Mesodynamics, field cycling, and SARS: an explanation

Part of the motivation for my previous post about the spectral density was the recent appearance online (and upcoming appearance in print) of my paper in the Journal of Biomolecular NMR, which is open access, so you can open it up from home and read along as I tell you about it. The obscure-sounding title "Mesodynamics in the SARS nucleocapsid measured by NMR field cycling" means that we were able to characterize an interesting fluctuation in a protein from the SARS coronavirus, and that we used a cool technique to do it.

In the previous post, I mentioned that NMR dynamics studies ought to use data collected at multiple static magnetic field strengths. This is typically accomplished by increasing the strength, because of clear advantages in sensitivity and resolution at high and ultra-high field. Corresponding author Alfred Redfield, however, created a device (left) to capture information about relaxation at lower magnetic field strengths while retaining the advantages of, say, a 500 MHz magnet. This is accomplished by field-cycling, which in this case means physically moving the sample from the center of the magnet's superconducting coil to a spot several centimeters away. If one has carefully measured the magnetic field gradient with respect to distance, one can reproducibly measure relaxation at a desired (lower) field within the bore of the 500 MHz magnet.

As you might surmise from the photograph, Al built the field-cycling device himself, often jury-rigged from whatever parts were convenient. For instance, as you can see at right, the push-rod that connects the sample in the tube to the motor assembly was made from an arrow purchased at a sporting goods store. I'll count myself lucky if I'm half as creative and active in my 50s as Al is in his 70s. Al has used this device to investigate the dynamics of nucleic acids and lipids, but he was interested to see what we could learn about proteins by examining relaxation at low magnetic fields. Relatively low, at any rate — the weakest magnetic field I used is higher than you would typically encounter in, say, a clinical MRI. In his ³¹P research, however, Al has gone near zero field during the relaxation period.

Elan Eisenmesser, now a professor at the University of Colorado Health Sciences Center, did some initial investigations using this technique on cyclophilin A, and edited the pulse sequences so they could control the field-cycling device. Unfortunately, the results in CypA were kind of boring because for that protein the dynamics on the ps-ns timescale are relatively homogeneous. At this time, Elan was also working on the N-terminal domain of the SARS nucleocapsid protein (henceforth SARSN). As you can see from the structure at left (explore it at the PDB), SARSN has a long β-hairpin (sticking out to the right) which is known to be flexible. The hairpin is thought to interact with RNA as part of the viral assembly process, as well as binding to several host proteins during the process of infection. As Elan prepared to move on, he passed the project to me, and with Wladimir Labeikovsky assisting for the first couple of months, I took a bunch of spectra under various conditions.

You can see what a low-field spectrum looks like at right: this is an HSQC from an R₁ experiment where excitation and acquisition were performed at 50.7 MHz (¹⁵N) and the relaxation period took place at ~17 MHz (blue). I've also overlayed a spectrum collected entirely at 50.7 MHz (red). The peaks are all in the same place and the sensitivity is good, but the signal/noise ratio is clearly lower for the 17 MHz spectrum, and we get some sidebands from the water on the right side of the spectrum. Getting the water signal to behave was a significant challenge for these experiments and took several tries to get right.

Besides the experiments I performed personally, spectra were collected by Elan and Geoffrey Armstrong at the Rocky Mountain magnet facility (the 900 MHz R₁ and NOE) and Karl Koshlap at the UNC Pharmacy School (500 MHz data). Karl's involvement was necessitated by a change in sample conditions and the unfortunate incident our spectrometer had with an HDTV channel (chronicled here, here, and here).

In the end I managed to gather relaxation data from four high fields (using standard equipment) and two low fields (using the field cycler). The R₁ data are shown in Figure 2, and if you read the previous post then they shouldn't surprise you very much. For most of the protein, R₁ decreases steeply as the strength of the static magnetic field is increased, but for a subset of amides this field dependence is substantially reduced. Most of these residues fall into a continuous stretch encompassing the β-hairpin of SARSN and an adjacent loop (shown on the structure in Figure 1). In addition, the heteronuclear NOE measurements for these residues show a very large R_NOE/R₁ ratio at 50 MHz that decreases substantially as the field increases (Figure 3). As I discussed in the last post, these patterns of field dependence are characteristic of flexible regions in a protein, but more specifically they indicate flexibility with an internal correlation time of around a nanosecond or so.

One might expect a large, relatively unconstrained feature like the hairpin to have flexibility on multiple timescales. In particular, it seems like the sort of structural element that might move with a time constant of microseconds or milliseconds. These slower motions can't be fit with great accuracy using the experiments performed here, but evidence of their absence can be found in the R₂ experiments performed at 500 and 600 MHz (Fig. 4). Assuming that they are correlated with changes in chemical shift, we would expect motions on this timescale to increase the R₂, but in the hairpin this relaxation rate is substantially reduced, consistent with high flexibility on the nanosecond timescale (low S²).

In order to gain a more complete picture of the dynamics, I fit the relaxation data to model-free formulations of the spectral density. For most residues, the classic Lipari-Szabo formalism worked quite well, although the S² are generally higher than I like. An analysis of the fits, however, indicated that many residues needed to be fit to a more complex model, called extended model-free or model 5. In this model the spectral density is given as:

where S²_f and S²_s are order parameters for a fast and slow internal motion, respectively, and τ_s is the internal correlation time for the slow motion (τ_f is assumed to be ~0). The residues that were fit to this alternative model happened to be those with anomalous R₁ and NOE dispersions, meaning they mostly belonged to the β-hairpin and the loop incorporating residues 60-65.

Ultimately I didn't include the low-field data in the quantitative fits. The large random errors in these rates (error bars in Fig. 2) meant that the more precise high-field data would dominate the fits, for one thing. For another, the low-field data were not entirely consistent with the high-field results. Although the general features of the relaxation at 17 and 30 MHz agree with predictions from high field, the observed low-field R₁ differ substantially from predictions. This could be due to a number of error sources, the two biggest being positioning error and interference between the CSA and dipolar relaxation mechanisms (because we cannot suppress this interference in the fringe field). Al also thinks some of the error may be due to the influence of a low-amplitude fluctuation in the globular portion of SARSN. Qualitatively the R₁ behave much as we would expect, but bringing them into line quantitatively will take more work.

The upshot of all of this effort to fit the dynamics is that the residues in the hairpin have an interesting duality. On very short timescales (< 10 ps or so) they are quite rigid, much like the rest of the protein. On a slightly longer timescale, however, they are very flexible, with S²_s of around 0.6, and similar internal correlation times across the entire feature in the range of 600-800 ps (Fig. 5). Because the correlation time of this fluctuation is significantly faster than molecular tumbling but much slower than typical backbone fluctuations, Al called them "mesodynamic", a word Dorothee seems to like. At any rate, these observations led us to propose that the hairpin fluctuates widely (based on S²_s and τ_s) as a coherent structural unit (based on S²_f), rather than having its strands fall apart and flop around randomly. The hairpin is both ordered and disordered, depending on the timescale of analysis and frame of reference.

The physical plausibility of this dynamic model was assessed using a pair of 15 ns all-atom molecular dynamics simulations performed by Ming Lei. What these found, shown in Fig. 7, was that the hairpin maintained its internal structure while moving freely with respect to the globular portion of the protein. In addition, the simulations suggested a reason the 60-65 loop had similar dynamics to the hairpin — transient hydrogen bonds formed between side chains in the hairpin and residues in the loop, causing their motions to be correlated.

The qualitative agreement between the low-field and high-field data supports our contention that this technique can be made to work and to give valuable data about certain kinds of fluctuations. Future work on proteins with this technique will require a rigorous approach to control for the systematic bias we observed. Additionally, this study re-emphasizes the value of taking relaxation data at many fields in order to fully characterize biomolecular dynamics.

As for the dynamics of SARSN, the finding is interesting but doesn't yet provide any specific insight. Disordered regions of a protein are often associated with promiscuous binding activity, and this hairpin is no exception. However, the existence of multiple binding sites in one of these regions is usually attributed to a significant ability to restructure itself. Here, that possibility would seem to be limited by the apparent persistence of the hairpin's intrinsic structure. The ability of the hairpin to move freely while maintaining a particular internal arrangement may have advantages in capsid construction, an idea that could potentially be tested by inserting prolines or glycines in the β-strands, which should disrupt the hydrogen bonding that preserves the hairpin.

Al and his collaborator Mary Roberts are currently continuing their investigations of ³¹P dynamics in nucleic acids and lipids using low field. They're even advertising:


Clarkson, M., Lei, M., Eisenmesser, E., Labeikovsky, W., Redfield, A., & Kern, D. (2009). Mesodynamics in the SARS nucleocapsid measured by NMR field cycling Journal of Biomolecular NMR DOI: 10.1007/s10858-009-9347-6 OPEN ACCESS

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Let's explore the spectral density!

The model-free formalism of Lipari and Szabo is a way to convert experimental NMR data into a limited number of generalized parameters describing the internal dynamics of a protein. However, the relaxation rates that are typically measured by NMR — the R₁, the R₂, and the steady-state nuclear Overhauser effect (nOe) — do not themselves appear in the model-free formulas. Instead we see a term, J(ω), and this constitutes the interface between the data and the model. This term refers to the spectral density, which is a measure of the power available to relax spins at a given angular frequency. The relaxation rates measured by NMR spectroscopists interrogate this density at known frequencies, which means that we can use those rates to assess general information about the shape of the spectral density function and thus constrain the model-free parameters.

In biomolecular NMR, these rates are most frequently measured on the nitrogen of a backbone amide group, in which case they fundamentally depend on the spectral density at three frequencies: 0, the Larmor frequency of nitrogen (ω_N), and the larmor frequency of the proton (ω_H). The precise relationships are as follows:

R₁ = D [3J(ω_N) + 6J(ω_N+ω_H) + J(ω_N-ω_H)] + C [3J(ω_N)]
R₂ = D/2 [4J(0) + 3J(ω_N) + 6J(ω_H)+ 6J(ω_N+ω_H) + J(ω_N-ω_H)] + C/6[J(0) + 3J(ω_N)]
steady-state nOe = 1 + R_NOE γ_H / R₁ γ_N
R_NOE = D [6J(ω_N+ω_H) - J(ω_N-ω_H)]
D = μ₀²ℏ²γ_N²γ_H²/64π²r_NH⁶
C = Δσ²ω_N²/3

where γ_H and γ_N are the gyromagnetic ratios of these nuclei, ℏ is the reduced Planck constant, and μ₀ is the magnetic constant (or vacuum permeability, if you prefer), and Δσ is the chemical shift anisotropy of the ¹⁵N nucleus (typically -160 - -170ppm).

I'm not going to cover precisely why they have these relationships today; instead I want to focus on how these relationships connect certain dynamic behaviors to particular observations about relaxation rates. The key to this is to think about how the spectral density looks. At right I have a simplified spectral density calculated for a rigid protein of reasonable NMR size (I only show the positive side of the function, the negative is a mirror image). While the particular shape of the spectral density function will depend strongly on the internal dynamics and overall size, certain general features will be the same for most proteins. It should be immediately evident, for instance, that J(0) >> J(ω_N) >> J(ω_H) (shown on the figure for a 500 MHz magnet). This implies that each relaxation rate reports on just one spot in the spectral density. R₂ should be proportional to J(0), R₁ to J(ω_N), and R_NOE to J(ω_H), keeping in mind that ω_H >> ω_N.

The shape of this curve derives in a fairly obvious way from the Lorentzian used to calculate it, in this case the Lipari-Szabo formalism, which if you'll recall is:


Where τ_m is the time it takes the protein to tumble through one radian in solution, S² is the order parameter for the bond in question, and τ_e is the correlation time of internal motions. The Lipari-Szabo model is not the only model of the spectral density, but most of the alternatives just add more Lorentzians or scaling factors. These models differ in the fine structure of the spectral density, but the overall shape (and the features I'm about to describe) is generally not affected.

It should be clear from examining this (and given that τ_m >> τ_e) that the point where ωτ_m = 1 divides the spectral density into two regions. Where ωτ_m <= 1, the first term dominates, and the spectral density is determined by S² and τ_m. Where ωτ_m >> 1, the second term dominates and the spectral density is essentially dependent on (1-S²) and τ_e. This being the case, you would expect highly flexible moieties (low S²) to have inefficient R₂ and R₁ relaxation and highly efficient NOE relaxation, and this we generally find to be the case.

Similarly, you would predict that increasing τ_m would cause R₂ to increase. The graph at right simulates relaxation rates for a typical, rigid backbone amide nitrogen (at 500 MHz) as the τ_m increases (note log scale on x). As you can see, the R₂ (red) does in fact get continuously higher as τ_m is increased; this is one of the reasons NMR spectroscopy of very large molecules is so difficult. Also note that R₁ (blue) goes through a maximum and then declines. This is because as τ_m increases, the point where ωτ_m = 1 shifts to lower and lower frequency. When |ω_N| > 1/τ_m, the spectral density at ω_N starts to fall off, reducing R₁. This might sound advantageous, but in fact it is another reason that spectroscopy on large molecules is difficult — their inefficient R₁ relaxation means that additional time must be scheduled after each transient to create a sufficiently sensitive steady state. Because even a simple spectrum can have 2048 transients, adding just a few fractions of a second per transient can rapidly amount to a significant increase in experiment time.

It's obvious that it would be questionable to map the spectral density based on just three relaxation rates, if for no other reason than that we have four unknowns and three pieces of data. This is typically addressed in three ways, which are often used in combination. The first is to reduce the spectral density, by making some general assumptions about the nature of the spectral density around ω_H and collapsing the J(ω_N +/- ω_H) terms into 0.87*J(ω_H). Another approach is to increase the number of relaxation rates measured, by incorporating R_1zz or other measurements, but many of these rates incorporate additional factors (such as ρ_HH) that must also be fit, so that their ability to reduce the dimensionality of the problem is sometimes limited.

The third approach is to take data at several fields. The Larmor frequencies ω_H and ω_N depend on the strength of the magnetic field in the spectrometer, while J(0) is obviously field-independent. As a result, each additional field of data taken improves the ratio between data and unknowns. This improvement is valuable even when the relaxation is being fit to a simplified representation such as the model-free formalism, and therefore dynamics experiments should always include measurements at more than one field if at all possible. Moreover, the field-dependence of relaxation rates can be very informative, in general terms, about the dynamics of the system.

In the simplified view it might seem that R₂ should be essentially independent of field strength, but observations show this not to be the case. R₂ increases at high fields primarily because of the chemical shift anisotropy contribution, which has a square field dependence and therefore increases with field to a greater degree than ω_N declines. As a result, R₂ has a sort of chevron appearance as you vary the field, with differences in dynamics primarily affecting the magnitude rather than the shape. This means that for R₂ the field-dependence is not particularly informative about the dynamics. However, if a residue has anomalous R₂ field-dependence with respect to the rest of the protein, this can be an indicator of a chemical exchange process on the μs - ms timescale.

Because relaxation due to chemical shift anisotropy makes a lesser contribution to R₁ (and depends entirely on J(ω_N) for this rate) the behavior of R₁ with respect to field is generally much simpler — for proteins, the R₁ almost always decreases as field increases. The degree to which this occurs, however, can be quite different depending on the dynamics behavior that is going on. The reason for that can be seen in the sample spectral densities to the left, calculated for a typical backbone amide (blue) and a flexible one (red). As you can see, the more flexible residue has a lower J(0) and a smaller slope between the flat portions of the spectral density than the rigid one. This means that the R₁ will be lower at high field and higher at low field, decreasing the field-dependence of the residue's relaxation. The exact magnetic field where this crossover occurs depends on the correlation times of the internal motion and global tumbling.

The gyromagnetic ratios of the hydrogen and nitrogen nuclei have opposite signs, so the heteronuclear NOE measured for these nuclei should be less than one. How much less depends on the relative ratio between J(ω_H) and J(ω_N). For flexible residues, the spectral density at large ω will be high (and that at lower ω will be low), this ratio will be large, and a low value will be measured in the hetNOE experiment. R_NOE typically has a steep field-dependence for flexible residues, and because this rate dominates the ratio, one tends to see greater field-dependence of the hetNOE for flexible residues. However, the situation for the hetNOE is more complex than for the other two rates because the spectral density around ω_H defines the relaxation. As a result, the internal correlation time (particularly if it's on the order of 100 ps - 1 ns) starts to dictate the shape of the spectral density, and hence the magnetic field-dependence of relaxation. For certain τ_e, the hetNOE will have no apparent field dependence, whether the residue is flexible or not.

Actually parameterizing the dynamics of a given group requires numerical fitting of the relaxation data, but for many questions a qualitative estimate will suffice. In these cases just examining the field-dependence of one or two relaxation rates (especially R₁ or NOE) can provide valuable insight into the heterogeneous dynamics of a given protein. In the next post I'll describe an example of a case in which this turns out to be true.

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Capsule: Lost Odyssey

Final Status: ~3/4 complete, and that's it for me

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Most intriguing idea: Subverting some boss battles into a game of protecting your putative opponent.

Best design decision: The ring system alleviates many of the attentional problems that come with the limited interactivity of round-based combat.

Worst design decision: The irritatingly high proportion of idiot mazes in the dungeons.

Summary: Lost Odyssey is a fairly interesting tale about an immortal, amnesiac (ugh), vaguely effeminate (hruagh) man named Kaim who is trying to find his family and prevent a cruel, selfish sorcerer from conquering the other cruel, selfish rulers of the world. The game has several good ideas built in it — in addition to those mentioned above, I liked its skill system and the lightly-interactive text stories that reveal Kaim's life story. The score was also pleasant. The key issue is that the flow of the game is constantly broken up by loading screens and long battle introductions. This is a particular problem on the third disc, where the party is split up into 3-4 groups and long pauses for loading seem to occur every 30 seconds. The delays get to be quite frustrating, especially in timed sequences where the countdown continues as you engage in a meaningless survey of the completely non-interactive battle space. Penalizing the player for the developer's delays is kind of insulting, an emotion accentuated by idiot mazes such as the Prototype Staff, where shallow cart tracks somehow become an impenetrable barrier blocking Kaim's path. Eventually I got to a point where I just wasn't willing to put up with the crap anymore.

If you can't say something nice... Lost Odyssey shows off a number of really sharp decisions, which might have made it a classic if someone had come up with a way to cut out the dead time.

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An unhappy medium

Given the ongoing struggles against insurgents in Iraq and Afghanistan, it's perhaps a bit shocking that Red Faction: Guerilla ever got made. Although his cause embodies a different philosophy, the game's ironically-named hero Alec Mason adopts the same tactics and general approach as the Taliban and Al-Qaeda in Iraq. While the game rigorously penalizes the death of almost every civilian, Mason uses hit-and-run attacks, exploding vehicles, and ambushes to push an occupying force off Mars. The game certainly doesn't lack for fun, but it's inappropriately ambivalent about engaging its subject matter. Red Faction's weakly-constructed fiction is too flimsy to hold up a serious consideration of real guerilla war, but too strong and topical to dismiss those implications.

The weaknesses of the game's fiction become apparent very early on. Although there are relatively few housing facilities on this version of Mars, and these are fairly small, the surface is crawling with vehicles. They drive aimlessly around the various zones, never really seeming to arrive at or depart from anywhere. The many cargo vehicles are all empty, which is hardly a surprise given that so much ore is left just lying around the ground, in plain view of the road. The industrial facilities seem to have been imagined by somebody who saw a factory one time on TV; they're full of buildings that largely lack machinery, and smokestacks with no origin for the smoke. Some buildings that would certainly be critical for any real colony on Mars and would make natural points of emphasis for an occupying force — hydroponics, water purification — seem to be totally absent. Instead we have refineries (refining what?) and countless tanks of indeterminate, highly-explosive gas. Mining operations appear to be confined to a single pit in the Dust zone, clearly explaining why Mars cannot meet Earth's demand for metal.

The occupying army, the EDF, wants to improve productivity but apparently learned labor relations from George Pullman. Rather than squeezing efficiency by increasing automation or improving equipment (or possibly opening up another mine) they have chosen the tactic of brutally repressing and murdering the colonists they shipped across the solar system at enormous expense. Morally ambivalent approaches such as using their vast army as cheap labor or importing workers who are willing to tolerate worse treatment apparently did not occur to them. For their part, the Martians react not by going on strike (fair enough, since the EDF would shoot them), but by reforming their freedom-fighters, the Red Faction, and blowing up the irreplaceable equipment that is their only source of wealth and leverage.

Even though all of this seems like a set of caricatures, part of an easily discarded entertainment, Red Faction keeps pulling towards a more intelligent take. The terraforming effort uses reasonable tools (such as the mohole), and has made only slow progress, rather than turning Mars into a tropical paradise. The collusion between industry titans and the twisting of events by propaganda outlets is all believably presented. Red Faction doesn't feature some absurd sci-fi sexpot — instead, we get Samanya, a rarity among female video game characters in that she is smart, strong, and plausibly dressed. Many of the main missions feel quite believably like the actions of a small band of fighters taking on an overwhelming force, including a sharp and harrowing sequence in which we hear a woman who was mutilated by EDF soldiers torture one of her abusers nearly to death.

Yet, though many of the missions individually feel like plausible guerilla actions, this view seems to fall apart when they're considered as a whole. The player gets no sense of strategy or a larger war — instead, the missions (especially the seemingly incessant emergencies) give the player the impression that Mason is a one-man army fighting the EDF. Although "guerillas" often show up when you need a hand, nobody ever attacks a convoy or hijacks a truck without your assistance, this despite the EDF's tactic of regularly sending lightly-protected, high-value convoys to destroyed bases in territories it no longer controls. When the EDF abandons one of the Mars zones, it's rarely clear why; their "control" level can often be reduced to at or near zero with very little damage to their military facilities. Moreover, the "control" level for a given territory doesn't seem to have any impact on EDF behavior until the final mission is won. It's a binary system disguised as a continuous gradient.

The control in a zone is reduced by carrying out missions or destroying particular buildings. While many of these are military facilities, a nearly equal number are economic in nature. Given the narrative of plutocratic collusion with a corrupt occupying force, it's natural that power plants and industrial sites are protected by EDF troops. However, the people of Mars will eventually need those facilities, too. Red Faction rewards wanton destruction and mayhem without consideration for the long-term economic effects. The Red Faction wants a free Mars, but surely it desires a free Mars where everyone can make a living.

In a tactical sense the game also falters. One of the keys to guerilla warfare is to keep the enemy off-balance and out of position, but the game's conventionality removes this tool from the arsenal. EDF soldiers and vehicles spawn in rather obvious fashion, without changing their distributions elsewhere in the zone. Moreover, they arrive near-instantaneously, whether you're at the Martian capital or in the furthest corner of the Badlands. While this prompt arrival has the beneficial effect of encouraging classic guerilla hit-and-run tactics, you never get the sense that you're fighting a real army, one that can only get men and materiel into position by taking them from somewhere else and sending them along a real route of attack. The EDF clearly has an inexhaustible supply of soldiers and weapons, a concept that's difficult to swallow given the obvious sparseness of human settlement, and one that's inconsistent with the idea that the Red Faction could 'control' any region of Mars at all.

More troubling than its habit of glossing over strategy and tactics, the game generally refuses to philosophically engage the ugly side of insurgency. When "collaborators" are targeted, they are always plutocrats or crooked politicians, never ordinary Joes who go along with the EDF out of necessity, apathy, or fear. EDF-controlled industrial facilities have plenty of soldiers on guard, but apparently no civilians to run them who might get hurt in the blast. The Red Faction safehouses are loaded with explosives, but these never go off prematurely and kill innocents while they're being smuggled. Even though the Red Faction's leader, Hugo Davies, has a fanatic's devotion to kicking the EDF off Mars, the game never acknowledges the danger he poses to civilians. For the most part it is only the EDF's response to the Red Faction that harms Martians, not the deeds of the Faction itself. When the war moves into urban areas the risk to civilians stays very low, even though we all know that's not how insurgency really works. Nor does the game ever wrestle with the possibility of a lenient or even friendly EDF commander: would the Red Faction try to subvert such a man, or assassinate him to prevent a reconciliation?

Perhaps a more serious, considered attitude towards this subject matter is too much to expect from Volition, the makers of the notoriously raucous Saint's Row games. Red Faction treads a fine and uncomfortable line, however — realistic enough that it seems like it wants to be taken seriously, but too conventional and simplistic to live up to that promise. Because of its thinly-constructed fiction and shallow take on insurgency, Red Faction degenerates into little more than a tale of mayhem on Mars. Thanks to the engaging gameplay, that results in a compelling hoo-rah entertainment built around an innately troubling and relevant idea, which is less than that idea, or the gaming public, deserves.

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Capsule: Flower, Sun, and Rain

I finish, or get finished with, more games than I write long essays about here or elsewhere. Sometimes I'm just not inspired to write a detailed critique, other times I'm just not inspired at all. This being the internet, and me being a blogger, however, I thought I would try to put together a short, snappy format for conveying my thoughts on the games I'm done with.

Title: Flower, Sun and Rain

Final Status: Finished

Put this on your box: This game is some tedious, boring bullshit.

Most Intriguing Idea: By using the guidebook as the key to almost every puzzle, the game includes its own little GameFAQs.

Best Design Decision: This game contains no good design decisions.

Worst Design Decision: The walking. My god, the unending walking.

Summary: Flower, Sun and Rain is an adventure-style mystery game in which you walk and walk and walk and walk and walk and solve puzzles using an in-game guidebook and your brain, in the rare cases where your brain is of any help. In typical Suda fashion, it is about a weird secret conspiracy which has cleverly been hidden behind something even more bizarre, namely an island on which a single day seems to repeat. The characters speak in riddles and are generally insufferable asses, and the plot is obscure. The graphics, with the exception of the face portraits, are easily the worst I've seen on the DS. The game seems to be rather transparently commenting on how stupid adventure games are (unnecessary), how stupid the main character and this particular game are (uninteresting), and how stupid the player is for putting up with all of it (unwise).

If you can't say something nice... Fortunately, this game did not end Suda 51's career or destroy Grasshopper Manufacture.

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Alpha Centauri's narrative tension

I still remember reading the PC Gamer issue in which Sid Meier's Alpha Centauri received the highest rating that magazine had ever given, in which it was implied that this might be the greatest PC game of all time. An examination of subsequent "best of" lists shows that their prediction or their memories failed, but it's worth asking what about the game so captivated its players. I think that in part — and this is something you would not expect to say for a game of this kind — this was due to the story. Other games in this genre, including subsequent entries in its sibling Civilization series, have no greater narrative arc than a grand national romance. Alpha Centauri rises above this tradition with truly differentiated factions that imbue the different societies with real character, an explicit historical story, and a implicit but definite narrative embodied by its technology tree. When these elements work together, this interwoven narrative stands as one of the game's unique strengths. Yet these different influences can also create an unhappy tension with one another.

Alpha Centauri has an advantage over its cousins, the Civilization games, in that the different playable factions represent ideologies rather than nations. As Alex Galloway discusses in Gaming, the "civilizations" don't represent anything more than stand-ins for particular assemblies of AI traits. Unique units aside, the different "civilizations" have few differences, for gameplay reasons and because most real civilizations have adopted very similar patterns of success. In Alpha Centauri this is true to a certain extent, but by using each faction to represent an ideology, the distinction can be taken much farther.

The factions have material differences between them from the get-go, strengths and weaknesses that encourage divergent modes of play. The ecological handiness of the Gaians rewards a priority on interacting with Planet, but hinders population growth. The academic prowess of the University encourages technology-first gameplay, but forces the player to work diligently to protect his secrets from being stolen. While most of the game's "Secret Projects" can benefit any faction or style of play, certain ones become crucial for particular factions — denying them the ownership of these projects can be every bit as strategically important as obtaining the benefits for yourself. In this way each faction truly develops its own character, assisted in this by the recurrent voices of the faction leaders, whose writings and sayings provide context for many of the discoveries and projects.

These "characters" enter an overarching narrative of a fractured society landing on a new world (called "Chiron" or "Planet") already inhabited by a semi-sentient collective intelligence. The various factions are competing with one another for territory and resources, and with the alien life form for dominance of planet. Relationships between factions remain mostly antagonistic, but the game imposes an arc on the interaction between the player and the lifeforms of Planet. Certain events are triggered by the player to make an explicit arc, while the game's technology tree enforces its own narrative of cooperation. Scientific advancement relies on developing a further understanding of Planet, which in turn allows the cultivation of its lifeforms to produce the food, energy, and minerals necessary to survive or dominate. The apex of the scientific approach to victory is the fusing of human consciousness with the collective mind of Planet.

The problem is that this doesn't seem like an outcome that would sit well with every faction, and would appeal to some for very different reasons. The Gaians would happily join with Planet because this idea lies at the core of their civilization anyway. The collectivist Hive would also appreciate the idea, but Chairman Yang would mostly enjoy the power it gave him over all life. Surely a collective consciousness would be anathema to the capitalists of Morgan Enterprises or the Survivalists — though for these factions one could just choose to pursue the economic domination or world conquest victory conditions. That would not escape the fact that these societies are forced to pursue scientific goals at odds with their philosophies along the way.

And what of the Lord's Believers? To say nothing of the theological difficulty involved in acknowledging the existence of an intelligent lifeform not contemplated in scripture (and this would be socially important), the pursuit of immortality by giving up one's soul to an alien creature must surely be horrific to them. If you assume their ideals are honestly held (and in all fairness, the game does not), conquering their neighbors by the sword or filthy lucre would be just as bad.

Of course, the tech tree holds just as much offense for the Gaians as it does for the Believers — surely a bunch of tree-hugging environmentalists would hate to develop synthetic fossil fuels and pollute a whole new planet! And why should they? This is, after all, science fiction... instead of supercomputers, why not let the Gaians learn how to engineer Planet's xenofungus into a problem-solving neural net? Instead of learning to build tanks, why couldn't they biologically engineer armored monsters from Planet's mindworms and locusts? Of course, allowing truly varied alternative technology trees one creates a more difficult problem in terms of balancing, especially if some of the branches are mutually exclusive. However, Alpha Centauri would have benefited, in a narrative sense, from allowing tech trees that were more friendly or more inimical to Planet. An alternate endgame (perhaps one involving a new colonization effort?) might have been of benefit also.

This, then, is the respect in which Alpha Centauri's narrative falters. The technology tree creates an implicit story for every faction, and the problem with this is that it is the same story, for societies that are radically different. A more flexible and varied technology tree, with defined, exclusive routes towards more varied endgames and units, would have benefited the game by allowing each of its "characters" a destiny that fit, rather than contradicted, their principles.

Note: I played Alpha Centauri as part of the fun at Vintage Game Club. We're starting our next game, Majora's Mask (available via Ebay for N64 and Gamecube, and through Virtual Console on Wii), next Friday. If you're interested, we'd love to have you join in the discussion.

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How do adamantane drugs block M2?

Vaccination plays such an important role in our seasonal influenza strategy in part because we don't have many medicines that can be brought to bear on the disease. The neuraminidase inhibitors (specifically Tamiflu) are widely stockpiled, and continue to work for now, but the specter of resistance is already lurking. If these drugs are too widely or too improperly used, there is a good chance that resistance mutations will eventually render these drugs ineffective. Universal drug resistance may already be the fate of the drugs amantadine and rimantadine, built on an adamantane backbone (1). The adamantane drugs inhibit the M2 proton channel from influenza A, a tiny tetrameric protein that equalizes pH between the virus and the endosome of the cell that has swallowed it. This process releases the virus contents so that they can do their damage to the cell, so these medicines can significantly retard the infection process. Or rather, they could, if so many influenza strains didn't harbor the S31N mutation that almost completely nullifies their effect. If we are to develop new drugs to attack the M2 channel, it would be helpful to know how this mutation causes drug resistance. Over the past few years a great deal of structural evidence has accumulated showing how adamantane drugs work on the older, non-resistant channels. The problem is that the evidence supports two different models of M2 inhibition, and so far it has proven difficult to determine which of them is probably correct.

How the question arose

The controversy is the result of two structures published in Nature early in 2008 (2,3). The first of these is a crystal structure of a tetramer of peptides encompassing the transmembrane (TM) region of the M2 channel reported by the DeGrado group at UPenn, which you can see at right (explore this structure at the PDB, noting that the numbering is off by 21). In the detergent used for crystallization, the peptides form a tetramer with a roughly conical pore, which amantadine (purple in these models) physically occludes, giving rise to the pore-blocking model (PBM). This model is consistent with previous results indicating that a single amantadine molecule is sufficient to inhibit the proton channel. In addition, in this model the drug binding site is adjacent to S31 (blue side chain), which is what we'd expect given that an S31N mutation is responsible for most amantadine resistance. The authors propose, given the position of the S31 side chain, that the mutant asparagines form a hydrogen-bonded network that is too constricted for amantadine to bind. Click on the picture for a larger view.

An alternative model was proposed by Schnell and Chou from Harvard University (3). They produced an NMR structure (left) of a 42 amino-acid peptide from M2 encompassing the TM region and an additional C-terminal helix (explore this structure at the PDB). In their structure, taken at pH 7.5 in detergent micelles, the tetramer forms a roughly cylindrical pore that is blocked by the side chains of the known gating residues W41 and H37 (light green in these models). Their structure shows rimantadine bound at four sites near the base of the helix but not in the pore. Using pH-dependent conformational exchange experiments, Schnell and Chou showed that a decrease in pH caused rapid structural changes in the channel, motions that rimantadine slowed. On the basis of this evidence, they proposed a mechanism in which protonation of the gating histidines destabilizes the packing of the TM helices and allows the conductance of protons. Rimantadine blocks the channel by stabilizing the helices, thus this is a dynamic quenching model (DQM). The position of S31 in this model is also somewhat different than the crystal structure, although these models were made at different pH conditions and so this may represent a difference between the closed and open states of the channel.

The distinction here is important. If Stouffer et al. are correct, then drug development should abandon the adamantane backbone altogether and start with a set of significantly different leads to address the resistance problem. The PBM implies that any molecule large enough to occlude the pore will be too large to fit in there following the S31N mutation that induces amantadine resistance. If the DQM is correct, however, then it is conceivable that further refinements to the adamantane base, or similar molecules, could improve affinity enough to overwhelm the mutational effect.

Unfortunately, neither result is unimpeachable. Although it agrees with a great deal of experimental evidence, the low resolution of the crystal structure means that the electron density called amantadine cannot be assigned unambiguously. It is also curious that a hydrophobic molecule like amantadine would bind tightly in the hydrophilic pore. In addition, the crystal form with amantadine bound contains a mutation, G34A (black side chain), which is near the drug binding site and could conceivably have altered the binding specificity of the protein.

The NMR structure has the advantage that it directly includes distance information in the form of NOEs. However, the authors used 40 mM rimantadine to obtain these results, meaning that there were as many rimantadine molecules in the solution as phosphate buffer molecules. Under these conditions, it is possible that the drug bound to a secondary, low-affinity site. Even if this is what happened, it is strange that the rimantadine never bound to the high-affinity site indicated by the crystal structure.

Both experiments use significantly truncated constructs and highly artificial systems to mimic a membrane environment. The structure of any membrane protein depends in often unexpected ways on the composition of the lipid bilayer in which it is embedded and on the structure of that bilayer. The intense curvature of the micelles may have distorted the structure in the NMR experiment, and possibly inappropriate lipids may have had effects on both structures. We know these considerations are relevant for this system, because Schnell and Chou report that the construct used for the crystal structure would not form stable tetramers in the micelle system. Also, as Chris Miller notes in his commentary on these papers (4), there were questions about both constructs with respect to their proton conductivity. Lacking significant stretches of the protein and placed in these environments, it is possible that both structures deviate from in vivo reality in significant ways.

Because the conditions diverge so much, it is difficult to weigh the mechanisms based on these structures alone. The binding site identified by Schnell and Chou is only at the very end of the construct used by Stouffer et al.. In addition, the inhibited crystal structure comes from a low-pH condition while the NMR structure exclusively represents a high-pH condition. Given these differences in conditions, it is not impossible that both models, in whole or in part, are correct. We must turn to additional experiments and alternative evidence to choose between them, specifically data on the stoichiometry of binding and the effects of mutations.

Binding stoichiometry

The crystal structure shows a single binding site for the drug, while the NMR structure implies four, and this is at odds with existing results that indicate that a single molecule of drug is sufficient to inhibit a single channel. Given the homotetrameric nature of the M2 channel, it is in principle not possible for the NMR experiment to distinguish between a single rimantadine binding event and four. That is, the NMR experiment cannot tell us whether the rimantadine-M2 inhibition occurs with a single binding event or requires four drug molecules to bind. Therefore, to argue that DQM is inconsistent with 1:1 stoichiometry overstates the case somewhat.

It may also be somewhat overstating the case to say that there is only one amantadine binding site on M2. Washing amantadine out of your buffer does not reverse inhibition, in part because of slow kinetics of leaving the binding site and in part because these drugs, being very greasy, preferentially partition into the lipid membranes and are therefore not readily removed from a system when its aqueous phase is replaced. It is difficult to measure a binding constant for the drugs because the equilibria under consideration will be quite complex. The studies often cited on the 1:1 stoichiometry (5,6) use structural and kinetic evidence to get at this question.

Czabotar et al. (5) measured tryptophan fluorescence in M2 as a function of pH and rimantadine concentration. They found that fluorescence from W41 was quenched by decreased pH, but recovered when 1 equivalent rimantadine per tetramer was added. This result implies that structural or dynamic changes caused by histidine protonation are reversed by rimantadine inhibition, but this is so general that it cannot be taken to support either the PBM or DQM.

Wang et al. (6) measured the reduction of surface currents in X. laevis oocytes after addition of various concentrations of amantadine. From these results they are able to construct a Hill plot with a coefficient of 1, showing that binding of amantadine is not cooperative. In further results, Wang et al. find that amantadine inhibits M2 channels slightly better at high pH (when the pore is closed) than at low pH, and that amantadine inhibits proton conductance in either direction (rather than favoring one). Both these outcomes are unexpected for PBM, but can be easily explained by DQM. However, the differences in the binding constants are relatively minor and the linearity of the current-voltage relationship may result from some other idiosyncratic feature of the M2 channel, so these results are not unequivocal.

Neither experiment refutes DQM because they do not measure the number of binding sites, but rather the number of efficacious binding sites. If there are four binding sites, but 95% or more of the inhibitory or structural effect is caused by the first drug molecule bound, then these experiments would be unable to distinguish DQM from PBM. Overall, the evidence on the question of binding stoichiometry does not eliminate the possibility of four binding sites existing, but it does place a requirement on DQM that the inhibitory effect of amantadine on the tetramer result from a single binding event. Because the proposed DQM binding site for rimantidine lies between monomers and is linked to the gating tryptophan, this is not unbelievable. Other evidence from these experiments is equivocal, but can be seen as somewhat more problematic for PBM than DQM.

Functional effects of mutations

A serious problem for DQM is that the mutations known to give M2 resistance to adamantane drugs are all located near the PBM binding site. In particular, S31 is adjacent to the drug in the crystal structure and quite distant in the NMR structure. As Miller notes in his commentary, mutational studies are substantially more difficult to interpret than is typically suggested, so this isn't absolutely probative. In general, however, one predicts mutations to have short-range rather than long-range effects, so at least some resistance mutations ought to evolve at the binding site. However, many of the residues surrounding the DQM site are almost absolutely conserved, presumably because they are essential to the function of the channel. As a result, it would be very difficult to interpret any studies on point mutants in this area. What would be ideal, however, would be to find a set of mutations that produced a functional protein and abrogated amantadine inhibition.

This is the basis for an interesting experiment conducted by the lab of Robert Lamb and reported last year in PNAS (7). In this case, the authors took advantage of the fact that the M2 protein from influenza B virus is not sensitive to adamantane drugs. They constructed a chimeric protein containing about a dozen residues from influenza A M2 — specifically, the dozen or so residues surrounding the PBM site. If PBM is correct, then we would expect that these residues, which define that site, would impart amantadine susceptibility to the influenza B channel. This is what happens, sort of. For your benefit, I have shamelessly stolen their figure (right), but you can check out this paper yourself because it is open access. In this assay, again involving X. laevis oocytes, the hybrid channel is sensitive to amantadine (bottom trace), but only half as sensitive as the wild-type influenza A channel (second from top). This result suggests that there is important context conferring susceptibility outside the PBM site. However, this could be something as simple as helix orientation, so the result does not necessarily imply that there is an external binding site.

Additionally, the authors made point mutations at residues (L38, D44, and R45) that were presumed to be important in the DQM mechanism or have long-range effects on amantadine binding. None of these mutations appeared to affect amantadine resistance. In contrast, experiments in liposomes reported by the Chou group this May showed that a D44A mutation prevented rimantidine from having an effect (8). This conflict in results is difficult to reconcile, but may result from the different constructs used (the Chou group used a truncated form of M2 while the Lamb group used the full-length protein) or from changes in ion specificity caused by the D44A mutation. It might be of value to repeat these experiments with the alternative construct: truncated in oocytes, full-length in liposomes. Because the D44A mutant does not appear to conduct protons as efficiently as WT, the proposition that the function of this mutant is too deranged to provide trustworthy information should also be considered.

Additional experiments in the Chou paper are meant to address the relationship between the DQM site and the mutations at the PBM site. They show that the S31N mutation prevents rimantidine binding to the remote site, and also that this mutation makes the protein generally more dynamic. From this evidence they propose that this mutation, at least, disrupts amantadine binding by destabilizing the helical packing of the channel and thus interfering with the organization of the lipid-facing pocket.

They also examine an S31A mutation and find that it is not rimantadine-resistant or destabilizing to the packing. This supports their dynamic model in a limited way, because it demonstrates that only certain mutations at the S31 site will generate resistance. It does not cast any doubt on PBM, however, because in that model resistance in the S31N mutant is explained by the idea that its side chain will partially obstruct the pore so that rimantadine will not fit. I do not think it was ever proposed that specific contacts between S31 and the drug stabilize the binding; in fact, the general absence of such contacts strikes me as a concern about PBM.

Chou et al. also examine the effect of rimantadine on the shorter construct used for the crystal studies. They find that the inhibition of this construct is substantially weaker. However, it also conducts protons at a much slower rate in this assay, suggesting that there may be additional serious problems with the function of this construct. It may be that it simply is not appropriate to use this construct for studies in solution or living membranes. That doesn't necessarily imply that this peptide will give incorrect information in the stabilizing environment of a crystal.

What do we know, and what do we need?

Very little of this evidence unequivocally prefers one model to the other. We know that a single adamantane molecule is sufficient to inhibit M2, and while this is most obviously compatible with PBM it need not be inconsistent with DQM. It is also apparent that various constructs of the M2 channel retain adamantane susceptibility after ablation of the DQM site, either by truncation, mutation, or the construction of a chimeric protein. In all assays, however, the adamantane drugs lose a considerable amount of inhibitory power, so these results are not entirely consistent with PBM either. And, at least in the Chou lab's assays, interference with the DQM site also reduces adamantane susceptibility and deranges function. Moreover, the NMR data from the Chou lab shows that mutations at PBM site have a long range effect on the DQM site, which mitigates the probative power of the S31N mutation.

How do we address this question? One important step would be to start comparing like to like. We are considering evidence from a plethora of constructs and conditions, and the evidence in conflict is often collected in very divergent experiments. Ideally we would like to have structures of the wild-type channel at low and high pH in a lipid environment that closely mimics the composition and curvature of a mature influenza virion. As this is unlikely in the near term, we must hope for NMR and crystal structures that at least use the same construct, minimally mutated, under similar conditions. NMR studies at low pH would be of value in assessing whether these studies in fact contradict one another. Additionally, it would be useful to make adamantane derivatives labeled with a free radical or other paramagnetic label; this would presumably allow the identification of a binding site at lower drug concentrations in an NMR experiment. Labeling the drug with a metal might also allow its identification in a crystal structure without any need to push the resolution significantly higher. Finally, actual structures of the S31N mutant, positively identifying the disposition of this side chain, would be of great value in judging the question.

Structural experiments can take a great deal of time and careful tuning, a requirement exacerbated by the often-fickle behavior of membrane proteins. As such, additional mutational studies could prove useful. Inverting the chimera experiment of Jing et al. to create a chimeric protein with the upper channel from influenza B and the lower channel from influenza A may be a helpful supplement to the existing experiments. If the C-terminal portion of the channel makes a contribution to adamantane inhibition this chimera will also be rimantidine-sensitive. In addition, new mutations at S31 could help distinguish the possibilities. The PBM supposes that the N31 side chains stick into the pore and form hydrogen bonds, while the DQM supposes that they stick into the interface of the TM helices and destabilize them. An S31L mutant should disrupt the helical packing but not form hydrogen bonds or extend the L31 side chains into the pore. If functional, such a mutant ought to be rimantidine resistant if the DQM is correct, but not if PBM is correct. Assuming the geometry of the longer side chain is wrong for formation of a hydrogen bonding network, an S31Q mutation might be useful as well. Similarly, mutations that increase the size of the L40, I42, or L43 side chains could prevent adamantane binding to the DQM site without degrading the channel's transport capabilities; the drug sensitivity of such a mutant would be a powerful argument either way. Any experiments of this kind would likely be easier to perform than to interpret, but could provide valuable insight. Obviously, it would also be important to establish that each mutant was competent at transporting protons.

The unspoken assumption of the debate so far is that these mechanisms are mutually exclusive, but there is no particular reason to believe this must be so. The structural experiments definitively show that binding to both sites is at least possible — even if one clings tenaciously to the idea that the density observed in the crystal is not in fact amantadine, that structure at least shows that the PBM site is capable of accommodating the drug. It might therefore be plausible that adamantane drugs inhibit M2 using both mechanisms simultaneously, or that DQM predominates at high pH and PBM at low pH. Redundancy in inhibitory mechanisms may explain the curious features of amantadine inhibition noted by Wang et al., and the inability of experiments specific to a single site to completely account for adamantane inhibition. In addition, the fact that S31N interferes with both mechanisms may explain why it is the primary resistance mutation.

An experiment with the alternate chimera mentioned above could test this possibility. In addition, if the mechanisms switch off in a pH-dependent fashion, then this should be testable with the hybrids: specifically, the A/B M2 used by Jing et al. should have lower susceptibility to adamantane drugs at high pH than at low pH. Similarly, the B/A M2 chimeric protein, if inhibited by amantadine, would be more resistant at low pH.

Doubtless these suggestions are nothing new to the members of the labs working on this perhaps unexpectedly hairy question. Membrane protein structure and function is one of the most difficult experimental subjects in biochemistry, and constitutes a critically important frontier in scientific efforts to improve human health. It is infinitely easier to propose most of these experiments than it is to perform them, and I would be remiss if I did not temper the persistently critical tone of this post with some praise for the efforts of all the scientists involved in this research, and for their commitment to getting the right answer. These papers represent years of work by incredibly talented people using some of mankind's most advanced scientific techniques. The lack of clarity on the question of adamantane drugs binding to M2, even in the face of this amazing effort, is a testament to the enormous difficulty of researching these critical systems.

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1. Deyde, V., Xu, X., Bright, R., Shaw, M., Smith, C., Zhang, Y., Shu, Y., Gubareva, L., Cox, N., & Klimov, A. (2007). Surveillance of Resistance to Adamantanes among Influenza A(H3N2) and A(H1N1) Viruses Isolated Worldwide The Journal of Infectious Diseases, 196 (2), 249-257 DOI: 10.1086/518936 OPEN ACCESS

2. Stouffer, A., Acharya, R., Salom, D., Levine, A., Di Costanzo, L., Soto, C., Tereshko, V., Nanda, V., Stayrook, S., & DeGrado, W. (2008). Structural basis for the function and inhibition of an influenza virus proton channel Nature, 451 (7178), 596-599 DOI: 10.1038/nature06528

3. Schnell, J., & Chou, J. (2008). Structure and mechanism of the M2 proton channel of influenza A virus Nature, 451 (7178), 591-595 DOI: 10.1038/nature06531

4. Miller, C. (2008). Ion channels: Coughing up flu's proton channels Nature, 451 (7178), 532-533 DOI: 10.1038/451532a

5. Czabotar, P., Martin, S.R., & Hay, A.J. (2004). Studies of structural changes in the M2 proton channel of influenza A virus by tryptophan fluorescence Virus Research, 99 (1), 57-61 DOI: 10.1016/j.virusres.2003.10.004

6. Wang, C., Takeuchi, K., Pinto, L.H., & Lamb, R. (1993) Ion Channel Activity of the Influenza A Virus M2 Protein: Characterization of the Amantadine Block J. Virol. 67 (9) 5585-5594 Available free from PubMed Central

7. Jing, X., Ma, C., Ohigashi, Y., Oliveira, F., Jardetzky, T., Pinto, L., & Lamb, R. (2008). Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel Proceedings of the National Academy of Sciences of the United States of America, 105 (31), 10967-10972 DOI: 10.1073/pnas.0804958105 OPEN ACCESS

8. Pielak, R., Schnell, J., & Chou, J. (2009). Mechanism of drug inhibition and drug resistance of influenza A M2 channel Proceedings of the National Academy of Sciences of the United States of America, 106 (18), 7379-7384 DOI: 10.1073/pnas.0902548106

The Scientific Activist and Discovering Biology in a Digital World also have interesting posts on this subject.

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Here there be dragonflies

Because we constantly interact with each other, we often forget that human beings are absurd, freakishly huge creatures. Of course you and I and any four-year-old can reel off the names of a dozen or so animals that are even larger, but that's misleading. The earth is inhabited by trillions upon trillions of animals, and almost none of them grow larger than our own hands. These creatures inhabit a world we can barely recognize, the objects we find familiar made exotic by the transition of scale, and yet their lives have drama and tension more than equal to our own. Deadly Creatures successfully sets up this atypical viewpoint, but falters because its narrative and gameplay adhere strictly to a human perspective.

You might expect a game about a tarantula and a scorpion to look like a National Geographic special, but Deadly Creatures eschews the crisp brightness of magazine shots in favor of a muted palette more reminiscent of the Metroid Prime series. The comparison seems even more apt in light of the almost alien character the desert landscapes take on, not just because of the changes in scale but also because of the variability of orientation. Deadly Creatures doesn't just put you in a scaled-up version of a pickup truck; it forces you to explore that space using every conceivable surface as a floor. The game also seems to share Metroid Prime's obsession with ruins, though here they are scaled down. The leftovers of human civilization — a cast-off boot, a broken lawn gnome — have become the homes of your dangerous enemies.

The comparisons go deeper than the aesthetics. Deadly Creatures comes across as a game of exploration, using the tiny grubs like Metroid Prime's copious power-ups to encourage you to see everything it has to offer. While it lacks something in variety, this approach fits relatively well with the game's fiction. Additionally, Deadly Creatures loves to return you to previously-visited areas with new abilities that allow you to take alternate routes across and out of them.

Where this comparison begins to break down is in the way the game tries to guide the player towards goals. Metroid Prime makes the player a dynamic actor in a relatively static world and lets him go where he will, his access limited only by the abilities he has acquired. While the player is not exactly free to choose his objectives, the motif of restricted access makes them feel like an organic part of the game's intrinsic fiction. Deadly Creatures artlessly blocks off particular exits with clouds of flies in an effort to push the player along in a set story arc.

The story itself is the most obvious example of the game's key failing, because it concerns human beings operating with specifically human motivations. Billy Bob Thornton and Dennis Hopper do a wonderful job with voice roles that simply don't fit in this game. What does buried treasure have to do with a tarantula and a scorpion? The game never bothers to make this clear, but it gives the player objectives that force him down a path towards confrontation. The goals may bee sensible to me, as a player who understands what the human characters are saying, but it's not clear why the deadly creatures of the game would choose to do these things. Unlike in Metroid Prime, the game's goals are not organically integrated into its universe. Here, the fiction of the narrative seems to be fundamentally divorced from the intrinsic fiction of the game world.

This also comes into play with respect to combat. The tarantula and especially the scorpion fight with a few simple attacks and elaborate (motion-based) finishing moves reminiscent of God of War. While motion detection is not ideal for some of these, the core mechanics of combat are suitably engaging. The problem is that they are repetitive, and this is a problem not only because stinging a rat in the brain stops being interesting around the fifth time you do it, but also because the number and organization of the enemies conflicts with the game's intrinsic fiction. Combat is too frequent, your enemies attack in waves, and they never attack each other. There is no recognition here of the dangerous line predators tread with respect to energy expenditure, or the flight instinct that protects most prey. Even the weakest beetle is always up for a fight. Nor is there any consideration for the idea that territorial spiders are not going to attack as an organized body, and even if they managed it they would be at least as likely to attack each other as the nearby scorpion. No, the heroes of Deadly Creatures must fight their way through an army of opponents in exactly the same way as you could see in any standard 3D brawler. The encounters and enemy AI are informed by human sensibilities, right down to the heartbeat sound that warns you of low health.

The game posits that the deadliest creature is man, and so the scorpion, at least, must confront him. Of course, arachnids are actually rarely endangered by direct encounters with humans, because they mostly have the good instinct to hide. Deadly Creatures breezes past this reality, and discards essentially everything worthwhile in its combat system, so that we may end the game on a low note with a boss battle marred by nonsensical mechanics and out-of-place toilet humor. Even if I'd found it funny to sting a guy on the nuts one time, repetition would have drained the exercise of its joy.

If geography is a matter of scale, then our absurd dimensions must deny us many of the world's most amazing vistas. They exist at a size we cannot reach, in a world we can barely begin to experience. Deadly Creatures walks up to the edge of that vast uncharted space, gazes longingly upon it, and then spins on its heel and returns to the map of tried-and-true conventions. The game turns its back on the powerful fiction implied by its setting in order to embrace a story about humans, enemies that operate with human sensibilities, and objectives that make no sense for its protagonists. Deadly Creatures does not ask the player to be a scorpion or a tarantula, it asks the player to be a dude on a couch controlling a scorpion or a tarantula, and that is how a fantastic idea becomes a merely passable game.

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The falling action

Most games, even particularly interesting ones, are hamstrung by the fact that they end too soon. I don't mean that the gameplay itself ends too quickly; indeed most games would be served by chopping out a few hours. What I'm talking about here is that the story gets cut short. Whether RPGs, shooters, or brawlers, the finale of most games goes like this: climactic boss battle, short non-interactive cutscene, The End. Sometimes the short cutscene gets replaced with a long one, but the basic pattern is widely accepted. This hurts game narratives because it forces the player to assess the implications of the game's climax while almost entirely outside of the game world. It is left to the player to imagine what the world of the game is like after, and while this occasionally fits a narrative design it does not always do so. Fallout 3 reached a positively manic climax with Liberty Prime's assault on Project Purity, then dove right into an abrupt (and almost inexplicable) closing sequence. The recent downloadable content, "Broken Steel", feels like an attempt to add a real denouement to the Enclave arc, and in this it largely succeeds.

Most quests in "Broken Steel" differ from those of the main game in that they are essentially devoid of meaningful moral choice. The events of the add-on represent a single infiltrator's actions in a military campaign, and as such most of its quests amount to extended dungeon crawls. While a number of new and extremely powerful enemies appear across the Wasteland, none of them take the form of Enclave soldiers — the new Hellfire Trooper is not significantly more effective than his compatriots — and when this is combined with the elevated level cap, the effect is a slow reduction of tension throughout the adventure. Sure, things can get a bit hairy during some segments (particularly the harrowing encounter with Reavers in the Presidential Metro), but every time you face the Enclave they feel weaker than before. In the final act of the add-on, I infiltrated the Enclave's mobile base and single-handedly took down their elite Sigma squad in the space of a few seconds. Things never really got any harder after that.

Broken Steel doesn't have any grand finale to match that of the main story, but that's not the point. The new content isn't about a heroic charge against overwhelming odds to conquer a fortified position; it's about stomping bugs. There's no new antagonist to confront, nor even an old one, as the new segments don't seem to notice if you let Colonel Autumn live. All you have are progressively less dangerous firefights, gradually easing off the pressure until you demolish the Enclave's last stronghold with fire from the sky.

The accessory quests don't add much in the way of gameplay or new areas, but they do allow the player to consider the non-military impact of freeing Project Purity. Notably, the Wasteland still has problems — cleaning up the water didn't immediately eliminate the widespread contamination, mutation problems, or raiders. Solving some of those problems simply created new ones: now the challenge is to ensure that the distribution of the pure water goes smoothly. These quests don't really have much in the way of interesting moral choices, but they help put the player's earlier decisions in perspective, allowing him to reflect on the implications of the choices he made earlier.

The weakness of "Broken Steel" is that it has little to offer beyond violence. The quests are mostly straightforward battles, and allow none of the flexibility inherent to some of the main game's more interesting missions. The player can't side with the Enclave, or even use his powers of persuasion to help destroy his enemies from within. The only choice the player can make is to shoot or die, and while that approach has its uses it's unfortunate that players who take advantage of the full range of character customization available in the game aren't rewarded with interesting alternative routes to victory.

The impression one gets from interviews is that the developers at Bethesda believe players reacted negatively to the fact that their game ended. For me, at least, this wasn't the case at all. It wasn't that Fallout 3 ended, but how it ended that felt unsatisfying. The assault on the purifier didn't feel like a personal triumph, the conclusion of the adventure came as a surprise, and the in-universe reason for ending it was contrived and ridiculous. After the events of "Broken Steel", however, I feel like the game could have stopped without any trouble. The powerful weapons and increased level cap slowly deflate the tension of the narrative throughout this episode, and the final crawl through the Enclave's mobile platform feels like the player's expression of personal dominance over their military engine. Despite its weaknesses, this aspect of "Broken Steel" is quite welcome: it makes the player feel like the story really is at a conclusion, by moving the end of the game away from the climax of the story.

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Go for the Overkill

While many publishers have largely decided to approach the Wii by speeding up their casual-game assembly lines, Sega has started to position itself as a go-to source for more adult fare on the friendly little console. This is the strategy behind the upcoming first-person shooter The Conduit, as well as the already-released games MadWorld and House of the Dead: Overkill. MadWorld was blessed with a striking cel-shaded art style and gameplay that had the potential to use the Wii remote in really interesting ways. So it's perhaps surprising that Overkill, a light-gun game that holds the Guinness record for most swearing in a video game, turned out to be so much better. There is a phrase in the previous sentence that tells you why that happened.

Of course, spewing F-bombs in a game is not a guarantee that it will become a great experience, but that's not the point. Overkill's amazing proliferation of profanity is important as an indicator of what the developers were trying to do and how they succeeded. The entire design of the game is meant to evoke memories of the bloody, profane, and ridiculous B-movies of yesteryear. And it does, from the moment you see an ammo-draped stripper dancing behind the credits. The soundtrack, dialogue, and obvious continuity (and projection) errors are all reminiscent of the bad old movies, as are the posters that designate each of the playable chapters. Even the bosses serve as a sort of parade of monster archetypes from these flicks. Of course, this is all unbelievably crass, and you'll only like the game if you can find it at least as hilarious as it is revolting.

The game fits its target well because the on-rails light-gun game is a second-tier entertainment these days anyway, long surpassed by the greater freedom afforded in first person shooters. Overkill embraces this heritage, relying on its scoring system to enhance the replay value (although the "director's cut" expands the game also). The Wii's graphical limitations aside, the enemy models are noticeably ugly and low on polygons, and while this may not have been intentional it slots into the overall aesthetic anyway.

This isn't to say that Overkill doesn't have any brains (the zombies mutants need something to eat, after all). It's certainly aware of how crass it is, to the point where one of the characters (who previously unleashed a hilariously chauvinistic tirade against the game's female protagonist) offers a cursory feminist critique of the game in the closing scene. One could also read a little commentary on the history of the genre into the episode that takes place in a carnival. Light-gun games are, after all, the progeny of midway amusements, by way of the arcade, something you can also find in the carnival episode. Overkill keeps its commentary light, however, mainly aiming to evoke our memories of films we'd be embarrassed to admit having watched. The game knows that we know those movies were terrible in every way, and if it's trying to make a point beyond that, it isn't trying too hard.

MadWorld evokes memories of a particular movie: The Running Man. A whole city has been transformed into a bloody amusement called DeathWatch for... somebody, and the protagonist, Jack, must kill his way through the other competitors in order to stop the evildoers who arranged the whole mess. Like Overkill, MadWorld aims to get laughs out of the absurdity of its violence and an accompanying commentary track. MadWorld, unfortunately, never really gets going and even if it did it hasn't got a coherent destination.

The key problem with the gameplay is pacing. Overkill has a tremendous advantage in that its on-rails structure means that the developers can completely control the tension and the player's viewpoint at all times. This has its downside — once a player has "learned" a particular area it can get boring — but it also keeps the action going almost constantly. MadWorld, however, is a series of open areas in which the player can roam freely. This allows him to control his experience to some degree, but it also means that the player can feel somewhat directionless. Local shortages of enemies force the player to move, but in many levels enemies and interesting locations are clustered rather than evenly spaced. As a result, the player sometimes wanders aimlessly and can stumble into stretches where there just isn't enough to do. The scoring system exacerbates this problem by generally keeping players in these arenas for too long, allowing even its over-the-top violent antics to become repetitive and tiresome.

That explains how MadWorld falters as a game, but it also collapses as an idea. The cheesy visuals of Overkill contribute to its overall aesthetic intentions, and the black-white-and-red cel shading of MadWorld seems like it will do the same, targeting violent comics. But while Overkill dispenses with reality almost immediately and starts taking its story in more and more bizarre directions, MadWorld keeps itself firmly under the top, embracing lengthy cutscenes where people look shocked or frightened in freeze frame as various characters painstakingly explain the history of DeathWatch and the ordinary, workaday motives of the rich men who have organized it. Oh, they did it to make money, who could have guessed.

MadWorld doesn't even surpass its inspiration. Decades have passed, so now we have a lot more blood and dismemberment, but do we have a lightning-slinging opera singer wearing technicolor armor? MadWorld eschews incongruous beasts like Dynamo, instead giving us exactly what we expect from a game like this. The bosses always are and appear every bit as dangerous as Jack himself, and most of them are huge, violent monsters who can happily survive having an arm chainsawed off. It's surprisingly conventional, and even a little cowardly.

This extends to the concept of DeathWatch itself. The Running Man has a virtue in that it points the finger at its own audience: a totalitarian government organized the events, but the people watching, cheering, and wagering on the murderous game show were very clearly us. The rising popularity of MMA and the recalibration of football broadcasts as celebrations of the hard hit are evidence that the thirst for violent sport is as great now as ever. The target is there, and MadWorld's bloody excess and sportscaster-like commentators are like a loaded gun aimed right at it, so it is almost infuriating that the game never pulls the trigger. The Running Man at least afforded the possibility of an uncomfortable moment, when the viewer attempted to reject the idea that he was like the spectators in the film and then realized he'd just spent 90 minutes cheering on bloody murder too. MadWorld goes to some effort to assure its player that ordinary people are victims, only the jaded rich think murder is sport, something about pharmaceutical companies, blah blah blah. The reality Jack is every bit as brutal and disgusting as the thugs and aristocrats he's opposing, and he is us. You could do something powerful with that, if you were so inclined. MadWorld's developers apparently weren't.

House of the Dead: Overkill succeeds because it takes its every weakness and makes it serve the overall aesthetic. It's a relatively ugly game built on an unpopular mechanic that's linear by its very nature, and it works magnificently because of the B-movie horror context. MadWorld has every advantage over the other game when it comes to artistic style and gameplay mechanics, but like its directionless players it simply loses its way. It's not just that it doesn't commit; the game doesn't even seem to know what it could commit to. Overkill seemingly aims lower than MadWorld, but because it is built into a coherent aesthetic experience, it hits higher.

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