Tell me if this sounds familiar. You study very hard for IELTS writing task two. You prepare for every single question and then on test day, you open up the paper and you have no idea what to do. And this is actually quite common. Students often do a huge amount of preparation but on test day, they get a topic they don't know or a question type that is very unusual and strange. But don't panic. This video is going to give you a step by step system that will allow you to understand and produce a very good essay for any question that you get on test day. So I asked our VIP students to help me out and I asked them for the most difficult question they've ever seen on the real task two paper and this is it. So in this lesson, we're going to show you how to analyze any question so that you fully understand it, then generate relevant specific ideas no matter what the topic and then structure your answer in a way that you can take those ideas and easily transfer them into a high level essay. And if you watch until the end, I'm going to give you a band nine sample essay so that you can see what we produced. So without further ado, let's get into the lesson. So let's look at the question first. So just pause the video if you want and have a read of this question. Now this is quite a strange question because we have three sentences, sentence one, sentence two, sentence three, before we get to our actual question statement. So the bit here that I've circled to what extent do you agree or disagree? This is the main thing that the examiner wants you to do. So this is one of the most important parts of the question because this part that I circled is going to influence your structure and the way that you answer the question. What's also quite unusual, as I already said, is there are three sentences before you actually get to the question statement. The question statement is here. So there is a bit about world peace. There is a bit about cheap and clean energy. And there is a bit about the benefits outweighing the disadvantages. So what I'd like you to do, if you've not already done, just pause the video and have a think about how you would go about answering this question. Think about what does the examiner want me to do with this question? What is the general topic? What are the subtopics or the microtopics, if you like? Then try and start thinking about how you would answer this question and then we'll get onto why this is quite a difficult question. So the first reason that this is a difficult question is it is an opinion question, not an advantages and disadvantages question. Lots of students, when they look at the question, if we go back, they see benefits outweigh disadvantages and this is the first thing they see and they automatically think advantages and disadvantages. So they just talk about all the advantages of nuclear power and all the disadvantages of nuclear power. But this isn't what the question actually asks you to do. If you go back and look at the bottom here, the most important part is to what extent do you disagree and do you? So it's asking you for your opinion. If you don't have your opinion, you're gonna find it very difficult to get a high score. Other thing is we must discuss two things. We must discuss both world peace and we must discuss clean and cheap energy. If you leave one of these out, you're really not answering the specific question that is asked. So if we go back here and we'll see world peace is our first sentence out and cheap and clean energy is the second sentence out of the first two sentences here. So if we just talked about world peace, say they give your opinion on world peace or you just talked about how nuclear energy is either cheap and clean or not cheap or clean, then you haven't really answered the specific question. And also, if you don't talk about the benefits and the disadvantages, your opinion on those, then you're not really answering the question either. So it's quite a tricky question. So the two things that you must realize here is that it is an opinion question, not a disadvantages and advantages question, and there are two separate things that you must discuss and you have to discuss these within the context of do the benefits outweigh the disadvantages. So next, we would go on to ideas trying to think of relevant ideas for this question. So we already know now that it's an opinion question. We know that we have to discuss world peace and cheap and clean energy. So now we have to think of relevant ideas. So the best thing to do would be just to take these separately, world peace and clean energy and try and think, do you agree that world peace within the context of nuclear weapons that the benefits of having nuclear weapons for world peace outweighs the disadvantages? Do you agree or disagree with that and why? And then go on to cheap and clean energy. Do you agree or disagree that the benefits of having nuclear energy outweigh the disadvantages within the context of it being cheap and clean energy? So just pause the video if you haven't done already and try and think of just one idea for world peace and one idea for cheap and clean energy. So if we go on to my ideas, world peace, I disagree that the advantages outweigh the disadvantages. Why?  So that's a pretty huge disadvantage. So I completely disagree that the benefits outweigh the disadvantages. And my main idea is that we will lose ourselves or human beings if something goes wrong. So the next one, clean and cheap energy. Again, I disagree. So I'm giving my opinion. Why? Because the disadvantage is the destruction of our planet. So if something goes wrong in a nuclear power plant, a nuclear making nuclear energy, it could completely destroy the environment. So that is a huge disadvantage. So that is my main idea. So you'll notice that I am talking about disadvantages, but it isn't an advantage and disadvantages question. I'm just saying and I disagree. So this is my opinion on whether the advantages outweigh the disadvantages. So the next would be the structure. So how would you put this into a four paragraph structure? So the introduction, I'm gonna state a state if you agree or disagree for both world peace and clean or cheap energy. So in the introduction, I'm gonna make it very clear if I agree or disagree for both of these. This is gonna make it very easy for the examiner to understand my point of view. And it's also gonna make me make it easy for me because I have already thought of my ideas and my opinion is clear. So main body paragraph number one, reason why you agree or disagree about world peace. So in main body number one, I'm just going to say give my opinion on world peace. Main body number two, and then I disagree. Reason why you agree or disagree about clean and cheap energy. So I'm gonna say whether I think the advantages outweigh the disadvantages, whether I agree about that for clean and cheap energy. Conclusion summary of main ideas and then give my opinion again. So it just makes it very, very easy for the examiner to see what my ideas are again and make my opinion very, very clear. So let's just summarize the common mistakes. Answering the question like an advantages and disadvantages question and linked to that is not giving an opinion. So if you just talked about all the advantages and disadvantages and didn't actually give your opinion about whether the benefits outweigh the drawbacks or the benefits outweigh the disadvantages, then you haven't answered the question. Also, if you don't mention both world peace and energy, then you haven't really answered the question. So I hope that helped you out, but I should say that this is an extremely difficult question. Questions are not normally this long. Questions don't normally have both advantages and disadvantages and opinion. In them, questions don't normally have two separate things that you need to talk about. So the reason why I picked this question is because it is particularly difficult. So don't get down, don't get upset if you didn't understand everything and you made mistakes. The purpose of this whole exercise is for you to make mistakes and to learn about it so that if you do get a question like this in the future or a question that's just not even this difficult, you will think about it logically and think about it in the way that the examiners want you to think about it. There are two choices for you now. Number one, you can go into the description of this video and it will take you to our website and it will show you the Band 9 sample essay. So you can read it and see what we did there and model your own answer on that. Or if you wanna continue your journey throughout our YouTube channel, just click this video and it will help you on your journey and help you improve your IELTS score. Thank you very much.


and here we are done for today well done in general I'm gonna redo that all right and good morning today is I don't know also asked us to change that lintel for a concrete one because you can tell how you tell for me it's quite we've got a new toy for it shall we yes happy fray at Wednesday happy fray at Wednesday there's a little new toy outside mortars a little triple charger but it's obviously got to be big out for us and the GoPro cable keeps burning up so we've lost two to partial fire so once I've got a spare cable nice anyway day three of a two-day job so let's take you around shall we we're up to I think it was after getting off a similar finish last night so got this left which is secure because I've done the block work on it as you can see it's a 9-inch wall as we've said before left two snap headers obviously so that's left to do once we've shifted bricks out this is basically all evidence come out of there the door was partially done just two blocks you go at the top this took a lot of extra time up because we took the concrete step out you see where the black brick are they're the soldier brick from around the corner which we salvaged and we've just that's used the clay commons underneath just to bring it up they are supposed to be in bricks but once they're painted they'll be okay obviously all the steps coming out they've got this to take out which I was going to do the bolster from above because this still sourced on three but because it's rendered the cuts a lot shallower just snap them off bricks are dead so if you just start hitting it they start breaking off like that and internally oh yeah tops all finished just once a bit of motor in top there all events are loose so you can either keep them or take them out whatever happens there's our rhino clamp needs taken out that was holding a piece of timber in there to stop the little tipping back we were built over as you can see there's the cover to treat you must job that's finished that bit so basically when it's all tidied up and done the French doors in the middle of the room well slightly off-center but it's because of these pipes anyway that's where the customers of them so I'm gonna put all the timber in here give it all a good push out nice and tidy so basically it's just finish all the little bits off have a good tidy up so yeah that's it doing its job tiny tiny little RSJ probably about six inches by three two and a half inches that's just holding the floor just above yeah nice and strong now plenty ties it and the block wall is now tied into this and plenty tie-wising so it's got a lot more strength in it now because all this was in as you've shown it was all tired in front to back so now we've got the we've got the thermal and the damp break now all the way through right the way through the back of the house so it should be a bit warmer and what less chance of getting damping so I'm gonna get a mixing and actually get tired you know the other that is door completely knocked out now happy days so actually came out a lot easier than that did and now it's just a case of I'm gonna get the rest of them beds off of those bricks and gets all that crap swooped onto this side and hopefully try and get out of here before dinner time or for dinner time so seen a bit let's see if you noticed where that window was I saw that mortars dried off you won't be able to tell I was even a window there has done a very nice job with that and then we have we say we dad it's finished that off I managed to get that little bit pointed up there too which need to be done doors all done everything's blocked up outside and in there's my little bit nearly bit I've done on the whole body job but and dad has like pressed up a few of the bits that came off there so that's a nice and there's some flush cleaned out all the cavities cut a nice cavity right through for the wall now so we were half a day over but this is one of those things it was just a lot of unforeseen circumstances such as how much effort was going into the door the steps coming out it was a lot but we've done at least that's the main thing so I'm gonna try and get all this stuff taken back to Hodgson's and get some stuff ready for another job so and good morning today we are at a old job you may recognize that wall the window which is ripped up proudly tell and here's our door way we did it's now had the door installed by Neil Neil was it Neil was it during an Instagram there you go check him out but yeah so there you go and we're coming today to take you around here obviously cut all this bit okay it's the main reason right oh yeah so I'm gonna get back in position so you can keep it clean at it so today we've got two little jobs to do first one these soldiers they just need cutting back a bit so the flush with the blocks because is it we're gonna render it aren't they getting rendered and I did say we do that because everything was propped up and loose I didn't want to start bashing the soldiers when we realized so now everything's gone off in the doors in everything everything's safe we can go like that now so there's all of them coming out and then take you around front we've also asked us to change that little for a concrete one because you can tell how I tell for me it's quite it's quite rotten yeah so we're gonna get a nice bit of a concrete one up there and and for all this we've got a we've got a new toy for it shall we yes so evolution have sent us this 300 mil grinder to have a little play with and it's electric but it's designed to be water suppression so it's a bit of a beast that isn't it thanks for the kids there's a lot of pressure yeah and I take it the electric motors well back to protect it from the water yeah so yeah we've got a little water connection for that there's the on-off switch for the water about the same weight as it's still so yeah yeah and then it's also got an rcd protection in actually in the wire oh obviously because you're working with water yeah so um yeah for today's the day give it a try so a big thank you to evolution and if you want to get your hands on one of these they've actually give us a link to it put in the description so if you want to see this to yourself be sure to head over to the description and check out the link so that's the blade there's the blade all on brand as well evolution blade very nice it's a premium diamond it's faster brick stone slate and it'll cut reinforced concrete as well so it's okay going through lintels and stuff go through the reinforced so that's ideal as is it wheels attach on the back here all right so read up on it properly but so you can lean on the wall with it and put a bit of weight onto it so you like it put all the air all color all in all on brand different like the colors black and orange very very bitter language to you now there's your instructions on your manual something to tighten up this with so as you can probably tell from there could actually need a change in so the good that this is coming out now um read the trestles up i've had to drop it down one because um it's a bit cramped up there couldn't get the reciprocating and put it the bar in so that's just muddle our way through the um the accrues they're just simply to stop anything falling if anything's loose but i chopped out the two two sides so we've took took it out a bit wider for the extra seating for our lintel because the brick to brick 900 and we'll put the 1264 in there's no cavity training there there's nothing like that no insulation blah blah blah so we're just putting the standard concrete uh concrete lintel back in and uh putting it back up because we've got the canopy above which goes a good foot above and above that i've got the soldiers and it's all rendered so there's no water getting into that brickwork so there's no point putting the cavities to train them so then we're just doing it like for like well timber for concrete i think i'll just show you the one but oh there you go there's the inside the cavity there's there's the outside so it's like filling in filling the two take out the bottom bit to put a nice new filling in so alex are just making a brew and then we're going to do another brick do a bit of a hand mix for this and go back up and once this is done we're going to drop this lock down and go around the back and get all these soldier bricks we're going to chop them with the stilt with the stilt so and the grinder and then knock them off because they're engineering bricks it's going to be a bit of a tough one so that's where we're at now so we'll catch up the break time right don't need much so it's a hand mix job which is very easy to grill at all but that's what our grill to be full of stone and fast we had one free one which i decided to fill up with stone you know that's going to go yeah we'll go on this later that's next job so yeah once this is mixed we'll turn the stiff sides out yeah because it's going to be it's a 150 lintel so it's going to be quite a big bed above the top of that because it's um that acro is there just so the loose so that nothing fell down while we were using the um titan vibrating the wall but nothing's loose so that's coming out in a second and then we're going to point it out there's no need to slice it up or anything like that so um that'll do catch you in a minute let's get up there let's get it in and then we can go make a proper mess around the back yep just a little big bed on top so let's see where we can put you welcome to inside the canopy had a pan for every time i bang my head doesn't it's in i'm pretty sure they'll be in your front door going on here but if not it just needs a trim putting on here and obviously the rendering needs fixing just needs a trim putting in here and obviously the rendering needs patching over this but this is absolutely solid i've put a slate in here slate in here yep there's a couple of roping ones that one actually fell out so that's got a slate under there that's just gogged up and there's a bit of slate there so this is all solid so we've got a bit of seating thicker lintel and it's concrete so time to tidy up get around the back and make a mess around the back so we'll see you around there so yeah that's it then thank you very much for watching if you enjoyed be sure to leave a like and if you're new to the channel or you want to see more from us be sure to hit subscribe and tap that bell that way you'll be notified when we post anything and it is a massive help we greatly appreciate it and we appreciate any support for the channel so thank you very much this has been bricklaying with Steve and Alex and we'll see you in the next video


So let me give you a bit of an overview. Just to whet your appetite a little bit. You're going to find out a little bit about me, a little bit about Kevin. And I'm spitting into this thing. Is that better? That's better. Then we're just going to go over five key things from a marketing perspective. So this is your top of the funnel stuff. This is your exciting stuff to get people interested about your business. So who has heard of the MX2 mobile mapping system? Yes, get in there. Brilliant. OK, so my family, my dad is a surveyor. He's a hydrographer and a mine surveyor. So I come from the survey background. So we actually manufactured equipment for very dirty environments like mining and quarrying and oil and gas. And then, of course, I needed to set up my own business in marketing and sales. So I set up my business 11 years ago. And I don't look back because I love it. I love everything about it. So my background is in marketing and sales to help geospatial businesses grow and become more profitable. And the profitable bit is the most important part. Kevin. Very good. Well, since Elaine started off with a question, who's heard of Landsat? Anybody, Landsat program? Well, I was the first person to market commercial Landsat data over 30 years ago. And we had the pleasure of trying to sell for the first time satellite imagery. Well, after I did that at a company called EOSat, I started my own firm in the Denver, Colorado area. And I've been helping geospatial companies market their data, boost their sales through strategic marketing, content marketing, market research for 30 years. So we're here today to help you all fine tune your marketing and hopefully increase your market share. He's only 26, maybe 30. I think my company is older than you are, Elaine. I'm only 21. OK, and my iPad has gone to sleep, which is always wonderful. So marketing disaster number one. Who has a landing page on their website? Specific. Nice. Tom's there, got it? Anybody else? Do you know what a landing page actually is? OK, so get your phones out. Everybody get your phones out. Come on, let me see phones. Phones, phones, phones. I want you to take a picture of the QR code. Take a picture. Come on, I'm watching you all. Go, phones, phones, phones, phones. I don't know if people can get that. There's a matter in my madness. You're in the way. You're in the way. OK, so the whole point of a landing page is to attract the right type of buyer. OK, so you want to. I bet you're thinking, if I could only have half a dozen more of these types of wonderful buyers, you'd be quite rich, wouldn't you? You'd be quite wealthy. You'd be like, yeah, give me more, give me more. One of the things that most geospatial businesses are really bad at, they don't have specific landing pages. And these landing pages are focused on that right type of buyer. It's all about first impressions. It's all about building your brand. It's also a lead generation. So if you look on the QR code, it will take you to a page. The page, of course, is promoting what we do. But it's interesting. So those people that we are targeting will be interested in that information. Yeah, OK, I'm interested a little bit more about that. I will fill in the details. A landing page doesn't have to have tons and tons of things in it. The whole point of it, it's a lead capture form that is light, OK? Light. You're not selling them anything. You want to nurture them. You want to get them feeling, OK, I'm interested to know a little bit more. What do I need to do? When people start then selling them stuff, you think, OK, I'm switching off now. And that's why marketing has got a bad remit in the whole world and in the geospatial. So you guys need to go in there and build on this. So the landing page is perfect for that. It also massively helps with SEO and stuff. So some quick tips for you. First one is testimonials on the web page. Great way to actually promote what you do. If you notice on my landing page, I actually didn't put a testimonial on there. But you can have a little nosy around on the website to find out what's happening. The other thing is great images, but make sure they load fast. Because how annoying is it when you go onto a website, you can't find what you're looking for, and then the damn thing takes forever to load. Uh-uh, it's a nightmare. And then, of course, sign up here. And as you see on the page, there'll be a little sign up.  You want that, even I want that. Kevin, do you have any comments about the landing pages? I do. If you're gonna walk away remembering four things, I want you to remember faster, better, cheaper, safer. So your landing page, sure it's gonna be about you, but it can't be totally about you and your company. It has to be about your clients. And to set yourself apart, you've gotta explain how your product or service is gonna help your clients or potential customers do their jobs faster, better, cheaper, safer. Remember those four things. And that's also what most great client testimonial stories will be about. They'll focus on one or more of those. Faster, better, cheaper, safer. Okay, the next thing. Now this is a biggie for you guys. Everybody in this room, I bet all of you guys are actually selling in here. This is where people go wrong. You need to start before the person you're selling to even knows they have a problem. Okay, this is really key because you want to nurture those potential buyers who's never heard of you, who doesn't know about your business, who doesn't know they've actually got a problem.  The issue for the foreman on the site is not that they've dug up the pipes, even though that's a big disaster, but it's how much downtime they're gonna have now on that construction site, which is gonna cost a fortune. Okay, so you could have some content pieces on your website related to that particular problem. Then it educates the buyer. The buyer is you all know it yourselves. When you're interested in something, you start delving into things and you start researching what's going on. This is key. So this is some of the things that you need to do having content along this whole customer journey. So quick tips for success. Draw out your customer journey. Draw it out before the buying journey actually begins. So the pre-buying journey and just think about your ideal customer. What are some of the things that they would like to know more about? What's important to them? Not about selling your product, but what's important to them? It's all about the customer. They don't care if you've got, hey, I've got the latest equipment or the GNSS or whatever. They don't care about that. They want to know how it's gonna help them. Any comments from you? Correct. Well, and a couple of things to add to that. Just some elements on the buyer's journey to help them and you along the way.  The case studies, we already talked about them. Case studies are stories about your customers and their success with your product. That's where Faster, Better, Cheaper, Safer comes in. Of course, you need product sheets that describe the pro or service, product or service. Make those downloadable if you can so they can take them with them. Blogs are great. Speak directly to your customers or potential customers. The great thing about blogs is they can be as short as you want or as long as you want. I recommend short. And then also, don't forget videos. Videos are fantastic. Not just for marketing, but how to? You've gotta constantly educate your clients even after they've bought from you. How do they use your product? How can they use it better and have a better experience with it? So those are all things that are part of the buying journey. Yeah, absolutely. Okay, who knows what a power base is? Anybody? Little names in there? Ooh, one person. It's all right, she's on my team. She knows this one. Okay, so a power base is all the people around you, like your friends, your family, your colleagues, your customers that tell you when it's great and when it's terrible. These are part of your power base. These are part of your network. We just call it a power base. And these people are gold. So one of the things I want you guys to do when you go home, write a list of all the people that is connected to you. Now, if you actually type in network or power base, or it actually comes up with six degrees of separation. And actually, you are all connected to some amazing people, people that can buy from you, buy your service, buy your product. So make a list of these things. And don't rule out your family because you don't know who these people know, okay? It's caught me out. I'm like, nah, but yeah. Maybe because my dad's a surveyor, he knows a lot of people, but it's a really good way of actually building on it, your network, your power base, Kevin. And one thing to remember about your power base, the people in that base and the companies can be kind of unofficial business partners, even if you don't have some kind of an official signed memorandum of understanding or something like that. If you do, that's great, but you can have unofficial business partners. And for me, we trade clients, oftentimes we might have conflicts of interest where we can't take on a client, and we trade them. We don't necessarily have any money change hands when we do that. So we share. We just, you know, I give something to one of my business partners, they give something to me, and it all works out in the end. So it's great to leverage that. And one of the best business partners I have, they provide a service that I personally don't provide. They do market research. Some of the biggest market research reports that you've read in the geospatial industry has been done by one of my business partners. So when somebody comes to me and needs that done, I can just sub it out, or in that case, just give it away to that business partner. So you never want to have to say no to work. Absolutely. Okay, who leverages their staff members in their company to do social media? Not sure. Okay, so this is a big thing, a big disaster that you guys are making. You need to think about your team members. Now, don't think of the negative on the poaching. They're gonna poach my staff. You need to build a team. People want to work for you because they love you, because they believe in what your mission is, what your vision is, where you're after. But actually, using and getting people involved from a staff member perspective is massively valuable. The reason why it's valuable is, one, they feel special. Two, you're building the brand. It's a cohesive brand. And three, it has massive organic reach. So when you're promoting stuff and different things and the staff members are doing some great work, it's a brilliant way to actually get the name out there and really grow it. But you need to have buy-in from everybody. If you've got a bit of a, oh, I'm not quite sure about what my business is doing and I really don't want to share what they're doing, well, you're in the wrong game. So you need to have a real think about, actually, we need to build on this, this amazing network of people. I work with a company in the UK, for example, called Stormgeomatics. Storm at Geomatics. Have a look at them. Have a look on their LinkedIn profile. They are phenomenal about supporting their staff members. They've just hired a load of young surveyors. They do hydrography, a lot of work with water-based solutions. And all their staff are actually talking about what they're doing in the field. And it's really interesting because you get perspective from the young ones, you get perspective from the old ones. And it's been really good for building their brand which then attracts more type of customers. So a big thing to think about. Kevin. Think of it as a force multiplier. Many of you probably have Facebook, use Facebook, and I hope LinkedIn. LinkedIn is one of the most powerful social media tools we have along with the platform formerly known as Twitter. But just start with the other people in your company and make sure every time your company puts out an announcement on LinkedIn or on a Facebook business page or on Twitter, you get the other people in the company to go and like that post, right? It's because what happens, LinkedIn works essentially the same way as Facebook. When somebody likes the post, the post gets moved up and it goes out into more people's feeds and more people will see it. So if you've got 10 people in your company, all 10 of them, or whether it's 100, should be liking every official post from the company. And it could be Instagram, could be Facebook, all those things are good, but that's the important thing. And make sure everybody's aware of this and let the company know. Say, hey, we just put out a big announcement on LinkedIn. Please, everybody go and like it over the next couple of days and that'll make it hang out there, more people will see it. Another little tip actually with LinkedIn is video. Video is the big thing at the moment. I was back at Social Media Marketing World in San Diego. Woo, exciting, especially for us marketeers. And it was all about video, organic reach and video. So small, little, sharp, short videos as well as the big stuff. Tell the stories, this is really powerful. Who pisses off the media? Oh my God. I says, should I have that swear word in there? It's really bad. Anyway, this is a big one. And I think I'm gonna hand this to Kevin because this is a big area that Kevin looks at. This is something I deal with all the time. So in the United States, there's a big trade show called GEOINT for the intelligence community. And my firm runs the press room at GEOINT. And so I'm always talking to the journalists, the media in the press room. And one of the complaints I hear from the media so often is that PR people, marketing people, when they're reaching out to journalists, and I'll talk about a couple different ways, when they do that, they don't do their homework. And they're just sending things inappropriate to the media. So a couple things to keep in mind. When you're sending out press releases, all right, you can send out a generic geospatial press release to all the geospatial media. But let's say you've got a project that, or maybe a product that just applies to oil and gas. So you're not gonna send that to the urban planning publications. So I use email for sending out press releases and I have all of my email lists for publications and bloggers and reporters. I've got them divided, categorized by their vertical market and what they do. So that's the first thing.  Here's the second thing. A lot of savvy PR people, they will pick up the phone or send an email or at a trade show, they'll pitch a story idea in person to reporters. And that's perfectly fine. That's reporters are used to that. But do your research first. Make sure you know before you approach a reporter. It could be they only are interested in news, legitimate news that's happening. And there's some others that might be more interested in features and products. And so be very careful about what you pitched.   So be very respectful, do your background research and send appropriate information to the right reporters and publications.  Now they're flipping their mindsets and they're also changing, which I've seen a lot of lately. And also working directly with the journalists and the editors has been gold because again, going back to your power base, that's free advertising to lots of media to take full advantage of it. So any questions? Thank you very much for having us guys.


Hi, my name is Karolina Majorek and I'm from Wadek Minor's lab at the University of Virginia in Charlottesville. I would like to encourage you to read our article, Double Trouble – Buffer Selection and Histide Presence might be responsible for non-reproducibility of biomedical experiments. It is the featured cover article of the October 2014 issue of Protein Science. In this article, we talk about the influence of polyhystidine affinity tags and buffer heaps on the outcome of protein functional and structural studies. Both of these are very important in the process of obtaining proteins for numerous experiments, but they have to be used with caution, as both might perturb the confirmation and activity of a protein. Reproducibility of experiments in other laboratories might be affected by these factors, even when changes between protocol seem to be insignificant. This might cause a ripple effect as subsequent studies are based on questionable data. Most in vitro biochemical and drug discovery experiments require large quantities of purified and active protein. Many technologies have been developed to increase the efficiency of protein production for in vitro experiments. Affinity tags provide a handle for easy purification of recombinant protein. Polyhystidine, or HIST tags, are especially popular since they are smaller than others, and thus should minimally perturb a protein structure and function. However, there is growing evidence to suggest that HIST tags may have significant effects on structural and activity measurements. Similarly, buffering agents are crucial for preserving a desired pH, but the correct choice of buffer agent is critical to avoid other unwanted chemical effects. Buffers have been shown to interfere with proteins by reacting with substrates or inhibiting enzymatic reactions. By structural and functional experiments, we observed the impact of both HIST tags and buffer molecules on three proteins in the GCN5-related N-acetyltransferase, or NAT, superfamily. Both the HIST tag and the buffer heapus influenced the confirmation and activity of these proteins. Our prior work showed that the pseudomonas aeruginosa protein PA4794 is a NAT acetyltransferase and determined the structure of the protein in complex with products of the reaction, coenzyme A, and acetylated peptide, shown here in pink. When PA4794 is expressed and crystallized in the presence of heapus, the heapus molecule binds in the substrate site in a position similar to that of peptide substrate. Heapus binding induces a conformational change between the apo form of PA4794 and the buffer bound form. The differences are located in the active site area and involve both main chain and side chains. These conformational changes are not observed in any of the other 18 structures of PA4794. If the heapus bound form was the only known structure, one might mistakenly conclude that this conformation was the apo form of the enzyme. When heapus was removed from purification and crystallization buffers, but the histag was not removed by proteolytic cleavage, the histag blocks the substrate binding site. The histag is not inserted as deeply into the active site as substrate or heaps, but occludes the site from outside. In kinetic experiments, the presence of the tag decreased the affinity of PA4794 for peptide substrate. We also analyzed the kinetic parameters of two other NAT proteins, SPEG, a spermedine-sperming acetyltransferase from Vibrio cholera and SA-COL-1063, an N-terminal protein acetyltransferase from Staphylococcus aureus. When both enzymes were tested with histag present, both exhibit decreased activity or affinity for both acetyl-coenzyme A and substrates, as compared to untagged enzyme. Even though we show just a few examples, the effects of buffers and histag are highly underestimated. In more than 18% of the hundreds of structures in the PDB containing heapus, the buffer molecule is bound in close proximity to the active site of proteins. Similarly, many structures of proteins contain evidence of histags bound in the proximity of active sites, but are either not discussed or are omitted altogether from the model, regardless of the crystallographic evidence of their presence. For example, in the structure 2QHA, the histag motif was not modeled, and it was claimed in the primary citation that the motif was not visible in the density map. However, there is clear density for the histag in the active site. When the glycerol and water molecules in this density was replaced with six histidine residues, a few cycles of refinement decreased the R and R-free of this structure by several percent. This shows how critical it is to adjust experimental protocols for each protein or family of proteins and investigate the influence of these factors to properly interpret protein activity and structure. Our results have shown the importance of these factors for members of the NAT superfamily, but may apply to interpreting results obtained for other proteins as well.