Storytelling in Silent Hill

My review of Silent Hill: Shattered Memories went up last week at gamecritics.com, and I really regret that I had to pan it. The game had some good ideas buried in it, but the overall experience was just miserable, oscillating wildly between deadly boredom and screaming frustration. I covered most of the gameplay issues I had in the review, but I only lightly touched on the positives and negatives of the game's storytelling. My reason for this is that it's difficult to talk about what's really going on in the story without revealing the game's central twist. I think it's unfair to even slightly spoil a game in a review (I felt I had to change the review's subtitle for just this reason), and like many Silent Hill games the twist at the end is profoundly important to the player's experience. Here on the blog I don't feel so much restraint, however, and I think it's worth discussing what the game gets right and wrong in terms of the characters, locales, and echo messages. That means the following discussion has massive spoilers, so be forewarned.

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Glitches in the Animus

I think it's fair to say that one of the things Assassin's Creed II is about is immersion. The fiction of the game concerns a completely immersive virtual world reconstructed from one character's DNA, which allows him to inhabit the bodies of his ancestors. The game world itself is constructed to resemble, with startling fidelity, famous locations in Italy. The game also adopts a 3rd-person open-world approach that produces at least a competent illusion of player agency in the game world. Genuine player immersion is, at least with present technology, an unreachable goal, and so of course Assassin's Creed II never manages to achieve it. What struck me during the course of play, however, was how many times the game simply gave up on the pursuit, falling back on old game tropes that betray the illusion, despite how easily this could have been avoided.

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Haiti

I can barely imagine a worse place for a devastating earthquake to hit than Haiti, one of the poorest countries in the western hemisphere. Even worse, the main shock originated very close to its largest city, Port-au-Prince, and nearby aftershocks are continuing. Thousands, perhaps tens of thousands, died in the earthquake itself, and given the lack of infrastructure even more may perish in the near future from disease and starvation. Many of the nation's most significant structures (including the National Palace) have been destroyed, its slums have been flattened, and many of the buildings still standing have suffered irreparable damage and will have to be demolished.

You can help the survivors by donating to the Red Cross, Medecins Sans Frontieres, or Partners in Health. Wyclef Jean's Yele Haiti is also taking donations for earthquake relief. I know as well as anyone how hard things are right now. I know a lot of my readers are unemployed, or living on a limited budget. Small donations add up, though, and much of what will be desperately needed in Haiti in the coming weeks and months comes very cheap. If you can spare $50, that's great, but if you can only spare $5, give that. No matter how bad your lot is right now, I can guarantee you it's better than living in a shattered country without power, food, medicine, or clean water.

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The ones I'll remember

It's the end of a decade, which means a vast array of retrospectives for video games and movies and whatever the hell else. Assembling a "best of" list for games is a tricky and probably futile task, especially for a one-man show like this site. Games take too long to play, are too diverse in their experiences, and are spread across too many expensive platforms for one man to sample everything that might be a classic, or even everything that other people think is classic. Besides that issue, many games that are awesome experiences at the time trickle out of your mind to be forgotten until you see the case sitting on your shelf. So this is not the definitive best-of-the-decade list, nor is it even my best-of-the-decade list. This is my best-remembered list. These are the games I think about when I play other games, when I'm reading books, when I'm watching television. They are the games that have stuck in my mind long after I removed the disc from the tray, the games I think of playing again at least once a week. I'm not sure they're the best games of the decade, or even the best games of their respective years of release, but they're the games I'll remember.

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A single residue dictates a fold

Anfinsen's dogma — that the amino acid sequence of a protein uniquely determines its structure — naturally leads one to the idea that identity between amino acid sequences means identity between structures. This has proven to be a successful paradigm: sequence similarity reliably predicts structural and functional similarity. Evidence accruing in recent years, however, suggests that for small proteins, at least, this assumption may not be entirely safe. Adding to this view, in an article in PNAS this week (it's open access, so open it up), a research team led by Philip Bryan reports that they are able to generate significantly different folds with divergent functions from sequences that differ by a single amino acid.

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Non-native hydrogen bonds mediate structural transitions

A few weeks ago, I wrote that the goal of a structural biology research program ought to be to "characterize the conformation and energy of key, functionally-relevant members of the protein's structural ensemble and identify the pathways between them." The Nature paper last week, among other examples I mentioned in the preceding post, described functionally significant minor members of the native-state ensemble, and this is certainly an area where structural studies are making a lot of progress. But what about the other part of that statement, the transition pathways? How are we to study them, and what can we learn about them? Experiments alone are unlikely to tell us everything we want to know about the intermediates between different native structures. We can, however, use simulations validated by experiments to investigate the mechanisms of structural change. Today in Cell, research primarily performed by my coworkers Alexandra Gardino and Janice Velos demonstrates that the bacterial signaling protein NtrC  rapidly samples its active conformation even when it is not phosphorylated. Moreover, they confirm predictions that the intermediates between these two states are stabilized by hydrogen bonds not present in either one.

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Alternate structures and catalysis in cyclophilin

Of all the sources of structural variability in proteins, the hardest to pin down is side-chain conformational heterogeneity. Side chains aren't always easy to model into their primary conformation in the first place — you need excellent crystal diffraction or NMR data to do it. Even if you pulled that off, it's not always clear how (or if) side-chain fluctuations relate to a protein's activity. If we carefully examine our data for the faintest signals, however, we can sometimes find evidence of secondary conformations that play an important role in function. In an article in this week's Nature, James Fraser, myself, and others show that in the case of the proline isomerase cyclophilin A (CypA), alternative conformations of side chains play a key role in catalysis.

Previous experiments on CypA had established that the backbone amide groups of many residues were sensitive to a conformational fluctuation on the millisecond timescale. Under conditions where this enzyme is saturated with a peptide substrate, the fluctuation rate for some of these residues is very similar to the catalytic rate, suggesting that the dynamics and catalysis are linked in some way (2). Later experiments also showed that this fluctuation was an intrinsic property of the enzyme, continuing even in the absence of substrate (3). What we didn't know, however, was how the dynamics of cyclophilin were related to catalysis. We couldn't know, because we had no idea what the motion we were detecting was.

In the case of enzymes like adenylate kinase, there is a dramatic rearrangement of structural elements, and the population of conformations corresponding to the "end points" of that motion can be significantly enriched by altering the amount of substrate present in solution. In the case of CypA, neither of these things seems to be true. Supplementary Fig. 1a (freely accessible from the article page) neatly encapsulates the problem. For this figure, 48 structures of CypA, some with ligand and some without, were aligned, and the variation between them was determined. While there is some variability in the chain conformation, it is primarily limited to a group of residues known to undergo fluctuations that are not related with catalysis (blue chain). The residues involved in the catalysis-related dynamics don't seem to have much variability, even across this fairly large group. So we can't trap the unknown, minor state of CypA by adding substrate, and there's no evidence of an alternate state that explains the NMR data.

Knowing this, we suspected that some kind of side-chain motion accounted for the observed dynamics, probably involving an aromatic group of some kind. Our efforts to gather evidence for this, however, ran into some typical NMR problems — resonance overlap and poor sensitivity exacerbated by chemical exchange. Fortunately, the crystallographers came to our rescue, in the form of Tom Alber and his super-talented grad student Jaime Fraser. Jaime had determined a crystal structure of CypA at cryogenic temperature and analyzed the data using their algorithm RINGER, which examines electron density below the threshold typically considered "noise" in order to identify possible alternative rotameric states of side chains. He found evidence of multiple conformations for a few residues, but nothing that would explain the NMR results. Jaime had the bright idea to redo the experiment at room temperature, which Tom was convinced would result in nothing more than a radiation-damaged crystal and bad diffraction data.

What actually happened was that when Jaime examined the electron density from that experiment he could identify a group of side chains that had more than one conformation in the crystal, which you can see in Fig. 1. These residues included serine 99, methionine 61, and the catalytic arginine 55. Right in the middle of this group was phenylalanine 113, a residue with an aromatic side chain capable of causing changes in chemical shift at relatively long range. For context, the image to the left shows a structure of cyclophilin (PDB code: 1RMH) in complex with the model substrate we used in our own experiments (succinyl-Ala-Ala-Pro-Phe-p-nitroaniline), with the side chains of S99, F113, M61, and R55 in red. As you can see, F113 and M61 form part of the floor of the binding pocket, with S99 rather remote.

So here we have an alternative structure of CypA, hidden below the threshold typically considered when determining a crystal structure. It was certainly plausible that fluctuations in this ensemble of side chains could give rise to the NMR observations, but plausibility isn't proof. One way to address this would be to try and force CypA to adopt the less-populated conformation. If you look at Fig. 1d you can see that the two conformations of S99 lie at the standard rotameric positions, and that the less-populated rotamer of S99 would run into the more-populated rotamer of F113. So, if you replaced one of the side-chain hydrogens of S99 with a methyl group (i.e. mutated the serine to threonine), that might push the other residues of this group into their minor conformational state. So, that's what we did.

To the right you can see an overlay of structures for wild-type (WT) CypA (red) and S99T (green), aligned using structural elements on the opposite side of the protein from the active site. As you can see, the backbone traces match very closely, except for the helix and loop on the right. These elements are involved in crystal contacts in the S99T structure, but not the WT; a lower-resolution structure of S99T shows no differences here. Another key difference between these structures, of course, is the position of the side chains (thick neon); as shown here (and more clearly in Fig. 2c) they seem to have adopted the minor conformation from the WT structures. Although this mutation inspires widespread chemical shift changes (Fig. 3a) consistent with the hypothesis that this concerted side-chain rotation gives rise to the NMR observations, the structures seem very similar. Yet, S99T CypA differs from WT in two important ways.

The first difference is that the conformational fluctuations are dramatically slower, but only for residues that showed catalysis-related dynamics in WT (Fig 3d). In fact, this rate is now so slow that due to a quirk of NMR we can only determine the slowest rate of the process. At 10 °C, this fluctuation in S99T is about 60 times slower than the slowest process in WT.

The second key difference between the mutant and the WT is that catalysis is dramatically slowed. Because CypA does not consume its substrate (it acts on both cis- and trans- proline bonds) its activity can be assayed by NMR, as you can see in Fig. 4. As with any enzymatic assay, the net activity is proportional to the amount of enzyme added, so just glancing at these spectra (and knowing the enzyme concentration) you can estimate that S99T has at least 40-fold lower activity than WT enzyme. If you actually perform the fits, it turns out that the reaction velocity for S99T is about 240 times lower than that for normal CypA, but this includes a contribution due to the fact that S99T does not bind its substrate as tightly either. If you correct for this, it turns out that S99T has about 70-fold less activity than the normal enzyme. Not only is this similar to the change in dynamics, it's also quite comparable to another mutation, R55K, that removes a group that performs some of the chemistry.

These results indicate that a conformational change in a group of side chains including F113 is primarily responsible for the chemical exchange behavior observed in WT. The S99T mutation stabilizing the minor conformation dramatically and similarly reduces both the conformational fluctuation rate and the catalytic rate. This suggests that dynamics and catalysis are linked not by happenstance but by some direct relationship. Unfortunately, these experiments do not provide any direct insight into the mechanism by which dynamics contribute to catalysis. They do establish, however, that in CypA coherent fluctuations of side chains, barely detectable in protein crystals, nonetheless make a critical contribution to function.

1) Fraser, J.S., Clarkson, M.W., Degnan, S.C., Erion, R., Kern, D., & Alber, T. (2009). Hidden alternative structures of proline isomerase essential for catalysis Nature, 462 (7273), 669-673 DOI: 10.1038/nature08615

2) Eisenmesser, E.Z., Bosco, D.A., Akke, M., & Kern, D. (2002). Enzyme Dynamics During Catalysis Science, 295 (5559), 1520-1523 DOI: 10.1126/science.1066176

3) Eisenmesser, E., Millet, O., Labeikovsky, W., Korzhnev, D., Wolf-Watz, M., Bosco, D., Skalicky, J., Kay, L., & Kern, D. (2005). Intrinsic dynamics of an enzyme underlies catalysis Nature, 438 (7064), 117-121 DOI: 10.1038/nature04105

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Don't look for "the" structure

It surprises me how often I hear students, postdocs, and even professors talk about determining the structure of a protein. A singular structure has the advantage of being relatively easy to interpret, but the cost of this is often the loss of functional data. It's easy to understand how this terminology emerges from the discipline of crystallography, which after all only works when the protein molecules adopt only a small number of conformations. Yet even when it comes to NMR, a technique that should be very sensitive to the fact of structural multiplicity, the language of researchers and the structural tools available to them are too often oriented towards the idea of a singular structure. But any representation of a protein as a single conformation is a simplification — every protein exists in multiple structural states.

Trivially, we are aware that a given polypeptide chain can adopt a number of different conformations — the "folded state" of any given polypeptide chain covers only a tiny sliver of the possible conformational space. A protein that is "unfolded" occupies not a single, well-defined state but a vast multiplicity of states, and this kind of statement is not controversial because we tend to imagine unfoldedness as a messy chaotic jumble of conformations. The reality is less cut-and-dried: although unfolded proteins may have no regular structure, many still have a propensity to form particular secondary structures or interactions. The reality of denatured proteins is that they have a complex and varied energy landscape, not an array of possible structures that all have roughly equivalent energy. The flipside of the popular view is that the a protein's native state draws down to a sharp energy well, and this conception is also misguided.

The most dramatic counterexamples to the idea of a neat, punctate energy well come from proteins that adopt several different folds in the native state. One relevant case is lymphotactin, which freely interconverts between an α/β monomer and an all-β dimer under physiological conditions. Lymphotactin may be unusual, but the principal message from that study is one that ought be paid attention to in others, particularly when the protein in question has functional conformational diversity. Consider α-synuclein, a protein implicated in Parkinson's disease. In the presence of some detergent micelles this protein is known to take on an α-helical hairpin structure, with two helices laying down on the charged surface of the lipid headgroup. In solution, however, it seems to take on a number of different forms, and may interact with true lipid bilayers in a completely different way than it interacts with micelles. For proteins that interconvert between several different physiologically-relevant folds, one is never pursuing the structure, but rather a structure.

Of course, we don't expect most proteins or domains to regularly adopt alternate overall folds. However, reorientations of domains or monomers is a relatively common behavior, and one that poses a sticky challenge for structural biologists because incidental properties of a particular arrangement may bias our experiments towards observing it. A minor member of the ensemble, if it has favorable packing geometry, may exclusively populate a crystal. Similarly, NMR experiments to determine domain arrangement via residual dipolar couplings must always be undertaken with an eye to ensuring that interactions with the aligning media do not bias the results. No single structure of adenylate kinase can instruct us about its catalytic cycle, and structures of the unbound state do not capture the reality that the protein continues to open and close in the absence of ligand. Single structures do not capture motions of domains or monomers relative to each other and that often means an incomplete understanding of function.

Domain motions are also an overly dramatic example, because simpler rearrangements of the backbone take place in many proteins, even when regular secondary structures are evident. Fluctuations of the main chain play a functional role in several proteins — as, for instance, in the flaps of the HIV protease. Additionally, rearrangements of the backbone have a significant role in signaling, as in NtrC, which I'll talk about more in two weeks. Proteins where the main chain rearranges in response to ligand binding or post-translational modification generally cannot be described by a single structure.

Even if the backbone is rigid, every protein will have flexibility in the side chains of its amino acids. One of course expects to see this kind of behavior in side chains on the surface of a protein, where it is usually dismissed as irrelevant. However, we also know that side chains can rotate and move in the core of a protein, and that on some protein surfaces they can undergo coherent rearrangements. I'll talk a bit more about the functional relevance of side-chain motions next Thursday. For now, suffice to say that side chain rotations cannot be so easily ignored and sometimes have functional effects. Structural studies that do not capture these rotations may be missing something important.

My point here is not that single structures are stupid or useless. A structure can be very informative about about a protein's function, and often has great power to explain the effects of mutations and ligands. However, we should not mislead ourselves into thinking that any single structure will have all the answers, or indeed any of them. Every protein is a constantly interconverting ensemble of structures, and there are many layers of structural diversity within that ensemble, reaching from whole fold rearrangements to "mere" side-chain adjustments. Determining the structure of a protein is not a coherent goal for a research program. The successful structural biology study will characterize the conformation and energy of key, functionally-relevant members of the protein's structural ensemble and identify the pathways between them.

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Chosen at birth

Last post, I mentioned that the tendency to choose segregation as a means to solve problems was a feature of many societies in the world of Dragon Age. Another, related motif appearing in many Thedan societies is the existence of a rigidly-defined social order in which a person's status and even his occupation are set at the moment of birth. To varying degrees this kind of social rigidity appears in almost every social group in the game (except the elves). Through its dialogue and plot, Dragon Age: Origins repudiates these systems, but in its mechanics it supports them.

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Out of sight, out of mind

Playing Dragon Age gave me a relatively frequent sense of déjà vu. Although the game portrays a number of different nations and societies, there are recurrent features that speak to underlying ideas about the psychology of its inhabitants. One such motif is the tendency for its denizens to solve their problems through segregation. At several levels, the people of the continent of Thedas like to resolve issues by pushing problematic groups into isolated areas and pretending, as much as possible, that they no longer exist.

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A few things

I promise there will be actual content in essay form later this week, but I have to kick the week off with some housekeeping. Some of this is bad, and some of it is good (or I hope it's good).

Bad news first: I've been getting pretty regular comment spam on this blog for about a month now, at a rate of about one post every half day or so. I don't know why anyone's bothering — most of the posts affected are at least half a year old — but it's still pretty irritating to click back to the blog every day to delete internet pharmacy ads written in badly formatted bbcode. Blogger doesn't give me a lot of options for dealing with this, and I'm not in the mood to migrate to some other platform. So as a first step I am turning on moderation for all posts more than 90 days old. The only kind of comment I will refuse is an obvious advertisement, but comments will obviously show up slowly on those posts. My other options are to block anonymous comments, which I definitely will not do, or to use a CAPTCHA, which I find to be annoying and have evidence is only moderately effective. Hopefully this will resolve the issue.

On the good side, my friend Ben Abraham, proprietor of the SLRC blog and organizer of Critical Distance, is going to be visiting GDC. Michael Abbott and David Carlton have already gotten him a pass, but since Ben is in Australia and GDC is in America, there's a rather expensive plane ticket to deal with. On the right side of the blog you can see a little widget for donating money to help cover the cost of that flight. I'd appreciate it if you could spare a few bucks to give him a hand.

If giving money to Ben doesn't sound like something you want to do, then I hope you'll find space in your budget to give a few dollars to Penny Arcade's annual Child's Play charity drive. Child's Play buys toys, books, and games for children's hospitals throughout the world to help make the lives of children with serious illnesses much less miserable. I spent a lot of time in hospitals myself as a kid, and the only parts of those stays that didn't suck terribly was the time I got to spend getting my ass kicked by the original Legend of Zelda. You can donate money, plain and simple, or you can choose your local children's hospital and buy something from their Amazon wish list. There are items on those lists that cost as little as $10 and will make a big improvement in what is usually a horrible experience, so please donate something if you can.

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The uncertainty principle

Although it uses conventional horror tropes, Condemned: Criminal Origins takes an unusual approach for a survival horror game. Survival horror often generates tension through resource scarcity, limiting the player's ability to fight or heal himself, but because of its melee combat focus Condemned is necessarily limited in this respect. The relatively plentiful checkpoints mean that the player usually isn't punished excessively for failure. Yet Condemned is a frightening, tense game because it artfully keeps the player from knowing what is coming next.

Condemned has a significant advantage when it comes to generating fear because of its visceral combat. While a few guns can be found in almost every level of the game, its hero Ethan Thomas can't carry ammunition for any of them. As a result, he is forced to improvise melee weapons from the environment, and fight his enemies up close and personal. Moreover, he's not fighting fantastic creatures, but ordinary people who have taken on (in most cases) a subtly monstrous aspect. The close quarters and melee brutality make the combat intrinsically frightening, but if that were all that Condemned had going for it, it would not be a truly scary game.

Video games pose a special problem with regard to creating fear because any game is a system that can be mastered. If the designer wishes to evoke a fear response through an attack on the avatar, he must contend with the player's ability to manipulate a game's combat systems. If the player becomes proficient at fighting off the enemies, or lowers the difficulty to compensate for his incompetence, then the onset of a fight stops being frightening and starts becoming a problem to be solved. Similarly, if a cue or activity is known to precede a fight loses its power to develop fear. These moments cease to be tense and instead become a time where the player prepares himself for a battle he knows is coming. Knowing that a fight is about to happen lets the player feel in control of moments where he is meant to feel vulnerable. To be frightening, a game must re-weight the power relationship in favor of the designer, rather than the player.

Condemned presents just this kind of activity in the form of crime scene investigation tools that Ethan must use to track down the serial killer who has framed him. The player often must use the tools in order to find vital clues, but Ethan cannot hold one of these gadgets and a weapon simultaneously. Thus, every use of an evidence-collection tool leaves Ethan vulnerable to attack. In any number of games this would form the basis of a recurring sequence: see clue → collect evidence → get assaulted. The player would enter the state just described, in which starting to collect evidence would signify the onset of a combat sequence and allow the player to feel control. Instead, the developers of Condemned chose to attack the player during the course of evidence collection only occasionally. Sometimes Ethan gets attacked while he collects evidence, and sometimes he does not. The player cannot develop mastery because these sequences have ambiguous outcomes.

Condemned plays with this idea in other ways as well. Environmental sounds sometimes signify an imminent attack, and sometimes do not. In one level, some mannequins turn into enemies and others, often indistinguishable, don't. The levels occasionally rearrange themselves, without regular signifiers. Condemned avoids predictability, and this denies power to the player.

The finest articulation of the skillful design that went into Condemned comes in its penultimate chapter, in which Ethan must search the (apparently) empty house of the serial killer for clues. The killer has written trails of words that lead to important spots, but these can only be seen if the player uses Ethan's UV lamp. The whole house becomes an evidence collection routine, but the developers resist the urge to have enemies burst out of every door and window as Ethan searches. The occasional attacks they employ instead support the tension of the sequence without letting the player view it as a combat routine. This level is so memorable in part because of the exquisite suspense it generates.

The most interesting thing about choosing to attack at only some of these junctures is that it doesn't come at any cost. From studies of conditioning, we know that using a variable schedule of positive or negative reinforcement (i.e. not reinforcing every time) is no less effective at generating a desired response than continuous reinforcement. In fact, research has shown that conditioned responses created using variable reinforcement are more resistant to extinction than those created with continuous reinforcement. By associating the fear of a surprise attack with just some of these evidence-collection routines, the developers guarantee tension in all of them, without granting the player any feeling of power over the game.

Condemned ultimately falls down because of its last level, an uninteresting romp across a farm, beating down mutant hillbillies on your way to a dull and conventional final boss. Up to that point, however, Condemned cleverly steals power from the player by avoiding routine. Because the player can never be certain when an attack is coming, the intrinsic fright generated by the game's brutal combat is never diluted by the player's mastery. The player's uncertainty perpetuates his fear.

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