Who's in the party?

Along with the Vintage Game Club, I have recently been playing Chrono Trigger, a game widely hailed as one of the best console RPGs ever made. While the game has many attributes worth praising (not least the continuity of world and battle screens), I was particularly interested by its combination attacks. Almost any two characters that are in the combat party can combine their unique individual battle techniques into more powerful "Dual Techs". For instance, Lucca can charge Crono's sword with fire while he performs his "Cyclone" attack to create the "Fire Whirl" tech. Frog and Ayla can combine their weak individual healing skills into the "Slurp Kiss" that slightly heals the whole party. I think it odd that this approach has seen so little use subsequently, since it seems like modulating abilities based on party composition has great potential for infusing characterization into the combat mechanics.

Of course, most role-playing games, especially in the JRPG genre, give the player characters with complementary abilities or specialized roles, so that certain rosters are somewhat more effective than others. Having the characters explicitly combine their attacks is much less common, and is typically reserved for high-powered finishing moves, as in Kingdom Hearts II or Tales of Symphonia. Chrono Trigger differs from these games in that its combination techniques span a spectrum of opportunity costs (also, the animations are short). Some of them do require a significant investment of MP and can only be used sparingly, but others have a low enough cost that they can be used almost every turn. As a result, the shared abilities are almost as important as the individual abilities to the tactical evaluation of a particular party composition.

This had an interesting effect later in the game, after I managed to get Magus to join my party. Magus is a strong and versatile character with hard-hitting magic attacks of several different elements. Considering just his individual abilities he's a good character to have in the party. Yet, I almost never used him because he didn't have any dual techs at all. He couldn't increase his effectiveness by teaming up with anyone, and rather than give up the tactical flexibility, I left him out. It felt mechanically like he just didn't fit in with the party, an appropriate note because he is a major antagonist for much of the game. The mechanics of the game isolate him in battle just as the story suggests he would be isolated in the group.

This seems to me like the sort of thing that should be done more often. The enormous cast of many JRPGs means that most of the playable characters and their relationships never really get fleshed out, despite the hours of cutscenes. Why not use the combat system to do some of this work? Envision a set of party dynamics, and use the availability of combination attacks to illustrate those dynamics. If you imagine that Marle is jealous of Ayla and Lucca, creating friction in the group, express this by limiting their combos or making them less powerful. Then, when the player needs Lucca in the party for her fire attacks, the tactical limitations would encourage the player to respond by removing Marle from the combat group. The player's choice of party members remains an act of play, but through the manipulation of the mechanics it becomes an act of narrative characterization as well.

We can push this further by making the available combos respond dynamically to narrative. Events in the story that bring two characters closer could make new combination attacks available to them. When the relationship between two characters changes, the set of shared techniques could be adjusted to reflect the difference. This need not be tied to the planned story: a relationship system like that of Tales of Symphonia could be used to guide the evolution of combination techniques.

I've seen a couple of excellent essays (from Jay Barnson and Nayan Ramachandran) recently trying to dissect what's wrong with the JRPG and suggest ways that the genre could be revitalized. My own advice is to let the gameplay tell the story, too. Persona 4 and The World Ends With You both did this, in different ways, but you can still apply the lesson even if you aren't so unconventional. Use the gameplay of party dynamics to reinforce the story of personal relationships. Make "these characters belong together" part of the player's thinking in every phase of the game, and the stories of those characters may gain more resonance.

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Drugs disrupt DHFR dynamics

One of the most-studied cases of the relationship between dynamics and catalysis is the bacterial dihydrofolate reductase (DHFR). DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate while oxidizing the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). As part of this catalytic process, a region of the protein called the "Met 20 loop" switches from a "closed" state that shields the active site from solvent to an "occluded" state that separates the substrate from the cofactor. NMR studies of DHFR structural dynamics have correlated the protein motions with the chemical changes. In a recent study appearing in Structure, researchers from the University of North Carolina show that the binding of inhibitors such as methotrexate (MTX) and trimethoprim (TMP) appears to uniquely disrupt the dynamic networks of DHFR.

Previously, seminal work from the lab of Peter Wright surveyed the dynamics of DHFR in every step of its reaction pathway. Boehr et al. determined that structural fluctuations in each complex represented motions towards the next step in the reaction. The conformational exchange rates they obtained from their relaxation-dispersion experiments closely resembled the rate constants that had been independently determined for the chemical steps. In almost every complex the conformational exchange was widespread, affecting residues in both the substrate and cofactor binding sites, as well as important distal locations such as the Met 20 loop.

Because the existing work from the Wright lab hewed as close to the natural substrates and products as possible, Mauldin et al. chose to examine the dynamic effects of inhibitor binding to DHFR. Like Wright's group, they used relaxation-dispersion experiments to identify conformational changes taking place on the μs-ms timescale. In the NADPH:DHFR complex the motions are widespread, encompassing the substrate binding site, the Met 20 loop, and distal locations. Binding of either inhibitor eliminates about half of this dynamic network and dramatically reduces the fluctuation rates of those residues for which conformational exchange continues to occur.

Based on their fits of the exchange rates, Mauldin et al. conclude that the substrate binding pocket moves in a way that mimics the enzyme's normal motions in the transition from its closed state to its occluded state. The long-range conformational changes that actually complete this transition, however, have been completely quenched. With the inhibitors bound, DHFR is like a car that's turning over but won't start. Part of the enzyme is still moving in exactly the right way to proceed along the reaction coordinate, but for some reason this motion doesn't catch on throughout the protein.

In order to gain a more complete understanding of the dynamic effects, Mauldin et al. performed experiments to identify the motion of the protein on the ps-ns timescale. Analyzing the dynamics of methyl and amide resonances using the Lipari-Szabo model-free formalism, the authors realized that inhibitor binding did cause long-range changes in dynamics, just in a faster regime. Where the natural substrate complexes have motions that occur hundreds or thousands of times per second, the inhibitor-bound forms have (smaller) motions that occur millions of times per second. Because these altered motions encompass the Met 20 loop and surrounding residues, the authors argue that they reflect abortive attempts by the protein to transition into the occluded state.

Although these inhibitors do not appear to change the protein's overall conformation, they produce long-range dynamic effects on short timescales and quench distal motions on intermediate timescales. The binding pocket appears to still be experiencing fluctuations related to the transition between the closed and occluded conformational states, but the mechanism that couples the binding site dynamics to the motion of the loop that defines these two states appears to be broken.

The million-dollar question is this: do drugs alter DHFR dynamics because they inhibit the chemistry, or do these drugs inhibit the chemistry because they alter DHFR dynamics? Quenching dynamics costs energy in the form of conformational entropy, and it may be possible to tune a drug for improved efficiency by blocking the binding site without altering the dynamics. This is only true, however, if the dynamics don't matter to successful inhibition. On the other hand, if blocking the conformational switching of the Met 20 loop inhibits the enzyme, then drugs can be designed for that angle of attack as well. In the case of a protein like DHFR, where the bacterial enzyme has similar activity but a very different structure from its human equivalent, drugs that target regions other than the active site may significantly reduce side-effects. As a result, protein targets that were previously off-limits due to shared chemistry may become tractable due to divergent dynamics and structure.

Mauldin, R., Carroll, M., & Lee, A. (2009). Dynamic Dysfunction in Dihydrofolate Reductase Results from Antifolate Drug Binding: Modulation of Dynamics within a Structural State Structure, 17 (3), 386-394 DOI: 10.1016/j.str.2009.01.005

Boehr, D., McElheny, D., Dyson, H., & Wright, P. (2006). The Dynamic Energy Landscape of Dihydrofolate Reductase Catalysis Science, 313 (5793), 1638-1642 DOI: 10.1126/science.1130258

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Critical Thinking Compilation: BioShock

BioShock is the rare game that really does change the way we think about video games, if for no other reason than that it has turned up as an example in almost every discussion of game style, mechanics, story, or design that has been written since its initial release in 2007. BioShock has received excessive adulation, a much-discussed backlash, and even a backlash to its backlash. Discussions of the game spawned the most popular jargon in games writing. So much has been written about BioShock that I could only put my hands on a fraction of the material without driving myself nutty as a splicer. Thus, this constitutes only the first draft of a survey of critical thinking on a game that will likely be regarded as a classic.

"Is a man not entitled to the sweat of his brow?"

One of BioShock's most compelling features is that it details an interesting philosophical system and then uses it to frame an ethical question. The underwater utopia of Rapture was founded by an industrialist named Andrew Ryan on a system of principles much akin to Randian Objectivism, so much so that John Lanchester argues in the London Review of Books that BioShock is the only popular work in recent years to give Rand a drubbing. Lorenzo Wang fleshes out this case in his rich and interesting essay "BioShock Explained". In his view, the game attacks two key flaws of Rand's philosophy: society can never sustain its ideal state (somebody must, after all, scrub the toilets), and free will, even in the land of plasmids, is limited. Jay Barnson felt that the game critiqued the intrinsic short-sightedness of the market, which is regarded as all-wise by the lovers of laissez-faire economics.

This interpretation of the game's attitude towards Objectivism was not universal, however. Shamus Young interviewed an Objectivist on the subject, who argued that BioShock really aims its criticisms at the idea of philosophical certainty. In his very interesting Marxist critique, Richard Terrell lays out a case that the game conditioned the player to accept the principles of Rapture's economy. In his view, the choice to attack the Big Daddies (and possibly the little sisters) makes the player part of the oppressive capitalist regime. Justin Keverne argued that the mechanics of the game suggest that one's goal is to acquire power in order to gain the ability to acquire further power.

At this point, designer Clint Hocking felt that BioShock went off the rails in a certain sense. In his essential essay "Ludonarrative Dissonance in BioShock", Hocking argues that the game presents the player with two conflicting contracts. The gameplay establishes that the player must serve his own interest in order to advance, while the story forces the player to serve others in order to advance. Our attempts to deal with or ignore this tension are them mocked by the game's central twist.

"How can you do this thing? To a child?"

The philosophy of rational self-interest provides context for the story, and also for a moral choice that the player makes. When Rapture's lumbering Big Daddies are defeated, the player may choose the fate of "little sisters" they protect. If he rescues a little sister, he receives a small amount of ADAM that he can use to purchase plasmids, tonics, and other upgrades, and the girl survives. If he chooses to "harvest" the girl, he gets a great deal more ADAM, but she does not survive. For Leigh Alexander, saving the girls is like saving Rapture's last bit of innocence. In her view, the whole saga with ADAM was like a child's wish, and saving the girls is a way of forgiving Rapture for making that wish, despite the destruction it caused. Yet many did not find this choice to be compelling. As Wes Erdelack notes, in BioShock as in many other video games, the moral choices are too simplistic and do not feature a sufficient challenge to "goodness".

One frequent complaint about the rescue / harvest choice is that if the player rescues enough little sisters, then the twisted Dr. Tenenbaum will leave a Teddy bear at a nearby vending machine, stuffed with plasmids, ammo, and extra ADAM she has presumably harvested on her own. In a thorough critique of this moral choice, D. Riley argues that the benefits of rescuing are sufficiently great that the whole system is neutered. Duncan Fyfe concurs, and wonders how the developers could have gotten this so wrong.

This opinion is not universal. Leigh Alexander believes that approaching the rescue / harvest choice with a cost-benefit analysis is too limiting. In her view the question to ask is not what you want to get but who you want to be. As she says, "The merit of choice in games may not be what we get from it, but when done this richly, how it feels." Bonnie Ruberg feels that the gameplay is really there to expose the selfishness of gameplay tactics in general. I also felt that the near-equivalence (in economic terms) of the two choices was making a point about the hidden values of games.

Some also argue that the harvest choice is not compelling because the player is spared having to watch the act or even see the body that remains. D. Riley's previously noted essay includes a comment on this point, and I make a similar case in my own essay, "Ecce, soror". Nels Anderson acknowledges that this may be so, but points out that on-screen child murder by the protagonist of a game simply could never get by ratings boards or the easily-outraged public. Nonetheless, he argues that the nursery scene provides some of the necessary emotional impact.

And perhaps the little sisters aren't even the real focus of the moral choice. Justin Keverne argues that the player's real moral choice is whether to attack the enslaved Big Daddies for his own personal gain. Unlike the other denizens of Rapture, the extremely dangerous Big Daddies won't harm you unless you attack them first. For Glenn Turner, the choice to put down a Big Daddy was harrowing because of the little sisters' reaction. Gene Koo felt that becoming one of these lumbering behemoths brought the game's emotional and philosophical threads close to each other. In his view, however, this didn't quite succeed, because the player has no choice about whether he becomes a big daddy.

Whether the rescue / harvest choice was compelling or not, BioShock at least invited a fresh consideration of the meaning of moral agency in games, according to Leigh Alexander. She also pondered whether our behavior in the game would change if our peers were aware of it (perhaps through achievements or trophies).

"A man chooses; a slave obeys."

Choice is the subject of BioShock's most compelling moment, the confrontation with Andrew Ryan. This moment twists the preceding exposition of the ideas of rational self-interest into a commentary on the nature of gaming itself. Wes Erdelack views BioShock as parable about gaming, highlighting the fact that the feeling of agency is always an illusion. The Graduate School Gamer notes in an essay comparing BioShock to Braid that "The player can only converse with the text within the confines of the game's design and always remains at the will of the designer." The illusion of choice is not a subject unique to gaming, according to Roger Travis. He notes that the non-choice of killing Ryan resembles Achilles' non-choice to join battle in the Iliad.

The first-person perspective heightened the impact of the climax. Matthew Gallant regards the first-person viewpoint as essential, especially in moments like the confrontation with Ryan. The personal choices at the center of the game couldn't be the same if the player's eye into the game world didn't seem to make him a part of it. Sinan Kubba shares Gallant's skepticism about a BioShock movie, feeling that the immediacy and immersion of the game's perspective could never truly be replicated in film. The praise was not universal, however: Brad Gallaway felt that the silent, first-person protagonist interfered with the narrative at several important points. He especially felt that the internal logic of the game collapsed when the player injected himself with a plasmid for the first time.

The more widely-expressed complaint about the plot, and specifically the encounter with Ryan, is that there is far too much game after it. The denouement of the game is widely recognized as its weakest segment, a fact that Josh Birk explains by pointing out that BioShock, like many other games, has more backstory than story. The confrontation with Ryan is the culmination of the fascinating backstory, leaving the rest of the game to become little more than the tale of a man with a gun out for revenge. Moreover, the game doesn't exactly free the player up to make his own choices after its climax. The player continues to obey a character, only now it is Tenenbaum rather than Fontaine. BioShock refuses to engage this dilemma, which is a significant source of frustration for Duncan Fyfe. Chris Dahlen also found this troubling, because the game's most compelling character (Ryan) has so much agency and the player has so little. As he puts it, "...within the game, you never become a man. The only choice you have is to stop playing."

Aside from Ryan, surprisingly few writers have gone in depth on the characters of the game. A worthy exception to this rule is Leigh Alexander's examination of the bizarre Sander Cohen. She relates his personality to people she knows from her theater background, seeing in him a metaphor for the whole backstory of the game. In her view, Cohen is "a brilliant character not only for his spot-on characterization, but for the way his endless wrestling with 'the muse' is a perfect metaphor for the consumptive nature of Rapture in general."

"I know why it has to be children, but why just girls?"

Without focusing a spotlight on particular characters, several interesting pieces have examined the role of women and femininity in BioShock. Although creepy little girls are a staple of the horror genre, as Leigh Alexander has noted, BioShock uniquely gives the player power over their fate. Bonnie Ruberg found the female enemies in the game particularly disturbing, and wondered whether their horrific power drew from the simple fact of their gender. She also found the power relationship between the player and the little sisters to be troubling, and agreed with a Penny Arcade comic suggesting that it had overtones of pedophilia.

Nels Anderson indicates that the design of the girls is intended to evoke sympathetic feelings, but what attitude does this imply on the part of the player and developers? In a comprehensive critique, Richard Terrell argues that BioShock pervasively trades in patriarchal values because it "depicts women as weak, emotional, submissive, and nurturing and men as strong, and protective...". The little sisters are portrayed as helpless human commodities, and for much of the story Diane McClintock equates her self-worth with physical attractiveness. Moreover, Dr. Tenenbaum's redemption comes through an acquiescence to patriarchal ideas of motherhood. Terrell's analysis encompasses the mechanics of interacting with female characters as well.

"I chose the impossible. I chose... Rapture"

It's easy to understand how Cohen, Tenenbaum, and so many others could have chosen Rapture. For Ed Borden, the environment was key to BioShock's immersion of the player. The crumbling city arrests the player's attention and inspires his curiosity. Glenn Turner felt the same way, arguing that the art design was perhaps the game's best feature. For Richard Naik also, the selling point of the game was Rapture's auditory and visual design, overwhelming all of the game's shortcomings. The art design unified the disparate levels, making the world of Rapture feel like a coherent whole and maximizing the emotional impact on the player, as Tom Cross explains in "Surviving Rapture".

Part of the power of the environment was the way in which it was used to tell a story. Steven O'Dell compared the player's journey through Rapture to a guided tour of a dying city, one in which the enormously detailed spaces tell a story through the way they are designed. Wes Erdelack points out that the much-loved environmental storytelling of Fallout 3 has some roots in the construction of Rapture's spaces. While the audio logs constitute the most powerful storytelling in BioShock, the game's spaces allow the player to play detective and reconstruct his own history for the game world. Careful construction of spaces and traversals played a significant role in player experiences as well, as Simon Cooke explains. Every time the player encounters a massive set piece, the developers use clever design to make sure he is able to see every bit of it.

"...bugger gets into his 'ead that he's gonna go down guns blazing."

Also making sure that the player can see every inch of the city is the fact that he simply can't die in it. In some respects, the Vita-Chambers that resurrect the player after every fatal encounter resemble a streamlined checkpoint system (much like Elika in the later Prince of Persia), and Scott Juster places them among a number of ways that BioShock used narrative elements to disguise common gameplay tropes. Nonetheless, the Vita-Chambers were widely criticized. This goes beyond the hardcore player's lament, articulated for us by Josh Bycer, that resurrection makes the game 'too easy'. Josh Birk complains that they interfere with the scare factor in the game and the logic of weapon collection. Justin Keverne points out that they encourage the player to take the path of least resistance, though for him the draw of using the plasmid powers was enough to keep him playing fair. Not so for Richard Terrell, who felt that the chambers too strongly encouraged the wrench / revive / repeat approach to combat, and demoted the central activity of the game, which he felt to be shooting. Moreover, he argues that the effective immortality of the player weakens the psychological impact of the game in his psychoanalytic evaluation.

Another way to conceive of the Vita-Chambers might be as a resurrection spell, an apt comparison since BioShock had so many RPG elements. In fact, Richard Terrell felt the game's mechanics tended more towards the role-playing side, in particular because the almost nonexistent cover system forced the player to behave like a bullet sponge. He compares tactics in BioShock to the attack / attack / heal approach common in RPGs. Justin Keverne comments on the oddity of this, as the Vita-Chambers actually make healing and health packs totally superfluous in all but the final battle. Writing for Eludamos, Matthew Weise connects BioShock with RPG roots originating in Ultima Underworld.

BioShock resembled RPGs in even less flattering ways as well, specifically because of its fetch quests. These quests added numerous objectives that muffled the story, in Duncan Fyfe's view. Rather than engaging in a breakneck pursuit of Ryan (or Fontaine), the player spends his time mucking about Rapture looking for 7 vials of bee spit. This quest temporarily scared Tom Armitage right out of the game, even though it ultimately led to some interesting exploration. He warns, "I was thrown by the instructions the game gave me..." saying that even a good game can be derailed by players' bad memories of similar quests.

And despite the "too easy" vita chambers and exciting plasmid powers, the conventions that BioShock embraces limit its audience. Although Lanchester praised BioShock's consideration of Randian philosophy, he criticized the game because even its modest difficulty would keep it from being experienced by a broader culture that might genuinely appreciate it.

Author's Note

If you go to any of these blogs and search them for the word "BioShock" you will come up with dozens of posts. The game has become a kind of yardstick by which we measure others, and a rich source of examples to illustrate points. I attempted to limit what I included here by stipulating that the post must be at least 50% about BioShock, and that it should be in fully developed paragraphs rather than bullet points. I'm not sure I actually ended up holding to that, but that was at least the approach I tried to apply. I am positive I left some excellent posts out, mostly by failing to find them in the vast wilds of the internet. If I omitted your dissertation on the semiotics of dentistry in the context of Rapture, it is probably because I didn't run into it while I was doing my survey. Would you kindly let me know about it in the comments, via twitter, or with an e-mail?

Last updated: 4/6/09

Future updates of this compilation have moved to Critical Distance. Look for this and other great critical compilations (by myself and others) there.

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You can't put a price on your soul

BioShock's most famous moral choice concerns the fate of young girls who are wandering through the undersea city of Rapture. The player gets to decide whether these girls live or die, and in a game that features a civilization built on the principle of laissez-faire one expects economics to play some kind of role in this decision. Many writers, however, have complained that it does not, and that the choice has too little of an effect on one's ultimate experience of the game. Leigh Alexander and Bonnie Ruberg, among others, have suggested that something more sophisticated is going on here, and in other aspects of the player's behavior in Rapture. The difficulty of resolving the rescue / harvest choice in BioShock by a cost-benefit analysis illuminates the little-discussed value of selfishness embodied by most gameplay.

Ludonarrative dissonance and the player's choice

Clint Hocking's famous critique of BioShock accused it of developing a case of "ludonarrative dissonance", or tension between the message of the mechanics and the message of the story. As he put it, the mechanics offer a "ludic contract" that tells the player to seek out power (in the form of weapons, ammunition, plasmids, and ADAM) in order to advance in the game. This contract encourages the player to adopt a philosophy of rational self-interest much like that espoused by Andrew Ryan, the founder of the game's undersea dystopia. The story, by contrast, offers a "narrative contract" that tells the player to help others (initially Atlas, later Tenenbaum) in order to advance. In Hocking's view, the player can choose to accept or reject the ludic contract by harvesting the little sisters (maximizing his take of ADAM) or rescuing them, respectively. However, because the overall narrative of the game is fixed, the player cannot really choose to accept the philosophy of rational self-interest, and the climactic confrontation with Ryan seems to ridicule the player for putting up with that limitation.

Subsequent discussions of the game took a curious turn, however, in that aspects of the rescue / harvest choice itself were accused of being the centerpiece of dissonance. Wes Erdelack's excellent discussion of ludonarrative dissonance is a case in point, although I believe this take on the matter originates with Jon Blow. The essence of the complaint in this case is that the story sets up the player's choice to save or kill the girls as crucial, the consequences of the choice amount to almost nothing in terms of the gameplay. Although the player obtains significantly less ADAM when he chooses not to harvest the little sisters, repeatedly rescuing the girls causes Tenenbaum to give him gifts containing additional ADAM, ammunition, tonics, and a unique plasmid. As a result, in terms of strict gameplay consequences, the choice the player makes with respect to the fate of the little sisters doesn't matter. In essence, the game doesn't allow the player to make a choice about the philosophy of rational self-interest because both options are compatible with that philosophy.

These discussions all assume that dissonance ought be avoided, but as Michael Abbott has noted, it has its uses. The odd discordant note may offend the ear, but it draws attention and forces the observer to consider the artwork more carefully. The seeming mechanical equivalence of choice may be the conceptual error of a foolish team, or an intentional effort on the part of the developers to make the player consider the conflict presented more carefully. If we interpret this dissonance in BioShock as signal rather than noise, BioShock indicts the value systems of games and gamers.

You are only a link in the great chain

The philosophy of rational self-interest lies at the heart of both kinds of dissonance in the game. Andrew Ryan intended Rapture to be a utopia with no governing principle other than man's own self-interest, and regards those who seek the protection of a government to be little more than parasites sucking society dry. He builds Rapture for himself. His primary antagonist, Frank Fontaine, embraces this philosophy in a more nuanced way. He too only acts in his rational self-interest, but rather than Ryan's reactionary hatred of charity, Fontaine sees the feeding and housing of the poor as a transaction in which he purchases loyalty. They are very different men — Ryan wishes to build a society, Fontaine aims only to plunder one — but their heuristics for moral decision-making are identical. Whether either of them fairly depicts Randian Objectivism is an interesting but ultimately pointless debate; their self-interested philosophy most resembles the implicit morality of video games.

Obviously I don't mean to attribute a moral system to games like Guitar Hero, but most games that feature a player-controlled avatar operate on a moral system much like Fontaine's, in which all the world is the player's for the taking. Certainly most games try to cover this with a veneer of a story that somehow involves saving the world, but this motivation rarely takes center stage in the player's mind. In almost every game in existence, the player is encouraged to kill everything he encounters and plunder every chest he sees in order to win the game. Role-playing games often acknowledge this, usually through some abstracted morality system as in the Fable games. Yet in these cases only the extent of the behavior is criticized, not the fundamental attitude. So long as a chest or trove lacks any obvious owner, the player will not be criticized for taking it. The primary value of these games is always selfish advancement motivated by hedonism.

"Never play a man for the short con when you can play 'em for the long one..."

In keeping with this value set, we expect our options in games to be clearly differentiated in terms of their utility with respect to our self-interest. Because this is not operationally true of the rescue / harvest choice, many players were frustrated or confused. This statement from Duncan Fyfe, is typical of the protests:

It's hard to understand why this is the case because it's such a fundamentally simple and classic philosophical debate. You can make the rational, self-interested choice to gain as much resources as you can from this one interaction. Or you can sacrifice/minimise your short-term reward in favour of long-term benefit. This is how it's presented to you and it's basic political philosophy. It's realism versus idealism. You'll get less Adam for rescuing the little sisters but there's the promise of a greater reward down the line.

The problem with objections like this is that delaying gratification doesn't put philosophical ideas into opposition at all. The debate over long-term benefit vs. short-term benefit is just an argument over what constitutes self-interest, or what is the more "rational" route towards actuating it. Reducing the rescue / harvest choice to long-term vs. short-term gains merely recapitulates the contrast between Ryan's short-sighted anti-altruism and Fontaine's "long con", but neither of those options entails a rejection of rational self-interest.

It's not the choice you make, it's why you make it

If you want the player to really consider the implications of the philosophy of rational self-interest in the rescue / harvest choice, there are only two real ways to do it. The first is to make the rescue choice truly oppositional to the player's expected self-interest, i.e. give him no ADAM or very little for taking this route. The obvious disadvantage of this approach is that the game now becomes highly skewed. Although the vita-chambers relieve difficulty in the typical sense of dying and thus being unable to continue, the game will certainly become a much more frustrating and challenging experience. A gamer who played through such an experience might come away with the impression that the game was designed to convey the idea that Ryan and Fontaine were right, that he should have chosen killing.

The other alternative is to arrange gameplay in such a way that "rational self-interest" no longer constitutes a reasonable standard for decision, and this is what the choice looks like. The point is not to give the player a way to reject the philosophy in the decision, but rather to draw his attention to the fact that this was such a large part of his decision-making all along. Rational self-interest cannot tell us what to do with the little sisters because both options fit this mindset. Thus, the arbiter of judgment is your conscience, as it is in real life. The fact that costs and benefits of the options you can take with the little sisters don't conform to our expectations about "moral choice" in a game invites us to examine the values that inform those expectations, and the ways in which games express or exploit them.

Games have implicit values — do as you're told, take what you will — that we might disagree with were we forced to acknowledge them explicitly. Because these values are both hidden and integral, it is difficult to force the player to confront them without a shock or a feeling of confusion. The dissonance in the rescue / harvest choice may be intended to inspire the latter. If so, then both the confrontation with Ryan and the choice for the little sisters are commentaries on the nature of video games. Despite the ever-increasing promises of freedom, games have always played you the same way Fontaine played Jack. And regardless of how you feel about Ryan and Fontaine, you have always played games as if you were them. BioShock elaborately displays the callousness and evil of these men, and through them, you. How do you feel about that?

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It's only temporary

Far Cry 2's Africa burns impermanently. The lifelike flames consume plants and buildings and men, leaving great black scars on the landscape of a nameless, war-torn nation. The rickety shacks will char, the rusted jeeps will explode, and gas pumps will erupt like volcanic vents. In the course of the game you will leave dozens of checkpoints and ersatz military bases as smoldering ruins in the wake of your bloody path. Come back after a few minutes, however, and these sites will be transformed. The grass regrows, the buildings repair, the vehicles return to working order, and the men stand as they once did, hostile, guns ready. It's as if you were never there — and all this is fitting, because Far Cry 2 is, at its core, about futility.

Far Cry 2 casts the player as a mercenary sent to some African powder-keg to murder a conflict-stimulating arms merchant named "The Jackal". This mission fails spectacularly within the first ten minutes of the game, and then the powder-keg ignites, leaving the main character a penniless, malaria-afflicted gunman in a country that fortunately has a great deal of demand for his talents. It's a decrepit nation, without any apparent functioning agricultural or industrial infrastructure, and nearly all the civilians have already fled. As for the forces tearing the country apart, they occupy territories with indistinct boundaries, and lack any kind of uniform, as their armies consist almost entirely of mercenaries. Black natives hold the leadership positions in these ragtag armies, but their aides-de-camp and chief lieutenants are all white soldiers of fortune — making war for money, because this land has nothing else left worth fighting over anymore.

And yet the fighting continues. Far Cry 2 makes a half-hearted effort at embodiment, but for all that your hands can do in the game, what they will do most of the time is hold a gun. Missions are available from the two warring factions, your friends, arms dealers, a civilian underground, and mysterious voices that talk to you through cell towers. No matter who you're fighting for, everyone will attack you — a serious concern since almost every major intersection has its own contingent of mercenary guards. Their habit of regeneration means you must kill them coming and going, because your missions are handed out in central locations, and you will traverse the fastest routes to these places repeatedly, in cars or on foot.

Off the roads, this world has its nooks and crannies, hiding scattered diamonds or traces of your elusive target. The Jackal, strangely, sat for a series of interviews with an ignored journalist. Speaking clearly, but with alacrity that reminds one of high school policy debates, the arms dealer indicts all sides of the conflict, from the warlords to the first-world nations that ineffectually police them. The Jackal is as raw and uninhibited a capitalist as Bioshock's Andrew Ryan, more dedicated to the pursuit of the dollar than any abstract moral ideal. He sells guns to both sides, then buys them back when the cease-fire hits and sells them again somewhere else. And why not? As he points out: "[Weapons] aren't biodegradable. Only the dead are biodegradable."

Yet your weapons do degrade, in frustratingly short order at times. They jam, misfire, or even just explode in your hand as you pull the trigger. In Far Cry 2's Africa, the guns are temporary, but the men are permanent. The only ones that stay dead are the ones whose names you know — Frank Bilders won't come back if you let the morphine take him. Eventually, though, another buddy will take his place in the foreigner's bar, asking you for favors while offering none.

Far Cry 2's petty annoyances become apparent almost immediately, and conspire to drive the player away. Walking or driving, the player must make a long, dull trek to receive his next repetitive mission, performing murderous chores at each checkpoint along the way. With time, however, one acclimates to these discomforts as one might become inured to the oppressive heat implied by the game's setting. The visceral combat, the Jackal's rantings, the selfishness of nearly every person who speaks, and the sheer futility of each bloody journey across the African wilderness impart an awful momentum to the game, an irresistible force driving it towards an inescapable conclusion.

Far Cry 2 departs from formula by accepting the dark finale its every moment implies. This game stars a murderer who comes to Africa and kills almost everyone he meets, and fittingly, he dies anonymously in the jungle. Are his few good deeds enough to redeem him? Does it matter that he manages to save a few civilians here and there? You can't know that. You are allowed no certainty but this: that the next man to drive down these roads will find the guard posts restaffed, the mercenaries rearmed, and the war continuing blindly, needlessly, mindlessly, until the guns at last biodegrade.

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Rare codons give domains time to fold

The ribosome produces proteins by matching tRNA that has been correctly loaded with an amino acid to a codon (triplet of DNA bases) in the mRNA that contains the gene sequence. The triplet code allows 64 combinations of nucleotide bases, but proteins are made from only 20 amino acids (plus a "stop" signal). This means that most amino acids are coded by multiple codons, and hence have multiple tRNAs. Not all codons are created equal, however; in bacteria some codons are found much less frequently than others that represent the same amino acid. The tRNA associated with these "rare codons" is also less abundant than other tRNA, and this means that when a ribosome hits a rare codon, it often has to pause while it waits to encounter a loaded tRNA. To structural biologists like myself, who do their work by overexpressing proteins in bacteria, rare codons can be a nuisance because they slow down protein production, or even prevent it entirely. In a recent paper in Nature Structural & Molecular Biology, however, researchers from Germany suggest that the slowdown due to rare codons may have a functional advantage in vivo.

As a first step, Zhang et al. used a bioinformatics approach to survey the sequences of bacterial genes so that they could identify patches that would be slow to translate (the Methods section appears to contain an error in the description of this technique). They found that for proteins longer than about 300 amino acid residues, nearly every transcript contained at least one cluster of slow-translating codons. When the authors used a cell-free E. coli expression system to make some of these proteins and allowed only one round of translation initiation per ribosome, they saw a pattern of translation intermediates that matched the sizes predicted by the location of slow-translating patches.

In order to find out whether these translation intermediates had any significance, the authors examined the multi-domain protein SufI. In their prediction of the translation speed, which is on top in this figure that I have shamelessly stolen, there are four slow spots. Aside from the first one, these appear to correspond to the boundaries of different structural domains in the protein (lower part of the figure). Experiments with proteases suggested that these domains actually folded during the pauses, as the ribosome-bound translation intermediates were resistant to proteolysis.

Interestingly, when two rare leucine codons were replaced by more common ones (the authors call this SufIΔ_25-28), the whole protein became vulnerable to degradation. Similarly, when extra tRNA for these rare codons was added to the cell-free expression system, the full-length protein became protease-sensitive. This suggests that the slow patches are actually necessary for proper folding of the protein. It's often the case that lowering the incubation temperature can improve the expression of certain proteins in E. coli. The authors of this study find that is also true for SufI, as the protease resistance of SufIΔ_25-28 can be restored by lowering the temperature, and thus the overall translation rate. When analogous experiments with SufIΔ_25-28 and tRNA supplementation were carried out in living E. coli, the translocation of SufI into the periplasmic space was reduced by a factor of 10 even though the overall protein concentration was not affected, indicating that the co-translational folding allowed by the rare codons is necessary for proper functioning of the protein in vivo.

Of course this is a single case study, and it would be premature to conclude that every patch of rare codons corresponds to an important co-translational folding event. Indeed, that doesn't even appear to be true of SufI, which folds properly when one of its other slow patches is removed. However, at certain key locations these stretches of rare codons may be an important part of the folding machinery in multidomain proteins. In addition, the more frequent appearance of rare codons in β-strands (as opposed to α-helices) may also be related to folding due to the slower kinetics of β-sheet formation. As the authors note, the intrinsic kinetics aren't everything — pauses in the translation process may also buy time for the complex to encounter essential chaperones or cofactors. Regardless of the mechanism, it appears that rare codons, in at least some instances, provide a way for the folding process to catch up with the translation process.

Zhang, G., Hubalewska, M., & Ignatova, Z. (2009). Transient ribosomal attenuation coordinates protein synthesis and co-translational folding Nature Structural & Molecular Biology, 16 (3), 274-280 DOI: 10.1038/nsmb.1554

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A deadly halo in Alzheimer's disease

The observation of plaques composed primarily of amyloid-β (Aβ) peptides in the brains of Alzheimer's patients long ago gave rise to a hypothesis that Aβ was the agent that caused the disease. The plaques themselves, composed of long, insoluble fibrils of Aβ, were believed to cause the synapse loss and nerve death characteristic of the disease, and some data supports this model. However, several experiments have suggested an alternative possibility: that the symptoms of Alzheimer's may be attributed to soluble Aβ oligomers. In this view the fibrillar deposits may be an incidental feature of Alzheimer's disease, or even a defense mechanism whereby the body tries to get rid of the oligomers by forcing them into insoluble aggregates. In the March 10 edition of PNAS, a team led by researchers at Massachusetts General Hospital claim to have reconciled these two models. Using fluorescence microscopy, they find that amyloid plaques are surrounded by a "halo" of Aβ oligomers that kill the surrounding synapses.

The authors of this studied used fluorescence labeling to identify plaques, oligomers, and synapses in thinly-sliced tissue sections and living brains. They performed their experiments in mice that had been genetically manipulated so as to develop amyloid plaques. When they examined the brains of live mice, Koffie et al. noticed that the fibrillar plaques were surrounded by a cloud of the oligomers, as you can see for yourself in the figure below. On the left you can see the plaque core labeled by a fluorescent dye, and the middle image shows fluorescence associated with an antibody that specifically binds to amyloid oligomers. When these images are merged, the diffuse "halo" of oligomers becomes obvious. The authors see a similar result when they perform a similar experiment using thin slices of brains.


The authors also used a fluorescent-conjugated antibody to identify elements of the post-synaptic density (PSD), so that they can identify healthy synapses in the brain. Experiments in tissue sections demonstrated that the number of healthy synapses was reduced not only right next to the plaque, but also in a region extending up to 50 µm away (a length comparable to the diameter of a human hair). Aβ oligomers are also enriched in this region, and the relative concentration of the oligomer roughly correlates with the loss of synapses. By comparing the pattern of Aβ fluorescence to that of the PSD, the authors determined that oligomers were associated with many synapses, and that interactions between PSD and Aβ oligomers resulted in decreased synapse size. The relationship between Aβ binding and reduced synapse size was also shown to hold in control mice expressing normal levels of native amyloid precursor protein.

The observation that the presence of Aβ oligomers correlates with synapse loss, and the apparent degradation of synapses by Aβ, indicates that the soluble oligomers are a significant cause of Alzheimer's symptoms, although this study does not rule out the possibility that the plaque itself is also toxic. Even if the plaques have no immediate toxic effect, the authors propose that they serve as reservoirs, releasing synaptotoxic Aβ oligomers into the surrounding neural tissue, increasing the size of the lesions beyond the extent of the plaque itself. In this way Koffie et al. believe they have reconciled the previous models — oligomers are directly toxic, plaques release toxic oligomers, so both can serve as causative agents in Alzheimer's disease.

If this model is accurate, it implies that Alzheimer's disease may be quite resilient to attack. Antibodies or drugs that break up the Aβ oligomers will be effective in mitigating the synaptic damage, but as long as the plaques persist they will continue to replenish the pool of oligomers. Treatments that successfully break up the plaques will probably result in worsening symptoms due to the release of toxic oligomers as the fibrils disintegrate. These possibilities reinforce the idea that the most treatment for Alzheimer's will involve reducing the concentration of amyloidogenic Aβ peptides to prevent them from forming plaques in the first place.

(1) Koffie, R., Meyer-Luehmann, M., Hashimoto, T., Adams, K., Mielke, M., Garcia-Alloza, M., Micheva, K., Smith, S., Kim, M., Lee, V., Hyman, B., & Spires-Jones, T. (2009). Oligomeric amyloid associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques Proceedings of the National Academy of Sciences, 106 (10), 4012-4017 DOI: 10.1073/pnas.0811698106

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Urea binds to the peptide group

I've mentioned urea and guanidinium (Gdm) before on this blog, usually with reference to questions about their mechanism of action. These small molecules cause proteins to denature, or lose their higher levels of structure and become unfolded chains. The complete unfolding of a protein typically requires a fairly high concentration of denaturant, almost always more than 1M, and the explanation for this is that the denaturant molecules preferentially associate with the polypeptide chain with low affinity. In a recent issue of PNAS, a paper from Walter Englander argues that urea, but not guanidinium, associates with the backbone of the protein via hydrogen-bonding interactions.

Lim et al. reached this conclusion using hydrogen-exchange experiments. Amide nitrogens in proteins freely exchange their covalently-bound hydrogens (protons) with the surrounding water. The rate of this process can be measured (among other ways), by placing a protonated amide group into a deuterated solvent and tracking the decline in proton signal by NMR; this is called an HX experiment. In the case of a folded protein chain the observed rate will depend on the intrinsic chemistry of the particular amide and the stability of the protein structure, because this structure excludes water from the backbone and makes hydrogen bonds that lock the protons in place. Rather than deal with all of that, the authors used a small peptide mimic that (probably) has no complex structure. This had the additional advantage that the simple spectrum could be tracked by 1-D NMR, substantially increasing the time-resolution of the measurements. The authors measured the rates as they varied the pH — because we're talking about D₂O, it's called the pD instead — and added various cosolutes that are known to denature or stabilize protein folds.

As expected, the dialanine itself had a V-shaped rate profile in these HX experiments, with a minimum at a pD of 4. The hydrogen exchange reaction can be catalyzed by acid or base, so the rate increases as you go up or down in pD from this minimum. When urea was added to the solution, the authors found that acid-catalyzed HX accelerated while base-catalyzed HX decelerated. The most reasonable explanation for the latter result is that a hydrogen bond between the carbonyl of urea and the amide proton protects it from water attack. The authors do some mathematical modeling to establish that the effect on rate reflects a bonding association between the peptide and urea, not just random collisions or thermodynamically neutral associations.

The acid-catalyzed result is interesting, because in theory one would expect that urea would accelerate acid-catalyzed HX more than it actually does, because under acidic conditions it can accept a hydrogen from the amide nitrogen. While there are some confounding factors, the most likely explanation for this result is that the NH₂ groups of urea form hydrogen bonds to the carbonyl of the peptide. Because acid catalysis of HX hinges on the favorability of protonating this carbonyl, a hydrogen bond would be expected to reduce the HX rate. The authors argue that the ability of urea to serve as an acid catalyst is therefore mitigated by its propensity to bind to the carbonyl.

The formation of hydrogen bonds between urea and the peptide group meshes well with evidence that it denatures proteins through interactions with the backbone, some of which I have mentioned before. From HX experiments under native conditions we know that even a folded protein chain regularly undergoes excursions from its water-excluded, hydrogen-bonded state. Urea may bind to the backbone during these fluctuations, preventing or slowing a return to the folded structure.

Lim et al. also tested a number of other cosolutes, and found that none of them had a similar effect on the HX rate. In the case of the stabilizing molecules (glycerol, sorbitol) this is entirely expected, as their action cannot be explained in terms of a preferential association with the backbone anyway. The surprise concerns guanidinium, which is a more powerful denaturant than urea. The authors noted that Gdm has a small effect on the rate, but not in a pD-dependent way, and one that was little different from an equivalent concentration of NaCl (ordinary table salt). Gdm has no groups that can hydrogen bond to the amide, so the absence of an effect on base-catalyzed HX is expected. However, it should be possible for guanidinium to hydrogen-bond to the carbonyl, so it should seemingly have an effect on acid catalysis. This is not in fact the case.

The authors note that existing evidence does not support the idea that Gdm forms hydrogen bonds with water (although urea is known to do so). Lim et al. suggest instead that the planar Gdm molecule forms favorable stacking interactions with other planar groups. These include the peptide bond and several side chains. They argue that the stacking of Gdm with these groups pries the protein apart without requiring hydrogen bonds.

As a means to investigate diseases that result from protein misfolding, many groups are now trying to structurally characterize the unfolded state of protein molecules. Many of these experiments model the in vivo denatured state by using chemical denaturants such as urea or Gdm. The possibility that direct interactions between the denaturant and the protein will give rise to experimental artifacts should be taken seriously. Urea's promiscuous formation of hydrogen bonds with the backbone, itself, and water, may give rise to loose networks of hydrogen-bonded molecules that act to condense the chain. By contrast, Gdm's stacking effect will likely act to artificially extend the chain by steric obstruction. Because of the difference in these mechanisms, it may be of value to cross-validate findings from structural studies on unfolded states by repeating experiments with alternative denaturants.

Lim, W., Rosgen, J., & Englander, S. (2009). Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group Proceedings of the National Academy of Sciences, 106 (8), 2595-2600 DOI: 10.1073/pnas.0812588106

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Activated caspases stick together

In a post last week I mentioned a technique for obtaining the high-resolution structure of a protein inside a living cell, but I also pointed out that this technique was difficult and expensive, and might not be applicable to large proteins. Techniques improve and become more powerful, of course, but you might not want to wait for NMR to catch up to your question. Fortunately, high-resolution in vivo structures may not be necessary if you already have relevant dilute-solution structures of your protein and merely want to distinguish between different known conformational states. In a recent paper in PNAS, researchers from San Francisco used conformation-specific antibodies to locate activated caspase-1 in cultured cells.

Caspase-1 is a cysteine protease that plays a role in the immune response, as well as being released during apoptosis. From crystal structures we know that this protein can adopt two different structures, of which only one represents a catalytically competent state of the enzyme (the on-form). Caspase-1 also possesses an allosteric site where an inhibitor can bind, locking the enzyme in an inactive conformation (the off-form). When it's not bound to anything (the apo-form) caspase-1 is presumed to have a conformation similar to the off-form. Like many proteases, caspase-1 has a large, inactivating tail when it is made (the pro-form) that must be cleaved off before activation is possible. The structure of the caspase-1 proenzyme is not known. Current models of inflammatory response propose that after processing, the on-form binds to scaffolding proteins in an "inflammasome". In order to confirm this proposition, the authors decided to generate antibodies that would bind specifically to the on-form or the off-form of caspase-1.

The key to this experiment was combining irreversible inhibitors that could essentially lock the caspase into one conformation with the phage-display technique for optimizing antibody recognition. The authors had the advantage that both the active site and the allosteric site have cysteines in them. In an oxidizing environment, small molecules can covalently bind to the protein via disulfide bonds, thus locking the enzyme into the on-form or off-form. The authors immobilized these "locked" forms of caspase-1 and used them to screen antibody fragments (Fabs) using phage display. In addition to the typical selection approach, the authors performed anti-selection at one point using the "wrong" conformation to increase the specificity. After several rounds of selection, and some controls to ensure that the antibodies were binding to caspase and not the inhibitors, Gao et al. had several candidates for further optimization and screening. After they completed that process, they had two antibodies, Fab_on and Fab_off, specific for the two conformations. Each antibody bound to its intended target with a K_D of less than 5 nM. The authors also made full antibodies (IgG_on and IgG_off) from these Fabs for expression in mammalian cells.

The authors took these new antibodies for a spin with the apo-form of caspase-1. One might naively expect that only Fab_off would bind to this protein, but in fact Fab_on bound as well, albeit with substantially reduced affinity relative to the on-form. One possible interpretation of this finding is that the apo-form is equivalent to the off-form, but that Fab_on can convert it to the on-form via an induced-fit mechanism. If this is the case, then we would expect Fab_off to have the same affinity for the apo-form as it has for the off-form. However, the authors find that Fab_off has reduced affinity for the apo-form relative to the off-form. This indicates that the apo-form is an ensemble of conformational states, most of which more closely resemble the off-form than the on-form. Consistent with this view, the authors found that they can activate or inhibit apo-form activity by adding Fab_on or Fab_off, respectively.

By contrast, IgG_on did not bind detectably to a model of the pro-form, suggesting that this form's conformational ensemble contains no members that are close in structure to the active form. The weak affinity of IgG_off for the pro-form suggests that there are substantial differences between this conformation and the off-form as well.

At this point we know that IgG_on will bind tightly to the on-form of caspase-1, weakly to the apo-form, but not to the pro-caspase. This means it will likely be an effective probe of active caspase-1 in cells. The authors performed this experiment in THP-1 cells that they differentiated into macrophages. While IgG_off produced diffuse fluorescence in these cells, IgG_on stained small, concentrated bodies in a fraction of the cells. This suggests that active caspase-1 is localized to supramolecular structures in these cells, which the authors argue are identical to a structure previously identified as the "pyroptosome".

Although this particular experiment took advantage of binding-site cysteines that are particular to caspase-1, it should be possible to extend this approach to other proteins. Even non-covalent inhibitors or activators should be useful in this approach as long as the concentration is held high enough to saturate the target site during the selection step. Of course, the conformational change must alter the structure enough that the antibodies have something to grasp — it may not be possible to get specific antibodies if the shift is too subtle. If this requirement is met, however, it should be possible to determine the distribution of specific conformational states in cells, or even (as the authors suggest) to use antibodies as activators or inhibitors in vivo.

J. Gao, S. S. Sidhu, J. A. Wells (2009). Two-state selection of conformation-specific antibodies Proceedings of the National Academy of Sciences, 106 (9), 3071-3076 DOI: 10.1073/pnas.0812952106

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Get by with a little help from your friends

Fog blankets the sleepy little town of Inaba, hiding a secret and a killer. On rainy midnights, the televisions in the village show strange images of citizens who have recently disappeared. Days later, when the fog lifts, these people turn up dead. Only a small group of teenagers know the truth: inside the televisions lies a perilous world where the hidden aspects of a person's soul become murderously real. Someone is kidnapping the town's citizens and forcing them into that world, and only the main character of Persona 4 can save the victims and nab the killer. By himself, this would be impossible, but he has an amazing skill that guarantees his victory: he's a good friend.

The world inside the televisions of Inaba is crawling with monsters, and in order to confront the powers that are behind the disappearances the characters must defeat these creatures in turn-based battles. Alone, they'd never stand a chance, but the teen heroes of the game are assisted by "Personas", spiritual manifestations of their personality that have enormous power. The main character, named by the player, can acquire hundreds of these in battle or by "fusing" other Personas, thus gaining access to new abilities and stronger attacks. His friends, however, have just one Persona each, and that can only be obtained by traversing a dungeon and confronting their own souls in the form of a "shadow" born from the suppressed or disliked aspects of their personality.

The characters' shadow selves lack subtlety and nuance — one reflecting a young man's uncertain sexuality runs around in a towel and lisps outrageously — but they're not supposed to have those things. They represent the clumsy stereotypes common to the worldview of teenagers, and the oversimplifications of mass culture. In a game that depicts television's numbing effect as a world-obscuring fog, they're completely appropriate, though they can (and arguably should) make a player feel uncomfortable. Typical forms of teen angst, from jealousy to power struggles, inform most of the shadows' behavior, though the early dungeons are dominated by expressions of uncertainty about sexuality. Persona 4 also reflects on the inadequacies of its own medium in these dungeons. One of the game's antagonists is a disturbed, game-playing teen; his shadow takes the form of an infant cocooned in armor representing the hero of an 8-bit era RPG.

Persona 4 requires a significant time investment due to the level grinding in these dungeons. The relative ease of escaping and re-entering them means this can be discretized into play sessions lasting an hour or less, but the repetitious trekking through empty, uninteresting hallways to fight the same enemies over and over again will wear on many players. Losing your focus can lead to a frustrating game over, however. Like most Shin Megami Tensei games, battles really depend on correctly identifying elemental strengths and weaknesses. High-level monsters can be toppled easily if you know their vulnerability, but routine battles can quickly turn into disasters if the enemy exploits yours. Selecting the appropriate Personas and allies for a particular area or battle is a critical strategic task — the right friends make the difference between a long, difficult boss fight ending in victory and a short, frustrating boss fight ending in defeat.

Persona 4 also invites the character to develop relationships and explore problems outside the TV world through the social link system. In deference to traditional RPG mechanics, each key relationship has a "level"; by spending time with your friends and talking through their problems with them your relationship levels up, bringing you closer to resolving their problems. In terms of the game mechanics, your reward for success in the relationships is that you are able to create more powerful Personas. In reality, the social link conversations become their own reward. They're believably written, moving, and diverse, though the majority are built around repairing relationships or coming to terms with events or situations that cannot be changed. The main character makes a pretty good emotional facilitator, however, and in their culmination most of the social links give the impression that he's done something good for these characters just by listening to their woes and being supportive.

The mini-plots of these relationship arcs work better than the central story, which plods along predictably for the first 2/3 of the game or so, coming across more as an excuse for changing scenery and enemies than an actual mystery. Late in the game the pace picks up, with layers of double-crossing and deception that actually manage to elicit the feel of a caper. The eventually-revealed culprit (who the player must pick from a list in order to avoid the game's bad ending) manages to surprise, but this is only because the clues to the murderer's identity amount to only a few sentences of dialogue out of a game that can last upwards of 100 hours. As a result, forcing the player to choose the solution feels a bit unfair. Yet this reveal has its own reward; the story the culprit tells about his exploits gives the impression that he's someone who never faced down the dark parts of his personality. One gets the impression that all of this could have been avoided if he'd had a friend like the main character to help him.

Don't let Persona 4's demons and monsters fool you — this is a game in which the mechanics and story are built around the positive power of friendship. The main character supports his friends through hard times and tough decisions. In return, they fight beside him and give him the strength to fight on his own. All the creepy imagery and uncomfortable situations disguise a core message that comes straight out of an after-school special, and you'll only realize it when the game's warm (and yes, somewhat hokey) finale puts it right in front of your face. As the train rolls out of the station, you may find that the fatigue of Persona 4's long hours fades away, and you'll find yourself wanting to play it again, just to spend a little more time with your friends.

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High resolution protein structure from a living cell

Virtually everything we know about protein conformation comes from experiments performed in environments that do not resemble the biological context of proteins in action. Our data generally come from solutions that lack the significant array of salts, sugars, and metabolites that fill the cytosol of living cells, and often these data are acquired at a pH far removed from cytosolic. In addition, the dilute solution conditions used in almost all structural biology experiments do not capture the crowding and excluded volume effects that are likely to play a role in determining protein conformation in densely-packed cellular environments. As techniques in biology and NMR have advanced, however, the determination of protein structures in living organisms has become possible, despite the significant challenges. This week in Nature, a research team from several institutions in Japan and Germany reports that they have solved a high-resolution NMR structure of a protein in the cytosol of living E. coli bacteria (1).

The authors chose a relatively small and simple protein to work out their technique, in this case a 66-residue metal-binding protein from a thermophilic organism. They expressed the protein in E. coli using standard methods, exchanging the bacteria into isotopically-enriched media once they reached the appropriate density for induction. At the end of the induction period, the bacteria were gently centrifuged, resuspended into a thick slurry, and put in an NMR tube. Samples produced in this way were stable for about 6 hours, which is generally not enough time to perform the kinds of 3-dimensional experiments necessary for NMR structure determination.

To get around this problem, the authors used non-linear sampling and maximum entropy processing. This approach allowed them to reduce the number of data points they took, without losing much of the frequency discrimination that is vital to successful NMR. In this way they were able to compress the essential assignment and structural experiments to about 3 hours, although they found it necessary to repeat experiments and add them together in order to get enough signal to proceed. In order to ensure that the data were not contaminated by sample degradation, they ran short two-dimensional experiments to check sample quality in between the 3-D spectra. With this approach they managed to take 9 assignment spectra, several relaxation spectra, and several NOESY spectra for structural data. Apparently, each spectrum required its own, new sample due to the short lifetime of the bacteria under these conditions.

The authors performed control experiments in order to address some of the problems that affected previous research on proteins in living E. coli. They found that removing the cells from the NMR tube eliminated most of the protein signal, and that lysates of the bacteria contained protein signal. These experiments showed that the data collected in their experiments genuinely came from protein inside the bacteria rather than protein that had leaked out.

After all this work, the authors were eventually able to solve a structure of TTHA1718 in live E. coli, which you can see to the right (explore this structure at the PDB). This result would not have been possible, however, had the authors not employed specific methyl labeling in order to get additional long-range restraints, a technique typically used for very large proteins. As you can see in the supplementary information, attempts to solve the structure without the methyl NOEs gave rise to a fairly disordered ensemble. Even this ensemble is nowhere near as tight as the in vitro structure that the authors also solved. Because the in vivo structure used many fewer NOEs than the one from dilute solution it is difficult to tell whether differences between these ensembles reflect real conformational changes or simple uncertainty. The loop near the metal-binding cysteines (shown as fat sticks in this image) is a case in point — it looks quite different from the solution structure, especially in the positioning of the critical side chains, but there are almost no NOE restraints for this loop in the in vivo structure (Figure 4e). Chemical shifts support the idea of a conformational change, and inside the cells many of the signals in that region are too broad to detect. This, in conjunction with some metal-enrichment experiments the authors performed, suggests that the protein is regularly binding to metals in vivo, but the structure of this bound state is essentially a mystery. There are also a few clear structural differences in well-defined regions of the protein, but their significance is also unclear at this time.

This experiment serves as proof of principle, but NMR spectroscopists weary of years of promising experiments that turn out to only work on ubiquitin might rightly question whether this approach has any further applicability. In order to address this, the authors expressed the protein to a lower level in order to demonstrate that the procedure could still work for less-concentrated proteins. In addition, they show spectra from calmodulin in the supplementary data, suggesting that this approach will at least be applicable to proteins up to the 20 kilodaltons. However, the relaxation data the authors show in the supplementary data indicate that tumbling in the bacteria is significantly slower than in dilute solution, and the T₂ of the protein is 5-6 times shorter in vivo. If this result is general, then structural work on larger proteins may not be possible.

Why did this experiment work when other experiments on globular proteins in E. coli have led to leaking protein or an absence of signal (2)? Part of this may be that not all E. coli are created equal: the authors of this study used the JM109(DE3) rather than the popular BL21(DE3) strain. Genetic differences between the cells used may be responsible for the altered outcome. This will be a difficult thing to nail down, however, as overexpression typically involves the introduction of foreign DNA, an antibiotic, and an exotic activator of some kind, not to mention that isotopic labeling requires nutrient-poor minimal media. The difference between CI-2 that leaks out of cells and TTHA1718 that stays in may be as simple as the amount of magnesium sulfate in the M9. Until the factors causing the excretion of overexpressed proteins are more fully understood, careful controls will be an absolute necessity of in vivo experiments.

Because of the difficulty and expense this is clearly not an approach to be taken up lightly. For the time being, at least, you want to save this sort of experiment for systems where there is a real inconsistency between structural data from dilute solutions and results in vivo. As we improve the NMR approaches and increase our ability to manipulate E. coli behavior, however, this technique will grow more powerful and broadly applicable. Moreover, the Japanese part of the team reports in the same issue of Nature that they have managed to acquire spectra from proteins transferred into cultured human cells (3). This suggests the possibility of purifying labeled proteins at high yield, transferring them into living human cells, and then monitoring their structural and dynamic properties in their biological context.

1) Daisuke Sakakibara, Atsuko Sasaki, Teppei Ikeya, Junpei Hamatsu, Tomomi Hanashima, Masaki Mishima, Masatoshi Yoshimasu, Nobuhiro Hayashi, Tsutomu Mikawa, Markus Wälchli, Brian O. Smith, Masahiro Shirakawa, Peter Güntert, Yutaka Ito (2009). Protein structure determination in living cells by in-cell NMR spectroscopy Nature, 458 (7234), 102-105 DOI: 10.1038/nature07814

2) Li, C., Charlton, L.M., Lakkavaram, A., Seagle, C., Wang, G., Young, G.B., Macdonald, J.M., Pielak, G.J. (2008). Differential Dynamical Effects of Macromolecular Crowding on an Intrinsically Disordered Protein and a Globular Protein: Implications for In-Cell NMR Spectroscopy. Journal of the American Chemical Society DOI: 10.1021/ja801020z

3) Kohsuke Inomata, Ayako Ohno, Hidehito Tochio, Shin Isogai, Takeshi Tenno, Ikuhiko Nakase, Toshihide Takeuchi, Shiroh Futaki, Yutaka Ito, Hidekazu Hiroaki, Masahiro Shirakawa (2009). High-resolution multi-dimensional NMR spectroscopy of proteins in human cells Nature, 458 (7234), 106-109 DOI: 10.1038/nature07839

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