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Changing Planet

South Africa’s Lion Bone Trade Disastrous for Wild Tigers

Posted by Environmental Investigation Agency

EIA is appalled that South Africa intends to export the skeletons of 800 African lions a year into a trade that stimulates consumer demand for the bones of more endangered big cats.

An attempt to put lions in CITES Appendix I with a zero quota on wild and captive lion bone trade failed at CITES CoP17 last October. Instead, a compromise document resulted in South Africa being allowed to continue with captive-lion bone trade.

From 2008-14, the bones of more than 4,900 African lions, from both wild and captive sources, were traded from South Africa to Laos, Vietnam, Thailand and China. This lion bone is marketed to end consumers as tiger bone and is indicative of the rapidly expanding and unchecked demand for “tiger bone wine”.

[Read the 2010 National Geographic Voices post: Lion-bone wine latest threat to survival of Africa’s big cats]

Legal big cat bone trade undermines enforcement and exacerbates tiger bone trade as elite consumers will continue to seek the authentic bones of wild tigers as a premium product, which in turn leads to poaching and illegal trade across Asia. Snow leopards, Asiatic lions, leopards and even jaguars are also poached for the big cat bone trade in Asia.

Some of the captive-bred lion facilities in South Africa are also farming tigers and sending body parts to Asia, completely in contravention of CITES decisions on tigers. The fact that South Africa’s regulations are insufficient to prohibit this should sound alarm bells for tiger and lion conservationists alike.

Debbie Banks, team leader of EIA’s Tigers Campaign, said: “There are fewer than 4,000 wild tigers remaining and all governments need to do everything they can to eliminate threats.

“We strongly urge South Africa to take into account the wider impact of the captive lion bone trade beyond its borders, issue a zero quota on lion bone exports from all sources and amend its laws to comply with CITES decisions to prevent the farming of tigers for trade in their parts and derivatives.”

What can you do?

South Africa’s Environmental Affairs department is holding a public consultation on the proposed lion export quota until February 2.

EIA urges all supporters and followers to submit respectful, written comments to Mr Mpho Tjiane via before the deadline.

More details are available on the EIA website.

main-logoEIA’s investigations are a trademark of our work around the world, but we tackle environmental crime and defend the natural world strategically, operating in a number of different ways. Our findings are combined with scientific documentation and representation at international conventions, creating the hard-hitting campaigns which have earned us a global reputation.

  • les

    how are these clowns going to regulate lion bone trade when they cannot protect the rhino or elephant or the citizens od south africa !!

  • Shelly Green

    Mr. Tjiane,
    What you are allowing Africa to do to its lions, tigers, whether wild or on farms is so inhumane.
    China has to be stopped, stop selling your animals for money.
    China should be banned from buying any kind of animal part from your country.
    The animals need your protection, your selling out your country at the cost of these beautiful animals. Because of you, poachers ,CHINA, trophy hunters these beautiful animals will cease to exist, what a horrible shame.
    They are helpless, humans are the worst animals of all
    Stand up for what’s right, the humane thing to do and join forces with EIA!!

  • liezel

About the Blog

Researchers, conservationists, and others share stories, insights and ideas about Our Changing Planet, Wildlife & Wild Spaces, and The Human Journey. More than 50,000 comments have been added to 10,000 posts. Explore the list alongside to dive deeper into some of the most popular categories of the National Geographic Society’s conversation platform Voices.

Opinions are those of the blogger and/or the blogger’s organization, and not necessarily those of the National Geographic Society. Posters of blogs and comments are required to observe National Geographic’s community rules and other terms of service.

Voices director: David Braun (

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5 Back To School Tips For Parents

As summer comes to an end, parents are preparing for the upcoming school year. From organizing the kid’s carpool schedule, to coordinating nutritious lunches, there is a lot to do before the big first day. To avoid wasting the last few weeks of summer stressing over the back to school basics, here is a checklist of five tips to make the process smoother for the adults and give you good grades in your kids eyes.

  1. Get a school supplies list! Don’t play guessing games when it comes to what your child needs for school this year. Teachers, or even the school offices, have lists to make tackling Staples with your family that much easier.
  2. Make sure your child is getting enough sleep. During the summer, bed times may have been a bit lax, so use the last two weeks of summer to gradually readjust your kids sleep schedule.
  3. Before you hit the clothing store for some brand new back-to-school gear, take a few minutes to evaluate what your child already has.  Research any school dress codes, donate what doesn’t fit, and make sure to get all the essentials your child may need.
  4. Take a look at your schedule, now. Don’t wait until last-minute to try to arrange transportation to and from school and other activities. Arranging a carpool for your community or neighborhood in advance, or coordinating with your spouse beforehand, is key to an easy school year.
  5. Much like how college kids use Rate My Professor to know what they’re getting into before a class, use professional reviews to get to know your children’s teachers before they step into the classroom. This can put you and your child and family at ease and reduce those first day nerves!

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Fashion Interviews

Chrome Store: modern vintage meets Berlin edge

Chrome Store is a vintage designer store selling a mixture of independent Berlin designers and handpicked vintage pieces. The store’s vibe is as chic and chilled as owner Jochen Drewes – we recently paid him a visit to talk about the store’s concept and hear Jochen’s style advice for winter.

Tell us a bit about Chrome Store?

Chrome Store is a high-end vintage designer store. But it’s also a place where you can find affordable unbranded items from the 80s and 90s, along with gifts and accessories made by local Berlin designers.

In other words we have a massive mix of products. We originally went for a cool, clean aesthetic, but this has evolved over time and our customers love that we change it up. You’ll find an & Other Stories top, a Miu Miu coat and a non-branded vintage piece all on the same rack.

When did you launch?

We opened in August 2015.

Whereabouts are you based?

We are based in the heart of so-called “Kreuzkölln”, the area lying between Kreuzberg and Neukölln in Berlin.

How did you get into fashion?

I was actually a psychologist working as a university researcher, but I got fed up of working in an office and being employed by someone else. My boyfriend at the time was a stylist and gave me the inspiration to open my own vintage designer store – not to mention a great list of contacts and a ready-made network.

 What’s your advice for anyone who wants to launch their own store?

You have to be consistent. You need to find a niche that makes it unique in some way, something special. And do your research to find the right area if you have a physical store. For instance, I chose an up-and-coming Berlin neighbourhood that’s not too expensive, but is popular with tourists.

Why do you think video is important for selling?

Video is really coming up in fashion. Designers now do their lookbooks in video, and will always capture behind the scenes footage from shoots. People crave moving pictures, they want to see more details on clothing – and get a true picture of how it fits. Photos can be deceptive in that way, heavily touched up with clothing pinned and modified to give it more shape. It’s harder to do that with video and you can also see more details on the clothing and get a better idea of how it moves and flows with the wearer. In some ways it’s more honest.

What are your top three style tips?

  • It’s all about layering – this year I dropped the big winter coat and chose to wear multiple layers with a lighter jacket underneath
  • It’s winter so go cosy with cashmere and merino wool
  • Accessorise with a great bag and gold jewellery

What’s on your Spotify playlist right now?

I keep alternating between Reggaeton classics and Christmas songs by Sufjan Stevens. It depends on whether I’m in the mood for something festive or in need of sun!

Shop Chrome Store’s vintage designer styles on YEAY. Download the app in the iTunes Store or on Google Play


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Merry Christmas, Happy Holidays, …

I hope the holidays are treating you well.  We’ve just finished opening presents at my house and the kids are all bouncing off the walls with joy.  As I said in my last post, it’s been a busy holiday season and I haven’t had much time to post.  After the new year, things should return… Read more

Busy as I can Be

Sorry for the light blogging lately.  It’s been busy the past couple of weeks and it’s not looking like it will be less so until after the holidays.  I spent two weeks in Europe in late Nov visiting customers, partners and attending Tech Ed.  It was a terrific trip and a great opportunity to understand… Read more

Visual Studio Team System Promotions

I wanted to give you an early peek at some great Team System cost savings opportunities.  The marketing campaign around it hasn’t started yet, but we are introducing a host of promotional Visual Studio and Team System price breaks to encourage people to try out new options.  There are 5 offers in the slate: Team Suite,… Read more

VSTS 2008 Database Professional GDR Ships!

The long awaited update for VSTS 2008 Database professional is now available.  You may have been following the many pre-releases (CTPs, RCs, etc).  This is the final update. The biggest new feature in this update is support for SQLServer 2008 but there’s a great deal more.  Among my favorite are support for offline database development… Read more

A new TFS add-on from Teamprise

Teamprise has released a new TFS add-on called Teamprise Remote Accelerator.  It is a solution similar to the TFS proxy but is designed for single user remote scenarios.  It functions exactly like a TFS proxy but can easily be run on your local client and polls the server to “prefetch” files so that they are… Read more

Coolest Team System Gadget

A couple of months ago, I mentioned that one of our MVPs (Mike Azocar) was running a “Coolest Team System Gadget” contest.  He has just announced the winner of the contest: Mathew Rowan for TFS Working On, a tool to help you track the time you spend on tasks.  The tool is availble on CodePlex… Read more

Community Translations of Team System Web Access

A few months ago some of our MVPs expressed interest in creating some translations of Team System Web Access.  We looked into it and concluded that enabling it would be pretty straight forward and seemed like a great way to experiment with a new way of interacting with the community.  Thus was born the TSWA… Read more

Extending the new TFS Team Members Power Tool

The October release of the Team Foundation Power Tools includes a new feature called “Team Members” that allows you to collaborate with your team even better.  One of the features is integration with instant messaging that allows you to see presence, IM, video, audio, etc from inside Team Explorer.  You can read more about the… Read more

Common Oct ’08 TFS Power Tools Questions

This post is going to be my periodically updated list of answers to questions/problems that I hear about.  I’ll get right to it… Last updated 12/2/08 1) Login problems with the TFS Windows Shell Extension – Right now the shell extension only supports contact TFS with your default Windows credentials (using integrated authentication).  This won’t… Read more


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AD FS 2.0 Claims Rule Language Primer

Hi guys, Joji Oshima here again. On the Directory Services team, we get questions regarding the Claims Rule Language in AD FS 2.0 so I would like to go through some of the basics. I’ve written this article for those who have a solid understanding of Claims-based authentication. If you would like to read up on the fundamentals first, here are some good resources.

An Introduction to Claims

Security Briefs: Exploring Claims-Based Identity

AD FS 2.0 Content Map

Claims Rules follow a basic pipeline. The rules define which claims are accepted, processed, and eventually sent to the relying party. You define claims rules as a property of the Claims Provider Trust (incoming) and the Relying Party Trust (outgoing).

Basic flowchart for the Claims Pipeline taken from TechNet.

There is also an authorization stage checks if the requestor has access to receive a token for the relying party. You can choose to allow all incoming claims through by setting the Authorization Rules to Permit All. Alternately, you could permit or deny certain users based on their incoming claim set. You can read more about authorization claim rules here and here.

You can create the majority of claims issuance and claims transformations using a Claim Rule Template in AD FS 2.0 Management console, but there are some situations where a custom rule is the only way to get the results you need. For example, if you want to combine values from multiple claims into a single claim, you will need to write a custom rule to accomplish that. To get started, I would recommend creating several rules through the Claim Rule Templates and view the rule language generated. Once you save the template, you can click the View Rule Language button from the Edit Rule window to see how the language works.

In the screenshot above, the rule translates as follows:

If (there is an incoming claim that matches the type "")

Then (issue a claim with the type "", using the Issuer, Original Issuer, Value, and ValueType of the incoming claim)

The claims "" and "" are URIs. These claims can be in the URN or HTTP format. The HTTP format is NOT a URL and does not have to specifically link to actual content on the Internet or intranet.

Claims Rule Language Syntax:

Typically, the claims rule language is structured similarly to an “if statement” in many programming languages.

If (condition is true)

Then (issue a claim with this value)

What this says is “if a condition is true, issue this claim”. A special operator “=>” separates the condition from the issuance statement and a semicolon ends the statement.

Condition statement => issuance statement;

Review some of the claims you created and look at the structure. See if you can pick out each part. Here is the one we looked at in the first section. Let’s break it down in to the basic parts.

The “if statement” condition:

c:[Type ==]

The special operator:

The issuance statement:

issue(Type = "", Issuer = c.Issuer, OriginalIssuer = c.OriginalIssuer, Value = c.Value, ValueType = c.ValueType);

For each rule defined, AD FS checks the input claims, evaluates them against the condition, and issues the claim if the condition is true. You probably notice the variable “C” in the syntax. Think of “C” as an incoming claim that you can check conditions against, and use values from it to add to an outgoing claim. In this example, we are checking if there is an incoming claim that has a type that is “”. We also use the values in this claim to assign the value of Issuer, OriginalIssuer, Value, and ValueType to the outgoing claim.

There are exceptions to this that are discussed later (using ADD instead of ISSUE and issuing a claim without a condition statement).

Issue a claim to everyone:

In the Claims Rule Language, the condition part is optional. Therefore, you can choose to issue or add a claim regardless of what claims are incoming. To do this, start with the special operator “=>”.

=> issue(type = "", value = "Adatum");

This syntax will issue a claim type “” with a value of “Adatum”

You could set similar rules for each Claims Provider Trust so that the Relying Party (or application) can know where the user came from.

Using a Single Condition:

In this example, we will look at a single condition statement. A basic claim rule checks to see if there is an incoming claim with a certain type and if so, issue a claim.

c:[Type == ""]
 => issue(claim = c);

This syntax will check to see if there is an incoming claim with the type “” and, if so, issue the exact same claim going out.

You can create this claim rule using the GUI. Choose the template named “Pass Through or Filter an Incoming Claim” and choose the appropriate incoming claim type.

Screenshot: Entries for a simple pass through claim.

You may also check for multiple values within your condition statement. For example, you can check and see if there is an incoming claim with a specific value. In the following example, we will check for an incoming claim with the type “” that has the value of “Editors” and, if so, issue the exact same claim.

c:[Type == "", Value=="Editors"]
 => issue(claim = c);

You can create this claim rule using the GUI as well. Choose “Pass Through or Filter an Incoming Claim”, choose the appropriate incoming claim type, select “Pass though only a specific claim value”, then enter the appropriate value.

Screenshot: Entries to pass through the Role claim if the value is “Editors”

Using Multiple Conditions:

Say you want to issue a claim only if the user has an Editor and has an Email claim and, if so, issue the Editor Role claim. To have multiple conditions, we will use multiple “C” variables. We will join the two condition statements with the special operator “&&”.

c1:[Type == "", Value=="Editors"] &&
c2:[Type == ""]
 => issue(claim = c1);

The first condition (c1) checks to see if you have an incoming role claim with the value of Editors. The second condition (c2) checks to see if there is an incoming email claim. If both conditions are met, it will issue an outgoing claim identical to the incoming c1 claim.

Combining Claim Values:

Say you want to join information together from multiple incoming claims to form a single outgoing claim. The following example will check for an incoming claim type of "" and “”. If it has both, it will issue a new claim, “”, combining the two values.

c1:[Type == ""] &&
c2:[Type == ""]
 => issue(Type="", Value=c1.value+" "+c2.value);

The resulting value is the value of the first claim (c1), plus a space, plus the value of the second claim (c2). You can combine static strings with the values of the claims using the special operator “+”. The example below shows a sample set of incoming claims, and the resulting output claim.

Example Incoming Claims:
"" is "Seattle"
"" is "Editor"

Example Outgoing Claim:
"" is "Seattle Editor"

Using ADD instead of ISSUE:

As mentioned in an earlier section, you can ADD a claim instead of ISSUE a claim. You may be wondering what the difference between these two statements are. Using the ADD command instead of the ISSUE command will add a claim to the incoming claim set. This will not add the claim to the outgoing token. Use this for adding placeholder data to use in subsequent claims rules.

This illustration was taken from a TechNet article. Here you can see that the first rule adds a role claim with the value of Editor. It then uses this newly added claim to create a greeting claim. Assuming these are the only two rules, the outgoing token will only have a greeting claim, not a role claim.

I’ve outlined another example below.

Sample Rule 1:

c:[Type == "", Value=="NYC"]
 => add(Type = "", Value = "East");

Sample Rule 2:

c:[Type == "", Value=="LAX"]
 => add(Type = "", Value = "West");

Sample Rule 3:

c1:[Type == ""] &&
c2:[Type == ""]
 => issue(Type="", Value=c1.value+" "+c2.value);

In this example, we have two rules that ADD claims to the incoming claim set, and one that issues a claim to the outgoing claim set. This will add a region claim to the incoming claim set and use that to create combine the values to create an area claim. The ADD functionality is very useful with the next section for aggregate functions.

Using aggregate functions (EXISTS and NOT EXISTS):

Using aggregate functions, you can issue or add a single output claim instead of getting an output claim for each match. The aggregate functions in the Claims Rule Language are EXISTS and NOT EXISTS.

Say we want to use the location claim, but not all users have it. Using NOT EXISTS, we can add a universal location claim if the user does not have one.

In Sample Rule 1, we will add a location claim with the value of “Unknown” if the user does not have a location claim. In Sample Rule 2, we will use that value to generate the “” claim.

Sample Rule 1:

NOT EXISTS([Type == ""])
 => add(Type = "", Value = "Unknown");

Sample Rule 2:

c1:[Type == ""] &&
c2:[Type == ""]
 => issue(Type="", Value=c1.value+" "+c2.value);

This way, users without the "" claim can still get the "" claim.

Claims Rule Language, beyond this post:

There is more you can do with the Claims Rule Language that goes beyond the scope of this blog post. If you would like to dig deeper by using Custom Attribute Stores and using Regular Expressions in the language, I’ve put up a TechNet Wiki article that contains these advanced topics and other sample syntax. In addition, some other articles may help with these topics.

Understanding Claim Rule Language in AD FS 2.0:

When to Use a Custom Claim Rule:

The Role of the Claim Rule Language:

The Role of the Claims Engine:

The Role of the Claims Pipeline:

Creating custom rules with the Claims Rule Language gives you more flexibility over the standard templates. Syntax familiarization takes a while, but with some practice, you should be able to write custom rules in no time. Start by writing custom rules instead of using the templates in your lab environment and build on those.

– Joji “small claims court” Oshima


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Tip 2: Updating Big Content Farms

tip is for Windows SharePoint Services 3.0 and Microsoft Office SharePoint
Server 2007 and may apply to SharePoint Foundation 2010 and SharePoint Server 2010.

your SharePoint farm has a large amount of content then detaching the content
databases from the web applications before running the SharePoint Products and
Technologies Configuration Wizard on the first server in the farm will speed up the installation
and upgrade wizard. Of course you will still have to
the databases
afterward, but if you are doing the upgrade after hours at least
you can get through the manual part of the install and monitor the attach

caveat, when you detach a content database, the next crawl of the site
collections in that database will be full even if they are scheduled as

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Healthy Snacking

You will not believe it but I am starting this post freaking early in the morning.No really. My manager managed to throw the keys to the building away. AND the spares. She’s now waiting for one of her assistant managers to hopefully wake up and get her message. >.< I feel so bad!

Anyway, it gives me the time to tell you all about yesterday!

It started with a really untrying picture of Kashi GoLean Crunch and half an organic gala apple. It’s the only fruit I had! =D Sorry my breakfasts are so disappointing lately. There’s just not enough hours in the day to make a hot breakfast. =/

After 4 hours of class lunch was well needed. I had a bowl of tomato soup with oyster crackers (which was super yum! My school knows how to make a good soup at least..) and a side bowl of a beet and carrot salad with a few pieces of marinated tofu.

Completely satisfying!

Around 4pm, my very last class of the day, I snacked on the other half of my morning apple with a little wedge of cheddar cheese. Mmm, I forget how much I love this combination.

A lot of the time when my cravings and crazy snacking is worst, it’s right while I’m trying to make dinner (or waiting for Chase to come home so I can eat it!). Taking the awesome advice from yesterday’s comments, I’d hit up the store for some Simply Dressed Ranch dressing to go along with some raw produce!

Holy moly. It’s funny how it’s the simple combinations one forgets. This was outstanding! I think I used to be turned off by veggies and dip because I was so afraid of the fat and calories in dip. Now, I don’t get some gross-ingredient low fat Ranch, but I don’t use very much at all so I’m not worried. ^_^

It was so good I even had a few pieces of broccoli along with a carrot while I waited for dinner.

Dinner was also delicious and veggie based: roasted delicata squash, rose fingerling potatoes, and onions, along with sautéed kale and black beans. Plus a hard apple cider because it was a rough day!

I really intended to head to the gym. Honestly. I even got my clothes and shoes on…but then I let Chase talk me out of it and relaxed and did some cross stitch instead. I definitely need to rework my workout schedule with how busy I am now. =/

For dessert I had two squares of Ghiradelli dark chocolate with almonds and sea salt. ❤

Anyway hope you all are having a happy Friday!

2 thoughts on “Healthy Snacking

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Heads Up Newsletter

As of June 2014 Heads Up moved to headsup.ucc.on.ca - for current issues go there. This page is retained solely as an archive of earlier issues.

Emailed to you every Thursday, Heads Up is a must-read publication. You'll find everything you need to know about your son's life at the school including forms, trip consents, class happenings, updates and upcoming events, and news about sports and academic acievements, service trips and more.

Just a reminder on how to navigate. Access news tailored to your division by clicking the colour-coded buttons on the left navigation bar.

  • For SK to Form 5 information, click on the Primary button.
  • For Forms 6 and 7, click on the Middle button.
  • For Year 1 and 2, click on the Intermediate button.
  • For Foundation Year, IB1 and IB2, click on the Senior button.

If your son is in the boarding program, Boarding Life will link you to news and happenings. The Future Ties button will link you to information on university placement. The Whole School section provides news of interest to the entire College.

Please remember to look in the download section each week for supporting information. Each download is also referenced in each divisional news item.


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BMC Bioinformatics

Open Access

Molecular dynamics simulation and bioinformatics study on chloroplast stromal ridge complex from rice (Oryza sativa L.)

BMC BioinformaticsBMC series – open, inclusive and trusted201617:28

Received: 1 September 2015

Accepted: 4 January 2016

Published: 12 January 2016

Rice (Oryza sativa L.) is one of the most important cereal crops in the world and its yield is closely related to the photosynthesis efficiency. The chloroplast stromal ridge complex consisting of PsaC-PsaD-PsaE plays an important role in plant photosynthesis, which has been a subject of many studies. Till now, the recognition mechanism between PsaC and PsaD in rice is still not fully understood.

Here, we present the interaction features of OsPsaC and OsPsaD by molecular dynamics simulations and bioinformatics. Firstly, we identified interacting residues in the OsPsaC-OsPsaD complex during simulations. Significantly, important hydrogen bonds were observed in residue pairs R19-E103, D47-K62, R53-E63, Y81-R20, Y81-R61 and L26-V105. Free energy calculations suggested two salt bridges R19-E103 and D47-K62 were essential to maintain the OsPsaC-OsPsaD interaction. Supportively, electrostatic potentials surfaces of OsPsaD exhibited electrostatic attraction helped to stabilize the residue pairs R19-E103 and D47-K62. In particular, the importance of R19 was further verified by two 500 ns CG-MD simulations. Secondly, this study compared the stromal ridge complex in rice with that in other organisms. Notably, alignments of amino acids showed these two salt bridges R19-E103 and D47-K62 also existed in other organisms. Electrostatic potentials surfaces and X-ray structural analysis strongly suggested the stromal ridge complex in other organisms adopted a similar and general recognition mechanism.

These results together provided structure basis and dynamics behavior to understand recognition and assembly of the stromal ridge complex in rice.

Rice (Oryza sativa L.)Chloroplast stromal ridge complex OsPsaC and OsPsaDMolecular dynamics simulationBioinformatics study

Rice (Oryza sativa L.) is one of the most important cereal crops in Southeast Asia and sixty percentage of the population regard rice as the staple food in China. Improving photosynthesis efficiency can greatly increase rice yield. Photosynthesis occurs in the chloroplast and it begins when the electron is raised to a higher energy in a special chlorophyll molecule P680 of the photosystem II (PSII). Afterwards, the energy is transferred by an electron transport chain and four thylakoid membrane protein complexes are involved in the electron transferring. These complexes are photosystem I (PSI), PSII, cytochrome b6f and ATP-synthase complexes. PsaC, PsaD and PsaE of PSI are termed as “stromal ridge complex” as they locate in the stromal side of the thylakoid membrane. The main function of the stromal ridge complex is to dock electron acceptors ferredoxin and flavodoxin by PsaC binding to electron acceptors FA and FB [1].

Genetic deletion experiments [24] showed PsaC firstly bound to the PsaA/B heterodimer, and then PsaD bound to the prerequisite PsaA/B-PsaC complex, and finally PsaE bound to the PsaA/B-PsaC-PsaD complex. Supportively, X-ray crystallography [5, 6] exhibited PsaC, PsaD and PsaE located on the top of PsaA/B, containing a pseudo C2-symmetry [7]. This spatial conformation allows PsaC rotate around pseudo C2 symmetry by its binding to PsaA/B [7]. As the second assembly stromal ridge subunit, PsaD is supposed to bind the correctly bound-region of PsaC, while undock incorrectly-bound PsaC. Additionally, functional experiments [8, 9] showed only if PsaD bound to stromal ridge proteins, the electron acceptors FA and FB were able to maintain their final magnetic properties [8] and to reduce the ferredoxin [9].

These experimental observations together stress the importance of the PsaC-PsaD interaction in the formation of stromal ridge complex. However, the dynamics behavior of the PsaC-PsaD interaction is still obscure. Currently, the in silico studies are good complementary to experimental approaches. All-atomic (AT) and coarse grained (CG) molecular dynamics (MD) simulations can reflect protein dynamic behaviors in detail such as conformational change. Free energy calculation had been used to explore molecular mechanism [10] and drug scanning [11]. For instance, Zhang et al. [12] computed the free energy profile of lipid PS6 along its dissociation path of aquaporin V by a non-equilibrium molecular dynamics method, with the Brownian dynamics fluctuation dissipation theorem. This method was also used to determine the free energy profile of O2 along its permeation path, helping scientists to understand the dissociation constant and binding mechanics. Zhang et al. [10] employed computational alanine scanning based on the molecular mechanics generalized Born surface area (MM-GBSA) approach to identify one complex model pSOD-OsTom20. Collectively, MD simulations combined with free energy calculations were efficient approaches to explore molecular binding mechanism in the protein complex.

In this study, we explored the recognition mechanism between OsPsaC and OsPsaD (Os: Oryza sativa) by MD simulations and bioinformatics analysis. By using MD simulations, important H-bonding interactions were identified. Free energy calculations suggested two salt bridges R19-E103 and D47-K62 were essential to maintain the interaction between OsPsaC and OsPsaD. Supportively, electrostatic potentials calculations and CG-MD simulations exhibited the importance of these two salt bridges. In addition, a bioinformatics analysis showed generality of these two salt bridges in other organisms.

Generation of atomic models of OsPsaCD complex

The modeling process was performed as our previous descriptions [10, 13, 14]. The amino acid sequence of the PsaC and PsaD (OsPsaC and OsPsaD) from Oryza sativa were obtained from the NCBI (ID: gi: 11466848 and gi: 115477831). In the first homology modeling step, template structures related to the PsaC and PsaD proteins were searched against the whole Protein Data Bank [15](PDB) using the Blast algorithm. We modeled complex structure OsPsaC-OsPsaD from Oryza sativa using the crystal structure from Pisum sativum (PDB id: 2WSC) as a template through the SWISS-MODEL [16].

In this work, residue numbering in the OsPsaD starts from the 66th residue because the first 65 residues of the PsaD are not in the recognition region. This means that the first residue of the OsPsaD (Q1) in the main text is actually referred to as Q66.

MD simulation with explicit solvent

The MD simulations were performed with the GROMACS 4.5.3 software package [17] using the OPLS force field [18] and the SPC216 water model. The protonation state of ionizable groups was chosen to correspond to pH 7.0. Counterions were added to compensate the net charge of the system. The initial structure of the complex was immersed in a periodic water box. The electrostatic interactions were calculated by using the Particle-mesh Ewald (PME) algorithm [19], and the van der Waals forces were treated with a cutoff distance of 10 Å. After 3000 steps of energy minimization using a steepest descent method, the system was subject to 300 ps of equilibration at 300 K and normal pressure, using harmonic position restraints with a force constant of 1000 kJ mol−1 nm−2. The system was coupled to an external bath by the Berendsen pressure and temperature coupling method [20]. The production run was performed under the same conditions except that all position restraints were removed. The results were analyzed using the standard software tools provided by the GROMACS package [17]. Calculation of free energy landscapes was performed with in-house scripts [10, 12, 13, 21, 22]. Visualization and manipulation of the conformations was performed using the programs visual molecular dynamics [23] and Swiss-PdbViewer 3.7 [16]. The distance analysis of the nonpolar residue-pairs was performed using the R statistical software package [24].

Calculation of binding free energies by MM-GBSA

The binding free energy of the OsPsaCD was estimated by the MM-GBSA approach [25, 26]. In this study, the implicit generalized Born solvation model was used (igb = 2). The temperature was set to 300 K. Non-bonded interactions were cut off at a distance of 12 Å. The ff99 force field (Parm99) [27] was applied throughout the energy minimization and MD simulations.

In the MM-GBSA implementation of Amber 11.0 [28], the binding free energy of A + B → AB is calculated using the following thermodynamic cycle:
$$ \begin{array}{l}\varDelta Gbinding=\varDelta Ggas-\varDelta {G}_{solv}^A-\varDelta {G}_{solv}^B+\varDelta {G}_{solv}^{AB}\\ {}\kern5em =\varDelta Hgas-T\varDelta S-\varDelta {G}_{GB SA}^A-\varDelta {G}_{GB SA}^B+\varDelta {G}_{GB SA}^{AB}\\ {}\kern5em =\varDelta Hgas-T\varDelta S+\varDelta \varDelta {G}_{GB}+\varDelta \varDelta {G}_{SA}\\ {}\varDelta {H}_{gas}\approx \varDelta Egas=\varDelta {E}_{intra}+\varDelta {E}_{elec}+\varDelta {E}_{vdw}\\ {}\varDelta \varDelta {G}_{GB}=\varDelta {G}_{GB}^{AB}-\left(\varDelta {G}_{GB}^A+\varDelta {G}_{GB}^B\right)\\ {}\varDelta \varDelta {G}_{SA}=\varDelta {G}_{SA}^{AB}-\left(\varDelta {G}_{SA}^A+\varDelta {G}_{SA}^B\right)\end{array} $$

Where T is the temperature, S is the solute entropy, ΔGgas is the interaction energy between A and B in the gas phase, and ΔG solv A , ΔG solv B and ΔG solv AB are the solvation free energies of A, B, and AB, which are estimated using a GB surface area (GBSA) method [25, 26]. That is, ΔG solv AB  = ΔG GBSA AB  + G GB AB  + ΔG SA AB , and so forth. ΔGGB and ΔG SA are the electrostatic and nonpolar terms, respectively. The bond, angle, and torsion energies constitute the intramolecular energy ΔE intra of the complex, while ΔE elec and ΔE vdw represent the receptor-ligand electrostatic and van der Waals interactions, respectively. Because of the constant contribution of − TΔS for each complex, we refer to ΔG binding for ΔG binding + TΔS in the discussion. To verify the quality and validity of the resulting OsPsaCD complexes, the relative binding free energy ΔG binding was calculated by using MM-GBSA for post processing snapshots from the MD trajectories. The computational alanine scanning method in MM-GBSA was used to evaluate the important residues. The key residues were mutated to alanine and subsequently the difference in the binding free energies between mutated and wild-type complexes was calculated based on the MM-GBSA approach.

Electrostatic potential calculations

Electrostatic potential maps were calculated with the Adaptive Poisson-Boltzmann Solver (APBS) [29] according to default parameters (physiological salt concentration of 150 mM, temperature of 298.15 K, solvent dielectric of 78.54, and solute dielectric of 2). Solute charges were distributed onto grid points using a cubic B-spline discretization. The molecular surface was defined by the interface between the radius of a water molecule (1.4 Å), and the solute van der Walls radii.

Bioinformatics analysis

To perform bioinformatics study, we followed our previous method [30] with minor modifications. The full-length PsaC (gi:743432912) and PsaD (gi:115477831) protein sequences from Oryza sativa were used as search queries. We conducted BLASTP [31] searches against the National Center for Biotechnology Information (NCBI) non-redundant database, with a cutoff E-value of 1e-10. To reflect evolutionary diversity of PsaC, we selected the following species (Table 1): four land plants (Oryza sativa, Zea mays, Blossfeldia liliputana, and Cuscuta obtusiflora), one charophyte (Chara vulgaris), one pteridophyta (Huperzia lucidula), one bryophyte (Syntrichia ruralis), one green algae (Bryopsis hypnoides), and two strains of cyanobacteria (Lyngbya sp. PCC 8106, and Chamaesiphon minutus). As to PsaD, the selected species for exhibiting evolutionary diversity were as follows: five land plants (Oryza sativa, Zea mays, Theobroma cacao, Medicago truncatula, and Glycine max), one bryophyte (Physcomitrella patens), two phaeocystis (Phaeocystis antarctica, and Phaeocystis globosa) and two green algae (Chlamydomonas reinhardtii, and Micromonas pusilla CCMP1545). To obtain the genes coding for PsaC and PsaD in these species, we used the OspsaC and OspsaD genes to conduct BLASTN searches against nucleotide databases among these organisms. Gene sequences were aligned using ClustalW [32].
Table 1

The distances between residues corresponding to R19C and E103D in Oryza sativa

Distance (Å)

Synechococcus elongatus

R18 NH1

E103 OE2

R18 NH2

E103 OE1

Pisum sativum

R19 NH1

E121 OE2

R19 NH2

E121 OE1

Pisum sativum

R19 NH1

E175 OE2

R19 NH2

E175 OE1

Pisum sativum

R19 NH1

E175 OE1

R19 NH2

E175 OE2

Pisum sativum

R19 NH1

E176 OE1

R19 NH2

E176 OE2

Spinacia oleracea

R19 NH1

E121 OE2

R19 NH2

E121 OE1

Synechocystis sp. PCC 6803

R19 NH1

E106 OE2

R19 NH2

E106 OE1

Construction of the phylogenetic tree

To construct phylogenetic tree, we followed the method of Wang [33] with minor modifications. Briefly, protein sequences were aligned using MUltiple Sequence Comparison by Log- Expectation (MUSCLE) [34] and the resulting protein alignment was subjected to manual inspection in MEGA6 [35]. Phylogenetic analyses were conducted by Neighbor-Joining (NJ) [36]. The NJ phylogenetic tree was reconstructed using protein Poisson distances [37] and the pairwise-deletion of gap sites implemented in MEGA6 and was evaluated with 1000 bootstrap replicates [38].

Coarse Grained representation

This atomistic model was converted to coarse grained (CG) model using the protocol described by Marrink [39]. The MARTINI forcefield [40, 41] was used for the CG-MD simulations. Briefly, the non-H atoms were mapping to the CG standard particles and CG ring particles by 4:1 and 2–3:1 mapping schemes. Eighteen CG particle types were defined and divided into four main categories: polar (P), charged (Q), mixed polar/apolar (N) and hydrophobic apolar (C). The Lennard Jones interactions were used for the inter-particle interactions, including 10 subtypes to reflect the hydrogen bonding capabilities or polarity.

The protein backbone was represented by a single particle, while the side chain was described by zero or four particles. The Elastic Network (EN) Modeling was built across the CG backbone beads, aiming to maintain the protein secondary structure. The EN scaffold component was based on the MARTINI molecular force field [40, 41]. Two backbone beads were linked by a spring with force constant and a predefined distance cutoff value. In this study, each monomer possessed its own separate EN scaffold, using the same value of RC and KSPRING. This design will let the monomer move independently with respect to the other one during the MD simulation.

Coarse grained MD simulations

All CG-MD simulations were performed using GROMACS 4.5.3 [17]. The Berendsen weak coupling algorithm was used to maintain the temperature (coupling constant of 0.5 ps; reference temperature 300 K) and pressure (coupling constant of 1.2 ps; reference pressure 1 bar), respectively. The nonbonded interactions were treated with a switch function from 0.0 to 1.2 nm for the Coulomb interactions, and from 0.9 to 1.2 nm for the Lennard-Jones interactions. The integration time-step was set to 20 fs and the neighbor list was updated every 5 steps. Each EN system was solvated in a rectangular box with a minimum of 1.0 nm between any protein bead and the edge of the box. After energy-minimization with position restraints (1000 kJ mol-1 nm-2) applied to all protein beads, a 50 ps MD simulation using a 1 fs time-step with the same position restraints was used to relax both the solvent molecules and the protein in the force field. The system was further relaxed with 1 ns long MD run, using a 20 fs time-step and position restraints on the protein “backbone” beads. Finally each system was simulated 500 ns for production without any restraints. In order to convert the CG protein representation, four steps were used according from the protocol described by Sansom and coworkers [42]. Firstly, the backbone of the protein was grown from the CG Cα particles using the Pulchra algorithm [43]. Secondly, the missing side chain atoms of the protein were added using in MODELLER [44]. Note that the advantage of these two steps is to preserve the original coordinates from the CG particles greatly [42]. Thirdly, the protein structure was energy minimized using the conjugant gradient and steepest descents approach to reduce the internal steric clashes of the model.

Stability of AT-MD simulations

To access the stability of the OsPsaC-OsPsaD complex, we monitored its structural drift with respect to the initial conformation just prior to the production MD simulation (Fig. 1a). The Cα RMSD values for OsPsaC-OsPsaD plateaued around 3 Å during simulations, exhibiting the stability of the protein complex. In addition, we monitored the secondary structure property of OsPsaC-OsPsaD during simulations, and compared it with that in S.elongatus and P.sativum (Fig. 1b). Our results suggest OsPsaC and OsPsaD were able to maintain their structural integrity during most of simulations, exhibiting our model had reached a conformational steady state.
Fig. 1

a System setup: top view of the model OsPsaC-OsPsaD complex. OsPsaC and OsPsaD were solvated in the water solvent. Shown in A was the protein in cartoon representation (OsPsaC in yellow and OsPsaD in gray), the waters in CPK representation (colored in red), and the ions in balls representation. Rendered with VMD [23]. b RMSD of the OsPsaC-OsPsaD complex with respect to the starting structure for Cα atoms of OsPsaC (blue), of OsPsaD (red), and of OsPsaC-D (green) during the 100 ns production simulation. c Time evolution of secondary structure elements in the S.elongatus, P.sativum, and O.sativa, in which β-sheets and α-helices were shown in yellow and red

Given that β-sheets were widely distributed in the structural architecture of PsaD, we evaluated the stability of β-sheets during the simulations. Fig. 2 suggested both H-binding interactions and hydrophobic interactions played important roles in maintaining β-sheets of OsPsaD. Observably, significant hydrogen-bonding interactions stabilizing the β1-β2 and β1-β3 strands were found in the residue pairs F27-V85, V29-Y83, T31-Q81, L57-I30 and L59-Y28 (Fig. 2b). In addition, we measured the hydrophobic interactions by calculating distance between the closest atoms. Fig. 2c exhibited the hydrophobic interactions in the residues pairs L56-I30, L57-V29, L57-I30, L59-F27 of the β1-β2 strand, and F27-V85, V29-F82, I30-F82 of the β1-β3 strand. Hence, the stability of β-sheets also supported our simulation system had reached its equilibrium state.
Fig. 2

(a) Residues maintaining the stability of β-sheets in OsPsaD. The number of H-bonds (b) in residue pairs F27-V85, V29-Y83, T31-Q81, L57-I30, L59-Y28, and the distance between the closest atoms (c) in the residue pairs L56-I30, L57-V29, L57-I30, L59-F27, and F27-V85, V29-F82, I30-F82 during 100 ns MD simulation

H-bonding interactions between OsPsaC and OsPsaD

Given that the H-bonding interactions play key roles in maintaining protein stability, we analyzed the H-bonding occupancy between OsPsaC and OsPsaD during MD simulations. Two charged residues R19C and E103D were able to form a salt-bridge, occupying most of the simulation time (Fig. 3a and b). Interestingly, previous study [1] showed the importance of salt bridges in the binding of PsaC to PsaD. Thus, we also calculate the energy contribution of these two residues in the following part. In the neighbor of E103D, V105D formed the hydrophobic interaction with L26C. Besides, a salt-bridge existed in the residue pair D47-K62, which was occasionally disrupted in the middle of the simulation. In addition, we also observed some H-bonding interactions in the residue pairs R53-E63, Y81-R20 and Y81-R61. However, these interactions were not pretty stable at the beginning of simulations.
Fig. 3

a A close view of key residues in the binding of OsPsaC to OsPsaD. b The H-bonds occupancy between the interaction residues during 100 ns MD simulations

Computational alanine scanning

To further explore the recognition residues in the OsPsaC-OsPsaD complex, we evaluated relative binding free energies by MM-GBSA, and compared interaction energy differences between the mutants and the wild-type by the computational alanine scanning (Fig. 4). We used in-silico method to construct mutants following our previous method [10] The residues were mutated to alanine, and subsequently the difference in the binding free energies between mutated and wild-type complexes was calculated based on the MM-GBSA approach. We conducted equilibrium MD simulations on the 11 independent snapshots extracting from the 100 ns simulation time. These 11 snapshots consisted of one snapshot at 0 ns, and ten snapshots from 90 ns to 100 ns.
Fig. 4

Binding affinities estimated from computational alanine scanning for mutations of the OsPsaC-OsPsaD complex ((ΔΔG binding  = ΔG binding(mutant)  − ΔG binding(wild ‐ type) ). ΔΔG binding results for 11 single mutations in either OsPsaC or OsPsaD in the initial structure at 0 ns (green) and averaged over 10 structures at the end of the 100 ns simulation (pink; error bars represent standard deviations)

The R19A and E103A mutants induced a loss of 9.5 and 14 kcal/mol in ΔΔG binding scanning results in the initial structure (Fig. 4). Importance of these two residues was further supported by the values of 8.5 and 10.6 kcal/mol in the ΔΔG binding scanning over the last 10 ns structures. These results showed a good agreement with previous H-bonding analysis, suggesting they significantly contributed to recognition between OsPsaC and OsPsaD. Besides, two charged residues D47C and K62D lost the energy 4.0 and 5.0 kcal/mol respectively in the initial structure (Fig. 4), and 4.5, 9.8 kcal/mol over the last 10 ns snapshots when they mutated to the alanine, suggesting their importance for the recognition affinity. Also, we noticed three residues R20D, R61D and Y81C corresponded to the loss of energy 10.4, 7.0, 2.6 kcal/mol in the initial structure, and 12.9, 10.5, 4.0 kcal/mol over the last 10 ns snapshots.

CG-MD simulations

To further investigate the effect of the E103A mutation on OsPsaC-OsPsaD, two 500 ns CG-MD simulations were conducted for the wild-type (WT) system and the E103A mutant (Fig. 5). Expectantly, the salt bridge R19-E103 was able to stabilize in the WT system during the whole 500 ns simulation. This is consistent with previous results from the AT-MD simulations. Significantly, we found domain rearrangements of OsPsaC in the E103A mutant. At the beginning of CG-MD simulation, R19 and A103 can still bind tightly. However, these two subunits deviated at around 100 ns due to the disruption of their H-bonding interactions. At 500 ns, the R19-A103 interaction was thoroughly disrupted and conformational change can be observed in OsPsaC. Results from CG-MD simulations verified the importance of the salt bridge R19-E103 to maintain the OsPsaC and OsPsaD interaction.
Fig. 5

Snapshots at 0 ns, 100 ns and 500 ns were extracted from 500 ns CG-MD simulations for WT (a) and E103A mutant (b)

Electrostatic potentials maps

The Electrostatic Potential Surfaces (EPS) of PsaD was claculated with Linearized Poisson-Boltzmann Equation (LPBE) mode in the APBS package. This method solves a molecule using an algorithm of single DH sphere boundary condition and generates a 3D surface skin model colored by electrostatic potential. Dielectric constants for protein and solvent were 2.0 and 78.0 in the calculations. We extracted OsPsaC-OsPsaD structures at 0 ns and 100 ns. Fig. 6 showed R19 with positive charge was embraced by negative electrostatic potentials. Similarly, the negative charged residue D47 was in the neighbor of electropositive potentials. Apparently, electrostatic attraction plays an important role in the process of OsPsaC binding to OsPsaD, which is consistent with previous energy analysis.
Fig. 6

Skin representation of the molecular electrostatic potential surface (EPS) of OsPsaD at 0 ns and 100 ns according to the electrostatic potentials. Red: ESP < −10 kT/e, blue: > + 10 kcal/e, grey: between −10 and +10 kT/e

Bioinformatics analysis

To investigate if the recognition mechanism between OsPsaC and OsPsaD is a general mechanism in other organisms, we conduct amino acid homology search and sequence alignments. Using BLASTP to search protein databases in NCBI, we found the protein sequence of OsPsaC had a high similarity to the PsaC proteins in land plants. Figure 7 showed R19 and D47 of OsPsaC were identical to those in other organisms such as Z.mays, B.liliputana, C.obtusiflora, S.ruralis, B.hypnoides, C.vulgaris, H.lucidula, C.minutus, and L.sp.PCC8106. Table S1 exhibited OsPsaC shares a similarity of 99 % identity to ZmPsaC from Z.mays, 95 % identity to BlPsaC from B.liliputana. The gene sequence of OsPsaC shares a similarity of 97 % identity to ZmPsaC from Z.mays, 89 % identity to BlPsaC from B.liliputana (Additional file 1: Table S2 and Figure S3). Supportively, the phylogenetic analysis exhibited OsPsaC can be clustered together with ZmPsaC and BlPsaC (Additional file 1: Figure S1). Besides, Table S2 showed the gene sequence of OspsaC shares a similarity of 79 % identity to CmpsaC from C.minutes and 76 % identity to LspsaC from L.sp. PCC 8106. This is mainly because C.minutes and L.sp. PCC 8106 are cyanbacterias, and land plants PsaC and cyanbacteria PsaC are with a different evolutionary origin (Additional file 1: Figure S1).
Fig. 7

Sequence alignments of PsaC ? (a) and PsaD (b) in different organisms. Four residues R19, D47, K62 and E103 were highlighted

Figure 7 also showed E103 and K62 of OsPsaD were present in all nine other organisms: G.max, Z.mays, T.cacao, P.patens, M.pusilla, M.truncatula, C.reinhardtii, P.antarctica,and P.globosa. Significantly, Table S1 exhibited the protein sequence of OsPsaD has a high similarity to the PsaD proteins in land plants. For instance, it shares a similarity of 82 % identity to ZmPsaD from Zea mays. The phylogenetic analysis showed OsPsaD was clustered together with ZmPsaD (Additional file 1: Figure S2), emphasizing the land plants share an evolutionary origin. In addition, we found OsPsaD was not clustered with PaPsaD from P.antarctica and PgPsaD from P.globosa. Supportively, the gene sequence of OspsaD only shares 32 % identity to PapsaD and 54 % identity to PgpsaD (Additional file 1: Table S2 and Figure S4). Besides, OsPsaD was not clustered with CrPsaD from C.reinhardtii and MpPsaD from M.pusilla CCMP1545 (Additional file 1: Figure S2). Thus, OsPsaD may have a different evolutionary origin from PaPsaD, PgPsaD, CrPsaD and MpPsaD.

Comparison of X-ray structures

By searching the whole Protein Data Bank (, we extracted seven different PsaC-PsaD complexes whose three-dimensional structures were known by X-ray technique. These seven structures were from four different organisms: Synechococcus elongatus (PDB: 1jb0), Pisum sativum (PDB: 2wsc, 4xk8, 3lw5, 4rku), Spinacia oleracea (PDB: 2o01) and Synechocystis sp. PCC 6803 (PDB: 4l6v). We calculated the EPS of PsaD using the APBS package. Figure 8 suggested the D47 binding region of PsaD was strongly electropositive. This region was widely distributed and had a tendency to form a positive pocket. Besides, the region of PsaD facing R19 of PsaC was electronegative, which was partially embraced by its neighboring electropositive region. These observations showed the charge distribution on the surface of PsaD played important roles for orienting residues E47 and R19 of PsaC, and the proper interactions between PsaC and PsaD was initiated by electrostatic forces.
Fig. 8

Skin representation of the EPS of PsaD extracted from the X-ray structures. Red: ESP < −5 kT/e, blue: > + 5 kcal/e, grey: between −5 and +5 kT/e. Residues corresponding to R19 and D47 of OsPsaC were shown in their mesh surfaces (R19 is shown in green and D47 is shown in magentas)

Besides, we also examined the salt bridge of R19-E103 in other X-ray structures, the formation of which is thought to be the main reason to stabilize the OsPsaC-OsPsaD complex. Figure 9 and Table 1 showed the NH1 and NH2 atoms of R19 were juxtaposed against the OE1 and OE2 atoms of E103, suggesting the salt bridge of R19-E103 widely existed in other organisms. Particularly in two higher plants Pisum sativum and Spinacia oleracea, the atomic distances between NH1 of arginine and OE2 of glutamic acid were 3.3 and 2.6 Å, which were similar with that in Orysa sativa. Interestingly, in two blue-green alagas Synechocystis sp. PCC 6803 and Synechococcus elongatus, the residual distances between arginine and glutamic acid were a little far. We noticed the atomic distances between NH2 of arginine and OE1 of glutamic acid were 5.9 and 4.8 Å, indicating evolutionary diversity between higher plants and alagas.
Fig. 9

A close view of the salt bridge between R19C and E103D in different X-ray structures. PsaC was shown in blue and PsaD was shown in red. Residues corresponding to R19C and E103D in Orysa sativa were shown in their stick representation

Validation of the MD model by protein docking

Given that our findings based on computational simulations, it is therefore necessary to apply the complementary method to validate these results. To this end, we performed the protein docking experiment using ClusPro 2.0 [31, 4547] due to its success in the CAPRI (Critical Assessment of Predicted Interaction). We used OsPsaD and OsPsaC as receptor and ligand for docking. Free energy scores of each cluster were used to screen candidates because they can provide good hints according to our previous work [10]. Significantly, we found two docking models exhibiting better scores. The scores for the docking model 1 and 2 were −723.9 and −824.9 kcal/mol, suggesting they were favorable binding modes. After superimposing the MD model and two docking models, we found these three models were able to fit well with each other (Additional file 1: Figure S5) and the C-terminal loop of PsaD can form a long arm to interact with PsaC. Taking a closer look at residues R19C, D47C, K62D and E103D, we observed two salt bridges R19–E103 and D47-K62 at the PsaC-PsaD interfaces (Additional file 1: Figure S5). This stressed the importance of these two salt bridges in maintaining the interaction between PsaC and PsaD.

A 2011 proteomic experiment [13] showed the stromal ridge complex of rice existed both in its PSI core complex and PSI super complex. Subsequently, this complex was modeled and refined by 15 ns MD simulation. Due to time limitation of simulations, the dynamics behaviors of the stromal ridge complex were still poorly evaluated. In this study, we combined a 100 ns AT-MD simulation and 500 ns CG-MD simulations to explore possible recognition mechanism between these two proteins. The most significant outcome of this study is that we identified two salt bridges R19-E103 and D47-K62, playing key roles to maintain the interaction between OsPsaC and OsPsaD. H-bonds analysis and free energy calculation suggested they contributed significantly to binding affinity between OsPsaC and OsPsaD. Specifically, electrostatic potential surfaces exhibited electrostatic attraction function in maintaining the interface of the OsPsaC-OsPsaD complex. In addition, CG-MD simulations emphasized the importance of salt bridge R19-E103 during the process of OsPsaC recognizing OsPsaD, the disruption of which would induce a significant conformational change of OsPsaC. Sequence alignments indicated the full sequence of OsPsaC’s was relatively conservative, and in particular, two residues R19 and D47 of PsaC in O.sativa were 100 % identical to those in other organisms. As to OsPsaD, its sequence exhibited little discrepancy in different organisms. For instance, PsaD in O.sativa, P.patens, P.antarctica and P.globosa lost partial of their N-terminal residues, indicating diversity of photosynthesis systems in different organisms during evolution. Interestingly, residues E103 and K62 of PsaD in O.sativa are 100 % identical to those in other organisms, strongly suggesting conservatively of two salt-bridges R19-E103 and D47-K62 in most of the organisms.

This study focused on the recognition mechanism of OsPsaC and OsPsaD since previous studies [3, 4] suggested the binding of PsaD to PsaC can greatly affect the assembly of the stromal ridge complex. Genetics experiments showed the in-vivo psaC deletion mutant lacked the subunit PsaD [4], while the psaD deletion mutant still contained PsaC [3]. This suggested PsaD was able to bind PsaC, only after the binding of PsaC to PsaA/B. It should be noted that residues distributing on PsaA/B heterodimer were highly symmetric, allowing PsaC rotate around the pseudo C2 symmetry of PsaA/B. This will lead half probability for the binding of PsaC to PsaA/B in its correct orientation, while also half possibility to bind incorrectly [7]. The binding of PsaD to PsaC is important because it can lock the region of PsaC correctly bound to PsaA/B, which is also supported by our in silico data. The binding free energies correlated well with previous structural analysis [1], emphasizing the H-bonding interactions play important roles in OsPsaD locking the correctly bound-region of OsPsaC and undocking the incorrectly bound-region of OsPsaD. Additionally, previous genetics experiments [2] also showed the psaE deletion mutant contained both PsaC and PsaD, indicating PsaE is the last stromal ridge protein to bind the PsaA/B-PsaC-PsaD complex and the PsaC-PsaD prerequisite complex is essential for the PsaE assembly.

Our simulations also explained how β-sheets of PsaD maintain their structure integrity and stability. The secondary structure of β-sheets was widely distributed in PsaD. Nuclear magnetic resonance (NMR) experiment [48] showed the free PsaD structure contained 11 % β-sheets. X-ray crystallography [5, 6] showed PsaD contained four β-sheets and one α-helix was connected by two neighboring β-sheets. Thus, it is interesting to understand how β-sheets maintain their stability. Our result stressed both H-bonding and hydrophobic interactions were essential to stabilize β-sheets, indicating the plausible mechanism of general distribution of β-sheets [5, 6, 48].

The correctly binding of PsaC to PsaD is a key step to form chloroplast stromal ridge complex, which plays important roles in plant photosynthesis. However, the recognition mechanism and dynamics behaviors of PsaC-PsaD are still obscure. This work aims to identify the key interactions between OsPsaC and OsPsaD by MD simulations and bioinformatics. Observably, a salt bridge R19-E103 is essential to maintain the stability of OsPsaC-OsPsaD. Its importance is determined in combination of free energy calculation and electrostatic potentials map, and is subsequently verified by two 500 ns CG-MD simulations.

On the other hand, we are also interested in if the recognition mechanism generally exists in the other organisms. To this end, we conduct amino acid homology search and sequence alignments. The results strongly suggest salt-bridges forming residues are relatively conservative. After comparison of different stromal ridge complexes from previous X-ray structures, we notice electrostatic forces provide proper interactions between PsaC and PsaD. Particularly, residues corresponding to R19C and E103D in rice are also able to form the stable salt bridge. This supports the generality of R19-E103 in other organisms. Collectively, this work will provide dynamics behaviors to understand recognition mechanism of the stromal ridge complex in rice.

ATomic Molecular Dynamics

Coarse grained molecular dynamics

Molecular dynamics

Molecular mechanics generalized born surface area

Oryza sativa

Subunits A and B of photosystem I

Subunits C, D and E of photosystem I

Root mean square distance

Wild type

The authors thank Junjie Yan, Jinzhen Shao for their insightful comments on the manuscript.

This study was supported by “973” Program of China (2013CB126900) and National Nature Science Foundation of China (NSFC 31471464).

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

State Key Laboratory of Hybrid Rice, Department of Genetics, College of Life Sciences, Wuhan University, Wuhan, People’s Republic of China

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© Zhang and Ding. 2016


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Masteries Issue

I just went into a Ranked match, just to find out i get a error when i try to switch my masteries from a empty page to my normal page. so i had to play a whole ranked game without masteries. now after 3 tries of trying to change my masteries in aram, still didn't work.
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What you can’t miss at the Progressive Miami Boat Show 2017

Miami Boat Show 2017

What you can’t miss at the Progressive Miami Boat Show 2017

On President´s Day Weekend, The Miami Boat Show, known as one of the largest boat shows in Florida, will take place at 3501 Rickenbacker Causeway, Miami, FL 33149.

When is the Miami Boat Show ?

Its premier day will be on February the 16th from 10am to 6pm. Those who attend on said day, will be the first to get to board more than 1,300 boats and shop 1,100 exhibitors from around the globe. The show will continue from February the 17th to February the 20th.

For the 2017 show, they will feature over 100 new luxury yachts, including dozens of debuts. Among the most recognized boat manufacturers are:

• Contender
• Everglades Boats
• Searay
• Riviera
• Marquis Yachts
• Marlow Yachts
• Prestige and many more.

VIP Boat show experience

One of the new features incorporated includes a VIP experience which provides exclusive access to a private lounge located aboard an 111-foot luxury yacht amidst some of the biggest boats and yachts ever displayed at the show. The lounge will offer beverage and dining service, live music daily and an open-air sky deck among other services.

However, for those who choose not to join de VIP experience, will also get to enjoy the food and beverage stands provided by the show, featuring more than 150 options ranging from luxe to casual fare.

Parking and transportation at the Miami boat show 2017

Also, this year the show includes more parking and transportation options. You can get to the show by land or water. Among the different transportation options are Water taxis, Shuttle Buses, and Miami-Dade Transit.

On the other hand, the parking options are: Downtown, American Airlines arena, Virginia Key and bicycle parking and special lanes.

Miami Boat Show 2017

On-land activities

The Miami Boat Show is also known for its on-land activities such as on-land boat simulator which consist in a simulated boat trip to practice boating skills such as docking, pivot turns, stopping and more. Alternatively, if you would like to receive professional and practical advice, you should head to Fred’s Shed Interactive Learning Center where you can learn what it takes to maintain, repair and upgrade your boat or engine.

On-water activities at the boat show

In contrast, the on-water activities include demo docks and sea trials, where they will feature more than 150 boats for demonstration and sea trials. Not only will you be able to look but also to board various types of vessels such as powerboats, cruisers, sport fishing and many more.

Boating skills classes

Also, if you are interested in improving your boating skills and learning new ones, you can opt for on-water boating skills classes. You will learn with practical hands-on instructions. All sessions are taught by USCG licensed captains who are professional, certified instructors. Advance registration is highly recommended. Same day registration will be available at the Discover Boating Registration Center during the show.
In addition to The Miami Boat Show, Strictly Sail Miami is returning to Miamarina at Bayside and displaying approximately 130 sailboats, seminars, and the newest sailing gear. Miami Boat Show visitors, can access Strictly Sail Miami by free water taxis or shuttle buses, running until 7 pm daily.

By the way…. Have you already tried Boatrax app? Check out the ultimate app for boat management: http://boatrax.com/download/

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Samsung launches new phone-to-phone ‘switch’ app to lure iPhone users

Samsung wants to make it effortless to ditch your old smartphone and switch to one of its new Galaxy handsets. To this end, the company is launching a new transfer app that enables users to freely move content from their old phone to any recent Galaxy device. What is particularly handy about the new Smart Switch technology is that it supports transfers from any phone and operating system.   Among other things, the new phone-to-phone service promises to make it possible to seamlessly transfer your custom setup to your new Galaxy device and move precious content like photos, videos and…

This story continues at The Next Web

Or just read more coverage about: iPhone,Samsung

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Pete’s Backcountry Report

Pete’s Backcountry Report – Matt and I headed up Bryant to the Cabin yesterday.  The water bars down low were definitely tricky to get around.  Once up higher it looked like mid-winter conditions.  While the water bars were still open they were much easier to navigate across.  As we got closer to Bryant Cabin we noticed that the trees were coated with some ice.  On our way up we took a look at JJ’s, while it looked really nice we were unsure of what we would find underneath, but thought that we would give it a shot.

Matt at Bryant

We stopped at the Cabin to be sure that it was ready to be rented, then headed onto Gardiner’s Lane.  A warning to those heading that way that there is a large birch tree across the trail just below JJ’s, which you can get around by going to the right.

Turns on JJs

We made our first turns down JJ’s and to our surprise that ice layer that we noticed earlier, in the trees produced a crust layer that we were not breaking through.  There was 6-8” on top of this crust.  While we felt a little rusty, the skiing was great.  Once back on Bryant we were able to cruise down, jumping over most of the water bars.  Not bad for a first day in the backcountry.

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Where will the Top-20 Sign?

In a wild, wild off-season littered with coaching changes and controversy, it’s only fitting that free agency will see a record number of free agents hit the open market in what will be an absolute frenzy. The off-season will finally reach its climax.

And as always, I’ve ranked the top-20 free agents (and more) and which team I think they’ll put pen to paper with. This was harder than usual, of course, with the vast amount of eligible free agents. This is the first free agency where we’re seeing the effect of the CBA’s now-allowed one-year contracts, which were agreed upon in the 2014 negotiations. It’s no guarantee that General Managers such as Jim Barker and Wally Buono, who typically sit back in free agency, will become players given the amount of free agents and the the low prices that the middle class of free agents will be forced to take. But I think they’ll come out of their shells and open up the payroll.

Last year, I went 9/18 – excluding the NFL departures – and am looking to improve this time around.

1. DT Ted Laurent, Hamilton Tiger-Cats

Laurent is undoubtedly the best nose tackle in the league – Canadian or not. As a 0-tech and 1-tech, the 6’1″, 301-pounder is an elite run-stuffer who pushes the pocket and attracts double teams in the pass-rush. He’ll likely be made the highest paid Canadian in the league, earning around $250,000/season. Laurent will receive interest from all nine teams, but the likely suit could be Chris Jones’ Saskatchewan Roughriders. The ‘Riders already have National depth to put behind Laurent with have money to spend.

Prediction: Signs with Saskatchewan

2. DE Justin Capicciotti, Ottawa REDBLACKS

Being only 26-years-old and having 23 sacks in the past two seasons, Capicciotti’s value may not ever be higher. He’s an elite, Canadian pass-rusher that’s been the anchor of Ottawa’s defensive line for the past two seasons. An alum of Simon Fraser University, Capicciotti has roots in British Columbia and would make a great fit in the Lions’ defense. BC desperately needs help with their pass-rush, as well as a Canadian starter to replace Andrew Harris in the ratio; Capicciotti makes the most sense.

Prediction: Signs with British Columbia

3. DB Aaron Grymes, Edmonton Eskimos

Grymes is arguably the best defensive back in the league and should get paid decently for an international. He’s been exercising all NFL options up to this point but I wouldn’t suspect he signs a deal. The ‘Riders only have one returning halfback in Tyree Hollins, while Grymes excelled in Chris Jones’ system in Edmonton. A return to northern Alberta or a new beginning in Regina are the likely options. Don’t rule out the Lions, either, if Wally Buono decides to be a player in free agency, as Grymes is a native of Seattle, WA.

Prediction: Re-signs with Edmonton

4. RB Andrew Harris, BC Lions

Despite being one of the best running backs in the league – and a Canadian, at that – Harris may not get paid as much as people think on Tuesday. And while, personally, I’d pass on Harris, he’s likely on his way to Winnipeg – his hometown where his daughter lives.

Prediction: Signs in Winnipeg

5. QB Trevor Harris, Toronto Argonauts

Harris and the Montreal Alouettes is a perfect fit. Montreal needs a franchise quarterback and there wouldn’t be much of bidding war to get Harris’ services. But I think he re-signs with Toronto, as Ricky Ray’s deal is incentive-laden, meaning the starting job is still very much open.

Prediction: Re-signs with Toronto

6. RB Jerome Messam, Calgary Stampeders

Messam was easily the most dominant running back last season, averaging 6.1 yards-per-carry. The 30-year-old managed to stay healthy throughout the season and wasn’t abused with too many carries. Contract negotiations have begun between Calgary and Messam, and are beginning to gain momentum. I think they’ll reach a deal.

Prediction: Re-signs with Calgary

7. LB Winston Venable, Montreal Alouettes

Venable had his breakout season in 2015 and likely earned a big pay raise. It’s no guarantee he re-signs in Montreal, however, as they already have Henoc Muamba, Bear Woods and promising rookie Kyler Elsworth as inside linebackers. With Chris Jones unable to recruit Sherritt to Regina, he’ll hit a home run with Venable.

Prediction: Signs with Saskatchewan

8. LB Keon Raymond, Calgary Stampeders

Considering the huge role he played within Calgary’s defense and how great he played, Raymond is still an elite player in this league. He can line up all over the formation and take on many responsibilities; he’s truly one of the most underrated player in the CFL. It would make a lot of sense for Raymond to follow his defensive coordinator, Rich Stubler, to Toronto, where he’d hold down a strong-side LB position that hasn’t be solidified in Toronto for a few years.

Prediction: Signs with Toronto

9. SB Ryan Smith, Saskatchewan Roughriders

The new regime in Saskatchewan appears to be moving on from Smith (which, by the way, doesn’t make a lot of sense). But he makes a lot of sense for the Bombers to sign – more sense than Weston Dressler – and would complete their receiving corps. The 24-year-old accumulated 991 yards in 16 games during his true rookie season and could be a slam-dunk pickup for the Bombers.

Prediction: Signs with Winnipeg

10. SB Kenny Stafford, Edmonton Eskimos

Surely, Kenny Stafford will look elsewhere of Edmonton where he’ll get a bigger role in another offense. He’s a legitimate deep-threat receiver that would fit in well in Winnipeg’s offense. But I think he’s Saskatchewan-bound, reuniting with Chris Jones and offensive coordinator Stephen McAdoo.

Pick: Saskatchewan Roughriders

11. DT Keith Shologan, Ottawa Redblacks

Having missed out on Ted Laurent, Cleyon Laing (NFL) and Justin Capicciotti, I think the Bombers will pick up Shologan, which would still be a big pickup. Jake Thomas provides good depth behind the 30-year-old, who’s been nothing but productive throughout his career.

Prediction: Signs with Winnipeg

12. RT Josh Bourke, Montreal Alouettes

Any time a Canadian offensive tackle hits the open market, he’s going to garner a lot of interest. But I re-watched a lot of tape on Bourke and he was the weakest link on a relatively strong Montreal offensive line. He’s now 33-years-old and Montreal could be letting him walk knowing 2016 is the season that he really falls off, similarly to Saskatchewan and Dominic Picard. With all of Hamilton’s offensive tackles slated to hit free agency, they could take a gamble on Bourke and start four Canadians on their offensive line.

Prediction: Signs with Hamilton

13. DT Euclid Cummings, Toronto Argonauts

Cummings emerged in his rookie season as one of the best three-tech defensive tackles in the league. He’s another one of Jim Barker’s many treasures from down south and I don’t think he’s going to let Cummings walk after just one full season in the league.

Prediction: Re-signs with Toronto

14. MLB JC Sherritt, Edmonton Eskimos

After a bounce-back 2015 campaign, Sherritt could test the market and check his value. But he’s been the heart-and-soul of Edmonton’s defense for a long time and I don’t think that’ll change. And Edmonton can’t allow that to change, either.

Prediction: Re-signs with Edmonton

15. K/P Swayze Waters: Toronto Argonauts

He’s just one year removed from dominating the CFL on special-teams and many pundits around the league seem to forget that. He’s an excellent kicker and punter that’s an impact player. Toronto can’t lose him.

Prediction: Re-signs with Toronto

16. DB Emmanuel Davis, Hamilton Tiger-Cats

Davis emerged in 2015 as a big-play defensive back, returning three of his five interceptions for touchdowns. He’s a good, not great, halfback, but would leave a big hole to fill in Hamilton’s defense if he leaves.

Prediction: Re-signs with Hamilton

17. DT Bryan Hall, Hamilton Tiger-Cats

Hall is a versatile three-tech that’s simply a bruiser in the trenches. He could garner a lot more interest in the market than people think, but Hamilton can’t afford to lose another key defender.

Prediction: Re-signs with Hamilton

18. SB Nic Moore, Winnipeg Blue Bombers

Had he missed maybe seven less games in is Bomber career, Moore would’ve never been on this list. But alas, he hasn’t been able to stay healthy and is now out of the Bombers’ plans. He’s still been productive when healthy, however, and likely would’ve had 1,000-yard campaigns in 2014 and 2015. The Stamps, who’ve lost Eric Rogers (NFL) and possibly Jeff Fuller, will be serious contenders.

Prediction: Signs with Calgary

19. WR Jeff Fuller, Calgary Stampeders

Fuller has the size and speed to be a Duron Carter-type player; he just needs to stay healthy. The six-foot-four, 223-pound boundary wideout is loaded on potential, and Jon Hufnagel knows that and won’t let him leave.

Prediction: Re-signs with Calgary

20. WR Shamawd Chambers, Edmonton Eskimos

Chambers will be a boom-or-bust pick for whoever picks him up. His tenure in Edmonton as a first-round draft pick was a bust – his 2-catch, 47-yard performance as Grey Cup Most Outstanding Canadian means nothing in the grand scheme of things- as he’s been constantly injured. National players almost always move closer to home in free agency, and that’s why I think the Markham, Ontario native will sign with either Toronto or Hamilton.

Prediction: Signs with Hamilton

21. K Justin Medlock, Hamilton Tiger-Cats

He’s an American that isn’t a great punter, which is rare to see, but Justin Medlock is still the best kicker in the CFL and that’ll make him a hot commodity on the open market.

Prediction: Re-signs with Hamilton

Best of the Rest:

SB Andy Fantuz, Hamilton: He’ll take a pay cut to stay in the QEW with Hamilton.
SB Chad Owens, Toronto: Ditto to Fantuz; re-signs with Toronto.
LB Taylor Reed, Hamilton: Cats can’t lose another defender.
RB/RET Chris Rainey: Dynamic player who’d fit in well in Ottawa or back in BC.
LB Cory Greenwood, Toronto: Serious concussion issues could scare a lot of teams off. He re-signs in Toronto.
SB Chris Getzlaf, Saskatchewan: Safe to say he’ll be back in Regina in a reduced role.
DB Otha Foster, Edmonton: Mediocre strong-side LB. Off to Saskatchewan.
C Cory Watman, Saskatchewan: Disappointing former first-round pick that’ll stay in Regina.

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Bombers ink deals with Cummings, Keeping

As reported by Sportnet’s Justin Dunk, the Winnipeg Blue Bombers have come to terms with international Defensive Tackle Euclid Cummings on a one-year deal.

The 24-year-old had a great first season with Toronto, amassing eight sacks from his 3-tech position. Cummings, six-foot-four, 281-lbs, has untapped potential and was already one of the best three-techs in the league last season.

This signing essentially spells the end of the tenure of Bryant Turner Jr. in the Blue & Gold. Turner Jr. served the Bombers for five seasons (2011-2015) and is a two-time division All-Star.

The Bombers weren’t done converting players from Double Blue to Blue & Gold, as earlier, they announced the signing of 10-year veteran Jeff Keeping.

Keeping brings some much-needed interior offensive line depth as well as mentor-ship for Winnipeg’s 2014 and 2015 first-round draft picks, Mathias Goossen and Sukh Chungh. The 33-year-old will likely serve as the club’s sixth offensive lineman, unless Winnipeg’s 2016 second-round draft pick – one of them will be an centre or guard – can take it on. Not likely; great signing.

That’s seven total free agents for Kyle Walters on day one free agency.

In other news, Nic Moore signed a one-year pact to return to British Colombia. The excellent route-runner completes what’s looking like a very scary receiving corps out in Vancouver. Manny Arceneaux, Andrew Hawkins, Moore, Austin Collie and Shawn Gore – not a group secondaries will want to see on a consistent basis.

Richard Lautens: Toronto Star

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Shona Ann Hill

Shona Ann Hill in truth is a way shower, a leader of leaders, in the city of York, UK. She is an Inspirational Speaker, Assertiveness Coach, Medical Intuitive Healer & Clairgnostic, here to help humanity repair and grow, through Worldwide web based Programmes, In person Inspirational Events, treatments and books.

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Thieves Stole $45 Million In Global ATM Cyber Heist [Photos]

- By Bossip Staff

A band of international cyber thieves stole $40 million dollars from ATM machines in a matter of hours back in February and $5 million late last year. Using two timed heist operations and accomplices on the street, the looting of the ATMs involved computer hackers, manipulated financial information and common street criminals. In New York, a group of men were the first to be arrested in this elaborate global scheme…. Continue

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