on top on the opening day of kiteboarding after he picked up a pair of bullets. He heads into the next stage as top seed. 2014 World Champion Maxime Nocher (FRA), European and Open Asian Champion Oliver Bridge (GBR) and 2013 World Champion Florian Gruber (GER) follow in places 2-4.
The 20 competitors will be divided up for Stage 2 heats on Friday and Saturday based on their seeding from Stage 1. The top ten boats will then progress to the medal stage.
Two semi-finals will be held with five racers in each. Places 1 and 2 will carry forward ten points, 3 and 4 seven points, 5 and 6 five points, 7 and 8 four points and 9 and 10 three points. Two races in each semi-final will follow with the top two placed sailors in each heading to the final.
From there, it's a four way single race shoot out for the podium spots.
Racing across the fleets is scheduled to commence at 12:00 local time on Friday 28 November.
Results are available here - www.sailing.org/worldcup/results/index.php


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Multiple Businesses Phone System
If you manage multiple small businesses based on different SkypeIn numbers, you can use PrettyMay Call Center for Skype to organize these phone lines easily.
With PrettyMay, you will be able to set up different IVRs (includes welcome greeting, extensions / voicemails, etc.) for each business based on dialed SkypeIn number.
- Multiple simultaneous lines supported - with multiple SkypeIn numbers, you will have up to 30 simultaneous lines supported in PrettyMay.
- Managing different business phone lines within one PrettyMay software.
- Set up different IVRs for each business.
- Full multilingual voice menu supported.


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Bengals Canned Food Drive
Media•on December 2nd, 2011•Comments Off on Bengals Canned Food Drive
The Cincinnati Bengals and the Freestore Foodbank are pleased to announce the results of the annual canned food drive which was held prior to the Cleveland Browns game on Sunday 11/27/2011. Bengal fans donated 8,120 pounds of non-perishable and canned goods and $14,021 in cash donations to help feed the hungry in our community. Fans surpassed last year's canned food drive by 1,000 pounds of food and exceeded cash donations by $2,600. Thanks to everyone for your support. If you weren't able to make it to the game, and would still like to donate…Click here.


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I don't agree with MacArthur, that there is any spiritual beauty in Christmas, because Christ-mass is the invention of the Roman Catholic church in its attempt to gain converts by syncretizing paganism with their dead orthodoxy. I cringe when I hear the story of Christ's birth referred to as "The Christmas Story", because the Christmas Story is the story of the history of the Roman Catholic church and its attempt to entice the whole world into a spiritually dead entity by creating a "holyday" (holiday) for some very unholy, and spiritually dead people.
The early church did not celebrate Christ's birth. Their focus was on His death and what it meant to them: new life in Christ and the death of their sinful flesh. But what MacArthur says about Christ's purpose for coming to earth is right on: Jesus Christ came into this world to save us from our sin. If you miss this truth, you miss everything. All four of the gospels mention Christ's death, but only two of them His birth. I think that says something about how we should spend our time. In our culture, the whole month of December , and sometimes even most of November is spent preparing for and joining in various Christmas festivities. I believe it is high time true followers of Christ take a careful look at their involvement in these activities, and ask themselves if it is a true reflection of their faith, if the time spent is a true reflection of their love for Christ. I can't help but believe that it detracts from the message of the cross, or rather eclipses it. Some will say that we have Easter for that. But once again, Easter (Astarte/Ishtar) is the syncretization of paganism and dead orthodoxy, and then it is only for the span of three days. How disproportionate compared to the time we focus on His birth each year. The true Christian church has been duped into following in the footsteps of the Roman Catholic church once again.
I was having a conversation with my husband today about how we and other Christians have allowed ourselves to be swallowed up by our culture. It is a very gradual process, being barely perceptible. It has been rather interesting for me to note that since I have abandoned the celebrations of Christmas and Easter in our home, that living the crucified life has become much more important; or maybe it is the other way around, that learning the significance of Christ's death for me, and the necessity of living a crucified life myself, that the attractions of these two seasons has been lost to me. I have actually experienced a real freedom in this; and not only that, I have found I have many more opportunities to witness to people I see everyday when they ask me if I am ready for Christmas (or Easter, if that is the time of year). The question we need to be asking people is whether or not they have dealt with their sin, and whether or not they are ready to face Christ. I see the majority of Christians these days la-de-da-ing through life as if there is no eternity, so caught up with this world that they have become ineffective in their ability to witness the truth to those around them. We are kidding ourselves if we think we can follow in the pattern of the world and have any true impact. I hate that phrase, "He (or she) is so heavenly minded he is of no earthly good". That is a phrase that contradicts Scripture. We are supposed to set our minds , hearts and affections on things above, not on things of the earth.
As this season is coming to a close, let us examine whether or not we are truly focusing on what is important about the Christian life. I have come to learn that the Christian life is first and foremost a cross-centered life. We must first of all come to the cross, and then we must stay there until Christ calls us home. We cannot lead others to the cross if we have wandered away from it ourselves. If you have found yourself in this situation, I call you to ask God to give you a fresh view of your soul. Look intently into the Word, let it be your mirror, and agree with God about what He says about your condition. Remember that apart from Christ there is no good thing that dwells within you.
There is a beauty in the cross, in that it deals thoroughly with our sin, the very ugliness of our own souls. When you understand this, you will cling to the cross, carrying it with you wherever you go. As we enter a new year, it is my prayer that you will carry the cross as your standard; that you will experience the true freedom and joy that comes from living the crucified life so that you can look forward to seeing Christ face to face with great anticipation, and upon seeing Him hear those wonderful words, "Well done, thou good and faithful servant".
For further reading: THE FOOT OF THE CROSS
by Octavius Winslow, 1864.
From the preface ~
"It is finished!" -but what mortal dare
In that triumph hope to share?
Savior, to Your cross I flee;
Say, It is finished! and for me.
"Then will I sing, The cross! the cross!
And count all other gain but loss;
I'll sing the cross, and to Your tree
Cling evermore, blessed Calvary!


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Oestrogens exert their physiological effects through two receptor subtypes. Here we report the three‐dimensional structure of the oestrogen receptor beta isoform (ERβ) ligand‐binding domain (LBD) in the presence of the phyto‐oestrogen genistein and the antagonist raloxifene. The overall structure of ERβ‐LBD is very similar to that previously reported for ERα. Each ligand interacts with a unique set of residues within the hormone‐binding cavity and induces a distinct orientation in the AF‐2 helix (H12). The bulky side chain of raloxifene protrudes from the cavity and physically prevents the alignment of H12 over the bound ligand. In contrast, genistein is completely buried within the hydrophobic core of the protein and binds in a manner similar to that observed for ER's endogenous hormone, 17β‐oestradiol. However, in the ERβ–genistein complex, H12 does not adopt the distinctive 'agonist' position but, instead, lies in a similar orientation to that induced by ER antagonists. Such a sub‐optimal alignment of the transactivation helix is consistent with genistein's partial agonist character in ERβ and demonstrates how ER's transcriptional response to certain bound ligands is attenuated.
Oestrogens play a critical role in the growth, development and maintenance of a diverse range of tissues. They exert their physiological effects via the oestrogen receptor (ER), which functions as a ligand‐activated transcriptional regulator (Tsai and O'Malley, 1994). Until recently, these effects were attributed to a single ER. The unexpected discovery of a second ubiquitous ER, termed ERβ (Kuiper et al., 1996; Ogawa et al., 1998), has added another layer of complexity to the action of oestrogens and prompted intense interest in the respective role of each isoform (Katzenellenbogen and Korach, 1997). The two ER isoforms exhibit overlapping but distinct tissue distribution patterns and differ in their ligand‐binding ability and transactivational properties (Kuiper et al., 1997; Barkhem et al., 1998; McInerney et al., 1998). Preliminary data from ER knockout studies demonstrate that each isoform has a separate biological role (Korach, 1994; Krege et al., 1998).
ER is a member of a large family of nuclear receptor (NR) transcription factors with a characteristic modular structural organization with distinct domains associated with transactivation, DNA binding and hormone binding (Tsai and O'Malley, 1994). ERα and ERβ share modest overall sequence identity (47%) with little or no detectable homology between their N‐terminal transactivation (AF‐1) domains but well conserved DNA‐ and ligand‐binding domains. The C‐terminal ligand‐binding domain (LBD) is multifunctional and, in addition to harbouring a ligand recognition site, contains regions for receptor dimerization and ligand‐dependent (AF‐2) transactivation (Fawell et al., 1990; Danielian et al., 1992). Hormone binding to ER‐LBD induces a conformational change in the receptor that initiates a series of events that culminate in the activation or repression of responsive genes (Tsai and O'Malley, 1994; Beato and Sanchez‐Pacheco, 1996). The precise mechanism by which ER affects gene transcription is poorly understood but, at least in the case of AF‐2 activation, appears to be mediated by numerous nuclear factors that are recruited by the DNA‐bound receptor (reviewed in Torchia et al., 1998).
ER is an important pharmaceutical target for hormone replacement in menopausal women and for chemotherapeutic drugs against certain reproductive cancers. A wide repertoire of structurally distinct compounds bind to ER with differing degrees of affinity and potency (Anstead et al., 1997; Kuiper et al., 1997; Barkhem et al., 1998). Some of these compounds, such as ER's natural ligand, 17β‐oestradiol (E2), act solely as receptor agonists, whereas others, typified by EM‐800 and ICI 164,384, function as pure antagonists (MacGregor and Jordan, 1998). A third category, termed selective ER modulators (SERMS), have the ability to act as both agonists and antagonists depending on the cellular and promoter context as well as the ER isoform targeted (Grese et al., 1997; Paech et al., 1997; MacGregor and Jordan, 1998). SERMS, such as raloxifene (RAL) and tamoxifen (OHT), are used clinically in the treatment of osteoporosis and hormone‐dependent breast cancer. RAL is a highly effective anti‐oestrogen in the reproductive tissues but acts as a partial ER agonist in bone and also lowers blood cholesterol levels (Draper et al., 1996; Gustafsson, 1998; Jordan, 1998). A variety of endocrine disrupters present in the environment also target ER. Phyto‐oestrogens are a diverse group of oestrogenic compounds produced by plants primarily as bactericidal and fungicidal agents. They represent the major natural exogenous source of oestrogenic molecules and may affect human health. The presence of such compounds in the human diet appears to be beneficial and may even confer reduced risk of hormone‐dependent breast and prostate cancer, and heart disease, and alleviate symptoms associated with the menopause (Adlercreutz and Mazur, 1997). Genistein (GEN), an isoflavonoid phyto‐oestrogen that is found at significant levels in soya beans and soy products, binds to both ER isoforms with moderate affinity but exhibits a preference for ERβ (Kuiper et al., 1997; Barkhem et al., 1998).
Previous crystallographic analyses of a number of unliganded and liganded NR‐LBDs have revealed a canonical fold for the NR‐LBD (Wurtz et al., 1996; Moras and Gronemeyer, 1998). Eleven major helices are arranged together in an antiparallel, three‐layered sandwich topology. In each case, the receptor's cognate hormone binds within a hydrophobic cavity buried within the core of the molecule. Agonist binding induces a conformational rearrangement in the LBD (Bourguet et al., 1995; Renaud et al., 1995) resulting in the formation of a specific binding site for the helical NR‐box module of nuclear coactivators (Darimont et al., 1998; Nolte et al., 1998). In the case of ER, bound receptor antagonists, such as RAL, sterically prevent the correct assembly of the AF‐2 region and the NR‐box binding cleft (Brzozowski et al., 1997; Shiau et al., 1998).
We have previously described the structure of ERα‐LBD in the presence of its natural ligand, E2, and the mixed agonist/antagonist RAL (Brzozowski et al., 1997). Here we present the first structural description of ERβ‐LBD in complex with RAL and the β‐selective partial agonist GEN. These structures, together with those already determined for ERα, provide a unique insight into the ligand‐binding properties of the two ER isoforms and their response to certain ligands.
Overall structure
The overall structure of the ERβ‐LBD framework is identical in the two ligand complexes and similar to that observed for ERα‐LBD (Brzozowski et al., 1997; Shiau et al., 1998; Tanenbaum et al., 1998). The core of the canonical NR‐LBD sandwich motif (H2–H11) is maintained with only minor differences in the length and relative orientation of the helices. A central layer of tightly packed helices, composed of H5, H6, H9 and H10, is flanked on one side by H7, H8 and H11 and on the other by H2–H4. A substantial hormone‐binding cavity lies immediately below H6 and sandwiched between the outer flanking layers. The position adopted by H12 is, however, very different in the two complexes (Figure 1).
Both human (GEN complex) and rat (RAL complex) ERβ‐LBD have been used in this study. These ERβs possess different size AF1 domains, but their LBD sequences are highly homologous and vary at only 20 positions between residues 254 and 504 {301–553} (Figure 2). Hereafter, the sequence numbering of human ERβ (hERβ; Ogawa et al., 1998) is used for both ERβ‐LBD complexes and the corresponding numbering of hERα is given in curly parentheses. The regions of high sequence variability between ERα and ERβ map to the N‐terminus prior to H3 and to a stretch of 35 residues in the vicinity of H10 at the top of the LBD. This distribution is similar to that observed when comparing ERα sequences from different species alone. Only two changes, at positions 336 {384} and 373 {421}, fall within the ligand‐binding cavity (Figure 2).
There are slight differences in the conformations of three regions of the structure that are induced due to changes in the crystalline environment. The short H1 observed in ERα is absent from both ERβ complexes as the first 7–8 residues at the N‐terminus are disordered. In addition, the loop regions connecting H2 and H3 (residues 282–289 {329–337}) and H9 and H10 (410–428 {458–477}) exhibit different conformations in the two structures (Figure 1A and B). In the (rat) rERβ–RAL complex, the H9–H10 loop packs tightly against an adjacent molecule within the crystal lattice. This has the effect of bending the N‐terminal end of H10 to one side so that it deviates by 20° relative to its position in ERα. Conversely, in the hERβ–GEN complex, where this region is not involved in intermolecular contacts, the loop connecting H9 and H10 is poorly ordered and the N‐terminal end of H10 is foreshortened (Figure 1B). The only significant structural difference in the framework region of ERα and ERβ concerns the relative positioning of H5. In both ERβ structures, the N‐terminal end of H5 (322–328 {370–376}) is shifted 1.5 Å away from H3 and towards H9 and H10 (Figure 1C). This has a subtle effect on the positioning of the side chains of Leu324 {372} and Val328 {376}, resulting in a slight widening of the hydrophobic groove between H3 and H5 in the region adjacent to Lys314 {362}.
In both ERβ complexes, the LBDs are packed so that the dimer axis coincides with a crystallographic dyad. The arrangement of molecules within the ERβ‐LBD dimer is identical to that seen previously for ERα (Brzozowski et al., 1997) and other NRs (Bourguet et al., 1995). The side chains of Met403 {451}, Ala456 {505}, Met460 {509} and Leu462 {511} form a central, tightly packed hydrophobic interface at the N‐terminal end of H11 that is stabilized on each side by a network of hydrogen‐bonding residues. The Cα r.m.s. deviation between the non‐crystallographic ERα‐ and crystallographic ERβ‐LBD–RAL dimers is 0.97 Å (393 equivalent atoms excluding H12).
Ligand recognition
The two compounds studied here are examples of the non‐steroidal class of ER ligands but they possess a diphenolic structural core that has similarities to the steroidal nucleus of E2 (Figure 3). The ligand‐binding cavity of ER is buried deep within the hydrophobic core of the LBD and surrounded by parts of H3, H6, H8, H11 and H12. Twenty‐two residues, predominantly hydrophobic in character, line the cavity and interact with the bound ligands. For convenience, the different regions of the ligand‐binding cavity are referred to with reference to the binding mode of the A‐, B‐, C‐ and D‐rings of E2 (Brzozowski et al., 1997).
Genistein binding
GEN binds across the cavity between H3 and H11 in a manner reminiscent of that observed for E2 (Brzozowski et al., 1997; Tanenbaum et al., 1998). The phenolic ring mimics the A‐ring of E2 and is clamped in the narrow cleft between H3, H6 and the β‐hairpin. The phenolic hydroxyl (O14) interacts with the side chains of Glu305 {353} and Arg346 {394} and a buried water molecule (Figure 4A and B). The flavone portion of GEN occupies a position similar to that adopted by the C‐ and D‐rings of E2 and is orientated so that the O2 hydroxyl (see Figure 3A for atom numbering scheme) makes a hydrogen bond with His475 {524} at the distal end of the cavity (Figure 4A and B). The imidazole side chain of His475 {524} is held in the correct position to act as a hydrogen bond acceptor by a second hydrogen bond between its Nϵ2 group and the main chain carbonyl of Glu371 {419}. The hydrogen‐bonding potential of the remaining polar moieties of the flavone ring is not satisfied by interaction with the protein. Instead, the ring oxygen (O9) is directed towards the unoccupied pocket on the β‐face of the cavity and the ring hydroxyl (O4) and keto group (O6) point toward the α‐face pocket (Figure 4B). The O4 hydroxyl, which occupies the same position within the cavity as 15α substituents of E2, presumably participates in an intramolecular hydrogen‐bonding interaction with the O6 keto group. Figure 4C illustrates the similarity in the binding modes of GEN and E2. The longer hydroxyl–hydroxyl length of GEN (12.1 Å), compared with that of E2 (10.8 Å), is accommodated at the D‐ring end of the cavity by a slight shift of H11 combined with the outward movement of His475 {524}.
Despite being entirely engulfed by the protein, direct van der Waals contacts between GEN and the residues lining the cavity are concentrated around the extremities of the ligand (Figure 4A). The binding cavity in the hERβ–GEN complex has a volume of 390 Å3 of which 236 Å3 is occupied by the ligand. This overall cavity size is slightly smaller than that of ERα–E2, which has a probe‐occupied volume of 490 Å3, and this reduction is due primarily to the replacement of the leucine at position 336 {384} in ERα by a bulkier methionine in ERβ. This conservative substitution reduces the size of the unoccupied pocket that lies above the ligand. It is noteworthy that Met336 is the only residue within the ligand‐binding cavity that exhibits static disorder and has been modelled in a major (70%) and minor (30%) conformation. In addition to this sequence change, the more planar profile of GEN compared with E2 allows the residues that line the cavity to pack slightly more tightly around the ligand in ERβ.
Raloxifene binding
RAL is accommodated within the binding cavity in the same manner as that observed in the ERα complex (Brzozowski et al., 1997). The phenolic hydroxyl of the benzothiophene interacts with Glu305 {353}, Arg346 {394} and a structurally conserved water molecule (Figure 5A). The hydroxyl of the distal ring hydrogen‐bonds with His475 {524}. However, the rigid nature of the aroylbenzothiophene core in combination with the architecture in the A‐ring pocket and the location of the channel exiting the cavity results in a different mode of binding compared with GEN. The distal end of RAL is thrust deeper into the D‐ring end of the cavity towards H8. The orientation of each ligand, relative to their major hydroxyl–hydroxyl axis, differs by ∼20° so that the respective hydroxyls that interact with His475 {524} are separated by 3.9 Å (Figure 5B).
The large basic side chain of RAL binds in a narrow channel that extends outward from the centre of the cavity. This channel is present in the hERβ–GEN complex but it is plugged by Val487 {536}. The end of the side chain is anchored to H3 by a hydrogen bond between the carboxylate group of Asp303 {351} and the piperidine ring nitrogen. The weak nature of this interaction, compared with that observed in the corresponding ERα complex, may be related to the low pH at which the rERβ–RAL crystals were obtained. Van der Waals contacts between RAL and residues that line the cavity are concentrated around the hydroxyl recognition sites and along the length of the side chain moiety (Figure 5A). The rERβ‐LBD undergoes a series of concerted changes, in both secondary and tertiary structure, on binding of RAL. The residues that define the 'neck' of the channel (Thr299 {347}, Ala302 {350} and Trp335 {383}) move outward to accommodate the pendant ring of the side chain. For the most part, this is achieved by the movement of the N‐terminal half of H3 (293–304 {341–352}), by between 0.8 and 1.4 Å, away from the ligand‐binding cavity. In an associated movement on the opposite side of the cavity, H7 and the H7–H8 loop (residues 367–373 {415–421}) are shifted in towards the bound RAL, presumably to compensate for the outward motion of H3 (Figure 5B). A similar, but more dramatic, movement of the H7–H8 loop occurs in the ERα–OHT complex (Shiau et al., 1998).
Cavity plasticity
While the residues within the cavity exhibit different spatial positions in the two ERβ complexes due to shifts in cavity scaffold, most of the side chains that line the cavity adopt identical rotamer conformations. There are, however, several notable exceptions. The different binding modes of each ligand at the D‐ring end of the cavity affect the orientation of Ile373 {421}, His475 {524} and Leu476 {525}. The imidazole ring of His475 {524} twists in response to the different distal hydroxyl positions of the two ligands in order to maintain an optimal position for hydrogen bonding. Leu476 {525}, situated on the β‐face of the cavity, adjusts its position to compensate for each ligand's different binding mode at the D‐ring end of the cavity. When GEN is bound, the side chain is flipped up away from the flavone ring but, in the presence of RAL, it is flipped down toward the distal ring. Similarly, Ile373 {421}, located on the α‐face of the cavity, rotates in toward the cavity in the RAL complex to pack against the distal phenolic ring (Figure 5B).
At present, RAL represents the only ligand for which structures are available with both ERα‐ and ERβ‐LBD, and their comparison provides clues as to the differences in the plasticity of the cavity of the two isoforms. The r.m.s deviation for all ligand atoms, after superposition of the hERα–RAL and rERβ–RAL complexes, is 0.52 Å. The benzothiophene and pendant rings occupy identical relative positions. However, in ERβ, the distal phenolic ring of the ligand is thrust less deeply into the D‐ring end of the cavity. The difference in the position of the distal hydroxyl in the two isoforms is 1.4 Å. As a consequence, the imidazole ring of His475 {524} maintains its positioning interaction with the backbone carbonyl of Glu371 {419} in the ERβ complex. In addition, the end of the basic side chain adopts a more extended conformation in rERβ–RAL, resulting in a 0.9–1.5 Å outward shift of the piperidine ring. This alteration in the basic side chain is presumably imposed by the different conformation of the H11–H12 loop and positioning of H12 in the rERβ–RAL complex. The residues lining the core of the cavity pack around the ligand in a similar manner, with only Phe377 {425} adopting a different orientation. In rERβ–RAL, the benzyl group of this residue is tucked up under the distal phenolic ring of the RAL core but does not make direct van der Waals contact with ligand. In contrast, in the hERα–RAL complex, the side chain is flipped away from the ligand resulting in the enlargement of the unoccupied pocket on the α‐face of the cavity. This difference is probably due to the amino acid substitution of an isoleucine (ERβ) for a methionine in ERα at position 373 {421}. In ERα, RAL binding induces the repositioning of the methionine side chain, towards Phe377 {425}, and displaces the ring. In ERβ, these positional adjustments can be accommodated without displacement of Phe377 due to the smaller size of the isoleucine side chain.
There are now sufficient structures of ERs in different space groups and at different pHs to permit a meaningful analysis of the relative mobilities of the ligand‐binding cavity. Analysis of the temperature factors of the residues that line the ligand‐binding site in the two RAL complexes indicates a similar degree of mobility within the cavity of ERα and ERβ. The A‐, B‐ and C‐ring regions exhibit very similar mobilities, but the residues lining the D‐ring region of rERβ–RAL have B‐values that are typically 1.5 times higher than those of the other sites. A similar pattern of cavity subsite mobility (A≈B≈C<D) is observed in ERα irrespective of the bound ligand (A.C.W.Pike, unpublished results), but the increase at the distal end of the pocket is less pronounced. The higher observed D‐ring mobility in the presence of RAL presumably reflects the greater degree of distortion imparted by the binding mode of the distal phenolic ring of the aroylbenzothiophene core.
Helix 12 positioning
Both ligands induce a conformation of the AF‐2 region that is distinct from that observed in the presence of pure agonists (Figure 6).
The binding of RAL is accompanied by a major reorganization in the tertiary structure of ERβ‐LBD. In particular, the bulky side chain of RAL physically prevents H12 from adopting its characteristic position over the ligand‐binding cavity. Instead, H12 lies in a shallow groove formed between H3, H4 and H5 (Figure 6A). As in the hERα–RAL complex, the loop connecting H11 and H12 (Met479–Pro486 {529–535}) is invisible in the electron density maps. H12 itself is highly disordered and the resultant modelled helix has high B‐values. In addition, only those side chains on its inner face (Leu491 and Leu495), that pack against H5, can be resolved. Nonetheless, the position occupied by H12 is similar to that observed for this region in the hERα–RAL complex except that it is shifted along the hydrophobic groove by between 1.9 and 3.2 Å. In the rERβ–RAL complex, Leu491 {540} packs against the side chains of Val328 {376} and Leu331 {379}. Leu495 {544} makes van der Waals contact with residues in H3 (Ile310 {358}), H4 (Phe319 {367}) and H5 (Leu324 {372} and Val328 {376}). The side chain of Lys314 {362} makes hydrogen bonds with the main chain carbonyl groups of Met494 {543} and Leu495 {544} and effectively caps the C‐terminal end of H12 (Figure 6A).
The groove in which H12 lies in the RAL complex also represents the binding site for the helical NR‐box module of nuclear coactivators (Feng et al., 1998). In fact, the spatial positions of Leu491 {540} and Leu495 {544} coincide with the first and third NR‐box leucine residues of the GRIP1 peptide (Shiau et al., 1998). This reinforces the previous observation that H12 in ER antagonized by RAL and OHT mimics the interaction of coactivator peptides (Brzozowski et al., 1997; Shiau et al., 1998). The anchoring of the end of the antagonist side chain by Asp303 {351} serves to direct H12 into the coactivator‐binding site. The importance of this tethering interaction is given further weight by the discovery of a drug‐resistant breast cancer cell line in which Asp303 {351} is mutated to tyrosine and the antagonistic effects of RAL are reduced considerably (Jordan, 1998). Together, these observations provide a structural rationale for the known interdependence of RAL antagonism and the length and positioning of the basic nitrogen within the side chain (Grese et al., 1997).
In the hERβ–GEN complex, H12 also lies between H3 and H5. There are, however, fundamental differences in the length, positioning and interactions made by H12 compared with the 'antagonist' orientation induced by RAL. H12 is longer, comprising four turns of α‐helix (residues 487–499 {536–548}), and is well defined in the electron density. At the end of H11 (Leu477 {526}), the main chain turns through 90° running along H3, past the entrance to the ligand‐binding cavity, and continues, as H12, in a direction parallel to H5 (Figure 6B). Val487 {536} rests in the gap at the entrance of the ligand‐binding cavity between H3, H6 and H11, and contacts Thr299 {347}, Ala302 {350}, Leu306 {354} and Trp335 {383}. The insertion of Val487 at the entrance of the cavity forces H12 to adopt a different orientation along the hydrophobic groove compared with that observed in the rERβ–RAL complex. There is a 25° difference in helix orientation between the two complexes. Rather than running along the hydrophobic groove, H12 projects away from the body of the protein in the GEN structure. The N‐terminal end packs close to the protein, with Leu491 {540} making van der Waals contact with Trp335 {383}. Met494 {543} lies on the edge of the groove against H3 and contacts Leu306 {354}, Val307 {355}, Ile310 {358} and Asp303 {351}. The position of Leu495 coincides with that of Leu491 in the RAL complex and it makes similar contacts. In addition to these hydrophobic contacts, this unique disposition of H12 is stabilized by a specific hydrogen bond between the phenolic hydroxyl of Tyr488 {537} and the carboxylate side chain of Glu332 {380} in H5 (Figure 6B).
In this study, we report the first description of the structure of ERβ's LBD in the presence of a partial agonist and full antagonist. These complexes shed light on the structural basis for each ligand's differential pharmacological effects through ERβ and provide some clues as to the determinants of isoform‐selective ligand binding. As expected from the high sequence identity between the LBDs of the two isoforms, their tertiary and quaternary structure are very similar. While both ER isoforms readily form homodimers, their overlapping cell and tissue distribution raises the possibility of heterodimerization. Formation of mixed dimers has been demonstrated both in vitro and in vivo (Ogawa et al., 1998), but the physiological role of the heterodimer is unclear. Nonetheless, the exclusive substitution of a methionine at positions 403 {451} and 460 {509} within the hydrophobic patch of H11 of ERβ has no impact on the dimer interface and suggests that the arrangement of molecules in an α/β heterodimer will be the same.
α/β ligand selectivity
The size and shape of the ER ligand‐binding cavity, combined with the plasticity of its component elements, allows ER to embrace a range of ligands containing a variety of structural motifs (Anstead et al., 1997; Brzozowski et al., 1997; Shiau et al., 1998). The generous size of the binding cavity coupled with the variety of known ligand‐binding modes (Brzozowski et al., 1997; Shiau et al., 1998) suggests that receptor selectivity in ER can be generated through a number of different interactions. This contrasts with the situation in other NRs, such as thyroid receptor (TR) and the retinoic acid receptor (RAR), where the cavity shape is well matched to that of the cognate hormone and the stability of the complex is dependent on a large number of hydrophobic interactions. For example, in RAR, the inherent flexibility of the receptor ligands allows them to adapt to the shape of the binding cavity, and isoform selectivity can be determined by a few key interactions (Klaholz et al., 1998).
In the case of ERα and ERβ, the relatively invariant tertiary architecture of the binding cavity explains why the majority of compounds bind to each isoform with similar affinities (Kuiper et al., 1997). The two conservative amino acid changes within the binding cavity have a direct impact on its overall volume and may also be responsible for the distinct ligand‐binding preferences reported for ERβ (Barkhem et al., 1998). GEN binds preferentially to ERβ with a 30‐fold higher affinity (Kuiper et al., 1997; Barkhem et al., 1998). The two cavity‐lining residues that differ between ERα and ERβ are obvious candidates for this selective effect. As mentioned above, the leucine present at position 336 {384} in H6 of ERα is replaced by a methionine in ERβ. This residue lies above the ligand and delineates part of the pre‐formed pocket on the β‐face of the cavity. In the hERβ–GEN complex, the side chain of this residue makes direct van der Waals contact with part of the flavone ring. While methionine is generally considered to be hydrophobic in nature, the sulfur has a weak polar character and participates in hydrogen‐bonding interactions in proteins (Stickle et al., 1992). The sulfur group of the methionine adopts two distinct, but unequally occupied alternative positions in the hERβ–GEN complex. In one of these positions, the sulfur is 4.6 Å from the keto moiety of the flavone ring but this interaction is unlikely to provide sufficient stabilization due to the large inter‐atomic distance. Furthermore, the static disorder exhibited by this atom indicates that such an interaction is unlikely to be responsible for GEN's higher affinity for ERβ.
The second substitution within the cavity replaces a methionine at position 373 {421} at the start of H8 (ERα) by an isoleucine (ERβ). This residue lies on the α‐face of the cavity immediately below the D‐ring of E2 and is sandwiched between His475 {524} and Phe377 {425}. Polar substitutions are poorly tolerated at the D‐ring end of the cavity in ERα (Anstead et al., 1997) and, in particular, at the 15α position where the O4 hydroxyl of GEN's flavone ring lies. Perhaps the migration of the weakly polar methionine from the α‐; to the β‐face of the cavity enables ERβ to accommodate more polar substituents at the distal end of the cavity. In summary, the distinct ligand‐binding preference of GEN for ERβ is difficult to reconcile convincingly on the basis of these sequence differences alone.
Determinants of H12 alignment
The reasons for the misalignment of H12 in the RAL complex are clear; the basic side chain protrudes from the cavity and physically prevents its positioning over the hormone‐binding site. In fact, the piperidine ring of the basic side chain occupies the same spatial position as Val487 {536} in the GEN complex. GEN, on the other hand, has a similar molecular volume (236 Å3) to that of E2 (250 Å3) and binds in an analogous fashion, yet in the hERβ–GEN complex, H12 has a preference for binding along the NR‐box cleft rather than over the cavity. What then determines the positioning of H12 in the hERβ–GEN complex? The amino acid sequence in the vicinity of the H11–H12 loop and H12 is highly conserved in both ER isoforms and differs at only two positions (Figure 2). This suggests that the differences in the helix positioning in the presence of these ligands is not due to any inherent difference in flexibility of this region between the two isoforms.
The primary determinant of H12 positioning appears to be the burial of its hydrophobic face against the protein and, in so doing, sealing the ligand within the binding cavity. The positions of H12 in the presence of pure agonist, such as E2 and diethylstilbestrol (DES) (Brzozowski et al., 1997; Shiau et al., 1998), and that seen here in the hERβ–GEN complex both fulfil this objective. In the archetypal 'agonist' orientation, typified by the ERα–E2 complex, the N‐terminal end of H12 appears to be stabilized by two hydrophilic interactions. The helix is capped by Asp303 {351}, which interacts with the main chain amide of Leu491 {540}. In addition, Tyr398 {537} makes a hydrogen bond with Asn300 {348}. These two interactions effectively anchor the N‐terminal end of H12 and position it so that Leu491 {540} can seal the ligand‐binding cavity (Figure 6C). In other NRs, the 'agonist' orientation of H12 is stabilized by similar N‐capping interactions and, in the case of TR and RARγ, by an additional interaction between the C‐terminal end of H12 and a lysine in H5 (Renaud et al., 1995; Wagner et al., 1995).
Functional analyses demonstrate that ERβ is easier to antagonize than ERα (S.Nilsson, personal communication). One explanation for this observation is that the 'agonist' orientation of H12 in ERβ is more unstable than in ERα. The asparagine present at position 300 {348} in ERα, which interacts with Tyr398 {537}, is replaced by a lysine in ERβ. Consequently, the 'agonist' orientation of H12 may be less favoured in ERβ due to loss of the hydrogen‐bonding interaction made by this residue to Tyr398. The slight widening at the distal end of the hydrophobic groove in ERβ compared with ERα (see above) is unlikely to be responsible for the different positioning of H12 in the GEN complex. Figure 6 clearly highlights the conformational adaptability of the H11–H12 region of ER‐LBD. Despite the radically different positions adopted by H12 in the various ligand complexes, several common structural features are maintained. For example, Val487 (ERβ) and Leu540 (ERα), two residues which are not equivalent in terms of sequence (Figure 2), play an identical structural role by sealing ligand within the binding cavity of the hERβ–GEN and hERα–E2 complexes (Figure 4C). Nevertheless, the origin of GEN's 'destabilizing' influence on H12 remains unclear, and further structural studies with full ERβ agonists are required to understand fully the subtle inter‐relationship between ligand binding and H12 orientation.
Implications for agonism and antagonism
Partial agonist activity of SERMs, such as RAL and OHT, through ERα is thought to be dependent on the N‐terminal AF‐1 transactivation domain rather than on AF‐2 (MacGregor and Jordan, 1998). However, the AF‐1 domain of ERβ is devoid of this functionality and SERMs act as pure antagonists via this receptor isoform when bound to EREs (Barkhem et al., 1998; McInerney et al., 1998). The partial agonism exhibited by GEN on ERβ (Barkhem et al., 1998) must therefore arise through a different effect. One clue is the sub‐optimal positioning of H12 in the hERβ–GEN complex.
ERs, and other nuclear receptors, recruit a variety of nuclear coactivators for optimal efficacy (Torchia et al., 1998). The recruitment surface is a shallow hydrophobic groove formed between H3, H5, H6 and H12 (Feng et al., 1998). In the complexes of ERα with the full agonists E2 (Brzozowski et al., 1997) and DES (Shiau et al., 1998), H12 is aligned over the ligand‐binding cavity so that the side chain of Leu491 {540} seals the entrance to the cavity (Figure 6C). In this orientation, the side chain of Glu493 {542} is positioned ideally to interact with the N‐terminal end of the helical NR‐box motifs of coactivators (Darimont et al., 1998; Nolte et al., 1998; Shiau et al., 1998). The observation that AF‐2 antagonists, such as RAL, prevent the correct alignment of H12 has provided a structural model for their effects (Brzozowski et al., 1997).
The observation that the partial agonist, GEN, elicits an orientation of H12 similar to ER antagonists is intriguing. The position adopted by H12 in the presence of GEN in hERβ is not sequence dependent or a crystal packing effect, as it is also observed in a tetragonal crystal form of rERβ in complex with the same ligand (A.C.W.Pike, unpublished results). H12 has been observed to interact with the NR‐box binding cleft in the structures of unliganded RXRα (Bourguet et al., 1995), PPARγ (Nolte et al., 1998) and Cys530‐cross‐linked ERα (Tanenbaum et al., 1998). However, in all these cases, H12 interacts with the binding cleft of a neighbouring molecule within the crystal lattice. The hERβ–GEN complex therefore represents the first time that this orientation of H12 has been observed in the presence of ligand and suggests that occupation of the binding cleft may serve a physiological purpose. Interestingly, the conformation of H12 in the GEN complex only partially mimics that of the NR‐box module. In fact, the position adopted by H12 is shifted some 4–5 Å from the position adopted by short peptides derived from the nuclear interaction domains of coactivators. The observation that H12 perfectly mimics the interactions made by NR‐box peptides in ER antagonist complexes, such as that described here with RAL, has led to the suggestion that H12 may act in an autoinhibitory manner (Shiau et al., 1998). Rather than an autoinhibitory response, such occupation of the cleft by H12 is more likely to represent a mechanism through which the receptor can attenuate the effects of certain ligands. In the case of GEN, which only acts as a partial agonist through ERβ (Barkhem et al., 1998), H12's preferential occupation of the NR‐box binding cleft sets up a direct competition for this site with ER coactivators. Consequently, potential coactivators must therefore displace H12 into an 'agonist‐like' conformation prior to binding.
Materials and methods
Protein expression, purification and crystallization
Rat and human ERβ‐LBD (residues 255–509; hERβ numbering) were overexpressed in Escherichia coli GI 724 cells using the pLEX expression system (Invitrogen). A short FLAG™ sequence (MDYKDDDDK) was engineered prior to the N‐terminus of the ERβ‐LBD to facilitate antibody‐mediated identification. Fermentation was carried out in fed‐batch culture (defined glucose/salt medium, 30°C) and expression of the recombinant protein was induced by the addition of tryptophan (0.25 g/l). After a 3 h induction, cells were harvested by centrifugation and frozen. Thawed cells were disrupted in a bead mill in lysis buffer [100 mM Tris pH 8.1, 300 mM KCl, 10% glycerol, 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 4 mM dithiothreitol (DTT)] and the bacterial lysate was centrifuged to pellet the cell debris. The supernatant was applied to an oestradiol–Sepharose fast flow column (Greene et al., 1980) and washed with 300 mM KCl, 2 mM EDTA and 2 mM DTT in 20 mM Tris pH 8.1. Bound protein was carboxymethylated in situ overnight with 15 mM iodoacetic acid. After carboxymethylation, the column was washed with 100 mM KCl in 20 mM Tris pH 8.1. For the GEN complex, hERβ‐LBD was eluted by including 50 μM ligand in the washing buffer. In the case of RAL, the column was washed with 250 mM NaSCN in 20 mM Tris pH 8.1 and the protein was eluted by addition of 50 μM ligand. Pooled fractions of each carboxymethylated ERβ‐LBD complex were concentrated and purified further by preparative PAGE (Bio‐Rad 491) using the Ornstein/Davies buffer system as per the manufacturer's instructions. Fractions were analysed by native PAGE and fractions showing a single protein band were pooled, concentrated and used for crystallization. The hERβ–GEN and rERβ–RAL complexes were crystallized using the vapour diffusion technique at 18°C. For the RAL complex, the reservoir solution contained 7.5% (w/v) PEG 4000, 0.1 M ammonium acetate, 3% (v/v) dimethylformamide in 25 mM sodium acetate pH 4.8. Drops were composed of a 2:1 ratio of protein (7–11 mg/ml) and reservoir solution. Tetragonal bipyramids, with unit cell dimensions of a = b = 67.55 Å c = 148.2 Å and containing one LBD molecule per asymmetric unit, appeared within 1–2 weeks. For the GEN complex, drops were composed of equal volumes of protein (8 mg/ml) and reservoir solution [6–9% (w/v) PEG 6000, 1.6–2.1 M NaCl in 0.1 M Tris pH 8.1]. The resultant hexagonal rods belong to space group P6122 and have unit cell dimensions of a = b = 63.12 Å c = 250.23 Å with one LBD molecule per asymmetric unit.
Data collection
Initial data sets to resolutions of 2.5 Å (RAL complex) and 2.3 Å (GEN complex) were collected at station 9.5 (SRS, CLRC Daresbury, UK) and station W32 (LURE, Orsay, France), respectively. Subsequently, diffraction data for the rERβ–RAL complex were collected to 2.25 Å resolution on station X11 at DESY (EMBL, Hamburg). Data frames, corresponding to oscillations of 1.4°, were recorded using an 18 cm MAR Research image plate placed at 190 mm from the crystal. Data for the hERβ–GEN complex were collected to 1.8 Å resolution on station ID14‐4 at the ESRF (Grenoble, France). Data were recorded in two sweeps using an ADSC Quantum4 CCD detector placed at 280 mm (55–3 Å) and 160 mm (1.8 Å) from the crystal, respectively, using 1° oscillations. In both cases, the crystals were cryoprotected with 25% 2‐methyl‐2,4‐pentanediol prior to freezing at 100 K. All data were integrated and reduced using DENZO and SCALEPACK (Otwinowski and Minor, 1997). X‐ray data statistics are summarized in Table I.
Structure determination and refinement
The coordinates of the ERα‐LBD–E2 monomer (PDB entry: 1ERE; Brzozowski et al., 1997), truncated to Tyr526 at the C‐terminus and without the ligand, were used as a search model in AMoRe (CCP4, 1994) to solve the structure of the rERβ–RAL complex. Subsequently, the refined coordinates of the rERβ–RAL complex (excluding the ligand and residues after H11) were used in AMoRe to determine the position of the LBD in the GEN complex. A single, clear solution was obtained for both structures using data between 15 and 4 Å and an integration radius of 25–26 Å. Initial electron density maps, calculated after rigid‐body refinement in AMoRe, highlighted regions where sequence changes were required and unambiguously indicated the position and orientation of each ligand within the cavity. Both complexes were refined with REFMAC (Murshudov et al., 1997) using all available data with no sigma cut‐offs. Bulk solvent contributions, calculated in XPLOR v3.843 (Brünger, 1992), were incorporated in the form of partial structure factors. In both cases, the structures initially were refined against the lower resolution data sets collected at the SRS and LURE prior to the incorporation of the higher resolution terms. For the RAL complex, successive rounds of refinement and manual rebuilding gave a final model with an Rcryst of 20.4 and an Rfree of 26.3 for all data between 25 and 2.25 Å. The final model comprises 1807 protein atoms, 34 ligand atoms, 128 waters and an acetate molecule. Residues prior to T262 at the N‐terminus and C481–Y488 (H11–H12 loop) are invisible in the electron density and have not been modelled. The electron density for the H2–H3 loop (S283–S293), H7 (D365–V370) and H12 (A489–A497) is weak but interpretable. H12 has extremely high B‐values but its inclusion in the model is justified as this results in an appreciable drop in Rfree. The side chains of seven residues (M295, S333, K353, K425, H428, S463 and H464) display static disorder and have been modelled in two alternative conformations. The final model for the GEN complex, comprising 1834 protein atoms, 20 ligand atoms and 139 waters, has an Rcryst of 21.9 and Rfree of 25.5 for all data between 55 and 1.8 Å. Residues S286–E291 (H2–H3 loop) and T415–D419 (H9–H10 loop) are poorly defined and have not been included in the final model. The side chains of 16 residues (E266, M309, S311, S333, M336, E337, E389, M403, S408, Q450, S463, S469, M473, L477, M478 and N496) and the main chain of two residues (P358–D359) display static disorder and have been modelled in two alternative conformations. All model building was carried out in the molecular graphics package QUANTA (QUANTA98; Molecular Simulations Inc., San Diego, CA). Dictionaries for the ligand molecules were derived from structures built and minimized in QUANTA. Water molecules that made at least one reasonable hydrogen bond to the protein were included as long as their B‐values remained below 70 Å2. A summary of the refinement and model statistics is given in Table I.
Superpositions of the different ligand complexes were carried out in QUANTA using all equivalent residues up to the end of H11. The remainder of the main chain was excluded due to the large differences in the position of H12. After this global superposition, the overlaps were fine‐tuned using the 'match closest residue' option in QUANTA. Cavity volume calculations and surface generation were performed in VOIDOO (Kleywegt and Jones, 1994) using a 0.4 Å primary grid and a probe of radius 1.4 Å. Figures 1, 3, 4 and 5 were produced using BOBSCRIPT (Esnouf, 1997).
Atomic coordinates have been deposited in the Brookhaven Protein Data Bank (accession codes 1QKN and 1QKM for the RAL and GEN complexes, respectively) and are 'on‐hold' for 1 year.
We would like to thank Julia Walton for help with crystallizations, Stefan Nilsson for discussion and comments on the manuscript and the beamline managers at the ESRF, EMBL‐Hamburg, LURE and SRS for technical assistance during data collection. The infrastructure of the Structural Biology Laboratory at York is supported by the BBSRC. We thank the European Union for support of the work carried out at EMBL Hamburg outstation through the HCMP access to large installations project (contract number CHGE‐CT93‐0040).
- Copyright © 1999 European Molecular Biology Organization


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Tutorial: How to paint Dwarf Grim Hammers from the Hobbit
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Small warehouse with low shelves: directional + Omni AP, for full coverage and stable signal. Large warehouse with high shelves: distributed AP and smart roaming, for improved wireless performance.
Converged Wi-Fi and IoT ensure internet access and automatic registration of goods in and out based on RFID, which improves efficiency and reduces TCO by 30 percent.
The cloud management Wi-Fi platform provides unified management of warehouse network equipment, simplified operations and maintenance, and easy network replication.
Huawei AP4050DN-E is an IoT-type of wireless access point that supports 802.11ac Wave 2, 2 x 2 MIMO, and two spatial streams. It is applicable to commercial chains, medical, warehousing, manufacturing, and logistics environments.
Huawei AP6050DN and AP6150DN are wireless access points that support 802.11ac Wave 2, 4 x 4 MIMO and four spatial streams, applicable to medium and large high-density scenarios, such as mobile offices and elementary and higher education.
Huawei AD9431DN-24X is a central Access Point (AP) applicable to areas with high density of rooms, complex wall structure, and high service bandwidth requirements, such as schools, hotels, hospitals, and office meeting rooms.
These 802.11ac Wave 2 RUs are used in Huawei's Distributed Wi-Fi Solution. With built-in smart antennas, 2 x 2 MIMO support and two spatial streams, they achieve 1.267 Gbit/s and are used for areas with complex structures or high-density rooms, such as schools, hotels, hospitals, and offices.
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