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Thoughts on Antichamber? Personally I think it's a fantastic piece of work. I can't think of any other game that I've played where my first reaction is to just try something instead of looking for the right thing to do. It's refreshing to just be given a space to explore with minimal hand holding. Amusingly, Link in a Chain Reaction is trivialized by the yellow gun as well. I actually spent 10 or 15 minutes forcing myself to do it the 'right' way after already having the yellow gun just so I could feel good about something after all my stupid with the green gun.
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If you aren't sure about a post,a. Donations.Retired threads will be removed.General GuidelinesCritical discussion about specific games, features, and topics is encouraged.In the event of a heated argument that has resorted to personal attacks/name-calling, moderator action will be taken against both parties regardless of who was the aggressor. We understand that it can be difficult to disengage from the aggressor, but we heavily suggest reporting the post and avoiding further interaction with the poster.Please report rule breaking behaviour.SpoilersTo tag something as a spoiler, format the spoiler like so: spoiler subject(#s 'spoiler details')This'll show up as Other subreddits.- Discussion, bar the Hivemind.- For news.- For memes.- Go here to help you find your next game to play.- AskReddit for games.- Find out what's worth getting.- Nintendo-specific subreddit for general Nintendo news and discussion. Personally I think it's a fantastic piece of work. I can't think of any other game that I've played where my first reaction is to just try something instead of looking for the right thing to do.
It's refreshing to just be given a space to explore with minimal hand holding.Also the menu room is a really nice touch. From a technical stand point menu selections are no different from throwing in-game switchs (it's all just adjusting variables) so it's nice to see the menu in-game.All in all it appears to really break down what a game actually is and what it could be. Space manipulation is one area where games have a distinct advantage over media and yet it seems to be so under-utilised as a tool.I'd be interested to hear what other people's thoughts on it. Probably the most fun I've ever had in a puzzle game.
I loved walking down a corridor and suddenly not being in it, turning round and that one too had gone. Mapping extra dimensions in my head to navigate was fun!:DI've played it twice now. First time was just before release (Yay for review copies) and finished it in just over 2 hours. Second time I went back and really searched for stuff and tried to break it as much as possible. I love that every time I thought I'd one-upped the game I just found out that I was in a secret room with concept art and stuff.It's a game for people who like to break games. I love it.:D.
I completed it the other day and after awhile I was sure it couldn't get any better but even that ending was both fascinating and interesting.Anyway, I'm really impressed by pretty much every aspect of it. We ask for new and unique things all the time and I can't think of anything that has achieved this to such extent, in recent memory.Exploring a non-euclidean space as, a concept alone, is something I couldn't be more excited about but he just kept introducing more unique and interesting mechanics as well. It's difficult for me to believe the amount of creativity he was capable of. Very stark use of colours but also very useful in puzzles.I'm sure he used at least one binaural audio track for one of the rooms which was neat to experience. I've felt that kind of technology is very sadly underutilized in anything, really. But the calm ambient tracks helped me remain in a fairly relaxed state where I was able to keep working through every challenge and concept at a comfortable pace.Overall I'd say my experience with it was very much like games used to feel back when I was less informed about them and they contained more wonder and I was excited to play them.
Not that I don't find enjoyment in a lot of today's games but I haven't felt this pleased with a game since Portal, and I have trouble comparing the two because a big part of Portal was narrative which Antichamber smartly avoids to focus on the robust set of core mechanics and puzzles.Antichamber is a rare and unique experience that I feel everyone should have and it saddens me that anyone would spoil it for themselves by following a guide for even one puzzle. There's never a point where you flat-out cannot progress because you can't figure out one puzzle. Just leave and come back to it, there's nothing that could possibly be wrong with that.TL;DR A rambling discourse of things I felt like saying about Antichamber. The best game I've played in a long time.
Type I modular polyketide synthases (PKSs) produce polyketide natural products by passing a growing acyl substrate chain between a series of enzyme domains housed within a gigantic multifunctional polypeptide assembly. Throughout each round of chain extension and modification reactions, the substrate stays covalently linked to an acyl carrier protein (ACP) domain. In the present study we report on the solution structure and dynamics of an ACP domain excised from MLSA2, module 9 of the PKS system that constructs the macrolactone ring of the toxin mycolactone, cause of the tropical disease Buruli ulcer. After modification of apo ACP with 4′-phosphopantetheine (Ppant) to create the holo form, 15N nuclear spin relaxation and paramagnetic relaxation enhancement (PRE) experiments suggest that the prosthetic group swings freely. The minimal chemical shift perturbations displayed by Ppant-attached C 3 and C 4 acyl chains imply that these substrate-mimics remain exposed to solvent at the end of a flexible Ppant arm. By contrast, hexanoyl and octanoyl chains yield much larger chemical shift perturbations, indicating that they interact with the surface of the domain.
The solution structure of octanoyl-ACP shows the Ppant arm bending to allow the acyl chain to nestle into a nonpolar pocket, whereas the prosthetic group itself remains largely solvent exposed. Although the highly reduced octanoyl group is not a natural substrate for the ACP from MLSA2, similar presentation modes would permit partner enzyme domains to recognize an acyl group while it is bound to the surface of its carrier protein, allowing simultaneous interactions with both the substrate and the ACP. INTRODUCTIONType I modular polyketide synthases (PKSs) are large, multi-domain complexes responsible for generating natural products with a spectrum of medically important activities, including antibiotic, anticancer, antifungal, antitumour and immunosuppressive properties.
Like type I fatty acid synthases (FASs), these systems consist of a series of covalently linked enzymes that extend a polyketide substrate by two carbon atoms and modify the functionality of the newly added building block via reactions at the β-ketone site. Reaction scheme and module organization for the mycolactone PKS system( A) Catalytic cycle for domains from the MLSA2 module. ( B) Module organization for the three subunits of the mycolactone PKS system (MLSA1, MLSA2 and MLSB).
The product of MLSA2 is shown attached to its carrier protein domain. The structure of mycolactone is colour coded to match the subunits responsible for synthesizing each segment. DH domains predicted to be inactive are shaded black.The intuitive, linear, assembly-line nature of modular PKS and similarly configured non-ribosomal peptide synthetase (NRPS) systems make them attractive targets for combinatorial biosynthesis and synthetic biology strategies ,. Although numerous new compounds have been generated by such approaches, engineered PKS multi-enzyme complexes often display reduced activity or result in undesirable product mixtures ,. A major limitation in overcoming such deficiencies is a poor understanding of the interactions that occur within and between modules. The role of the ACP is central to this question, since it must present covalently tethered substrates to the active sites of each enzymatic domain within its module, as well as a KS or a TE in the subsequent module ,.
Co-expression with Sfp to make 15N-labelled holo mACP 9The mACP 9/pET28 plasmid and a pSU2718 plasmid with the Sfp gene cloned between the NdeI and SalI sites were transformed into competent E. Coli Tuner (DE3) cells (EMD Millipore). Apart from addition of 34 μg/ml chloramphenicol (Sigma) to growth media for selection of the pSU2718 plasmid, expression and purification of the holo protein was identical with that of the apo protein described above. The extent of modification of mACP 9 was monitored by ESI MS (Supplementary Figure S2), and was complete in all cases taken forward for further study. Sfp-based addition of 4′-phosphopantetheine and acyl-phosphopantetheine groupsIn vitro loading reactions were performed on 500 μM apo ACP 9 samples by incubation with 5 μM recombinant Sfp , 2 mM coenzyme A or its derivatives malonyl, butyryl, 2-butenoyl, β-hydroxybutyryl, acetoacetyl, hexanoyl or octanoyl CoA (all Sigma) in pH 7.5 phosphate buffer with 10 mM magnesium chloride at 20°C for 1 h. 10 mM DTT was added when handling coenzyme A and holo mACP 9 to prevent disulfide bond formation between exposed thiol groups. Samples were subjected to size exclusion chromatography as described above prior to further analysis.
In each case, the identity and extent of modification was monitored by ESI MS (Supplementary Figures S3–S9). Methanthiosulfonate-based modification of the 4′-phosphopantetheine thiol(1-Oxyl-2,2,5,5-tetramethyl-∆3-pyrroline-3-methyl) methane-thiosulfonate (MTSL) or (1-acetoxy-2,2,5,5-tetramethyl-∆3-pyrroline-3-methyl) methanethiosulfonate (ATSL) (Toronto Research Chemicals), both dissolved in DMSO, were added to holo mACP9 in a 10-fold molar excess at less than 1% of the sample volume, then incubated at 20°C for 16 h. N-Propyl methanethiosufonate (PMTS) oil (Toronto Research Chemicals) was added directly to holo mACP9 at a 20-fold molar excess and at less than 0.2% of the final sample volume. All samples were incubated at 20°C for 16 h and then subjected to size exclusion chromatography as described above prior to further analysis.
Full labelling was confirmed by ESI MS (Supplementary Figures S10–S12). 15N nuclear spin relaxation experiments15N nuclear spin relaxation experiments were recorded using standard procedures at 283K on a Bruker DRX500 spectrometer. 15N T 1 delays (ms): 10, 50, 100, 150, 250, 400, 550, 700, 850, 1000. 15N T 2 delays (ms): 14.4, 28.8, 43.2, 57.6, 72.0, 86.4, 100.8, 155.2. The heteronuclear NOE reference and saturation experiments were carried out in duplicate to allow an estimation of the error. An initial τ c estimate was obtained from the R 2/ R 1 ratios for each residue ; the same procedure was used to make site specific estimates of the local rotational correlation time τ eff. The relaxation parameters were analysed with version 4 of the Modelfree program using the strategy described by Mandel et al.
H N─N bond vectors from the solution structure of apo mACP 9 were used for anisotropic diffusion tensor modelling of relaxation data for both the apo and holo forms. Expression and purification of mACP 9 speciesmACP 9, a protein fragment spanning residues 2050–2140 of the MLSA2 subunit, was expressed with an N-terminal His 6-tag, as described in Materials and Methods. Following purification using nickel affinity chromatography, the fusion tag was removed by thrombin cleavage, leaving four non-native amino acid residues originating from the expression vector (GSHM-) at the N-terminus of the construct. Analytical size exclusion chromatography indicated that apo mACP 9 is monomeric in solution (Supplementary Figure S13). Phosphopantetheinylation of mACP 9 was carried out in vivo by co-expression with Sfp, a broad specificity phosphopantetheinyl transferase , resulting in full conversion from the apo to the holo form according to ESI MS (Supplementary Figure S2).Coordinate precision, Ramachandran statistics and Z-scores were determined between residues 2050 and 2140.The core of the calculated structure contains closely packed hydrophobic side chains, with no evidence for the interior cavities observed in type II ACP structures.
The exterior surface is predominately composed of hydrophilic side chains, the only exception being a small nonpolar patch adjacent to the attachment serine, produced by Phe 2120 from turn α3′ and two solvent-exposed leucine side chains from helix α2 (Leu 2097 and Leu 2100). The nonpolar surface of the α2 ‘recognition helix’ is a common distinction observed between type I and type II ACPs, for which the equivalent region, proposed to be critical for interactions with partner enzymes, is rich in acidic residues. In the mACP 9 structure, helix α2 contains only one acidic residue, Glu 2101; the majority of charged residues, which are predominantly basic, are located on a face created by α2, α3′ and α3, whereas the opposing α1 face is relatively uncharged (Supplementary Figure S14).
Holo and acyl-loaded forms of mACP 9Backbone assignments for the apo form were transferred to experiments recorded on a holo mACP 9 sample and verified using NOE connections. As noted for the apo form above, the number of signals detected was consistent with population of a single conformational state. Two additional peaks in the 1H, 15N-HSQC spectrum of holo mACP 9 were identified as amide signals from N 4 and N 8 in the Ppant arm (Supplementary Figure S15).
The profile of average 1H N/ 15N chemical shift differences shown in A demonstrates that the majority of backbone amide sites experience only minor perturbations in their electronic environment (. Solution structures of octanoyl-ACP speciesRibbon representations, coloured from blue at the N-terminus to red at the C-terminus, for ( A) octanoyl-mACP 9 (5HV8); ( B) octanoyl-actACP (2KGC); and ( C) octanoyl-ACP from the S. Coelicolor FAS (2KOS). Stick representations of pantoate, β-alanine, cysteamine and octanoyl sections are coloured orange, red, magenta and blue, respectively.Experiments following the change in mean residue ellipticity at 222 nm as a function of temperature demonstrated that apo, holo, hexanoyl- and octanoyl-mACP 9 species all undergo two-state unfolding transitions, with melting temperatures of 326 K, 332 K, 331 K and 330 K respectively (Supplementary Figure S17).
These results confirm earlier work on the type II FAS ACP from P. Falciparum, which showed that priming with Ppant enhanced the thermal stability of the domain. For mACP 9, it is interesting that further modification with hexanoyl and octanoyl groups had minimal effects, rather than causing the melting temperature to increase. 15N nuclear spin relaxation studiesTo investigate the solution state dynamics of mACP 9, nuclear spin relaxation properties were measured for 15N-labelled amide sites in both the apo and holo forms and analysed using the Lipari–Szabo model-free approach (; Supplementary Figure S18). The data fitted best to an axially symmetric diffusion tensor model with a D par/ D per ratio of 1.30 and overall rotational correlation times of 10.3 ns and 10.4 ns for the apo and holo states, respectively, consistent with a monomeric, globular 90 amino acid domain at 283 K. In both the apo and holo states, backbone sites were found to be predominantly rigid (mean order parameter S 2 of 0.87±0.10 for both), with the same five residues possessing more dynamic S 2 values (. Paramagnetic relaxation enhancement studiesThe chemical shift perturbations observed on conversion of mACP 9 from the apo to the holo form could be interpreted as evidence that the Ppant group prefers to populate conformations that are oriented towards the α3′ turn, similar perhaps to those captured in the ensemble of structures for octanoyl-mACP 9.
By contrast, our 15N nuclear spin relaxation measurements suggest that the prosthetic group is highly flexible, implying that the arm samples multiple conformations in an isotropic fashion. To investigate this issue further, the Ppant arm of holo mACP 9 was modified with the paramagnetic spin label reagent MTSL. Two reference samples were also prepared: one modified with ATSL, with the nitroxyl moiety replaced by an acetyl group (Supplementary Figure S16); and one in which the nitroxyl radical of MTSL was reduced to a diamagnetic hydroxy group via treatment with ascorbate. The ascorbate-reduced reference sample showed only minor chemical shift perturbations compared with non-MTSL-labelled holo mACP 9 (B), confirming that any interactions between the MTSL label and mACP 9 must be similar to those observed for C 4-loaded species. Modification with disulfide-linked groupsUnexpectedly, labelling of mACP 9 with ATSL yielded a 1H, 15N-HSQC spectrum in which several resonances were doubled (Supplementary Figure S19), rendering the sample unsuitable for use as a reference state for PRE experiments.
ESI MS revealed that the sample contained a single species at the expected molecular mass (Supplementary Figure S11), suggesting that the two forms detected by NMR correspond to distinct conformational states rather than alternative modification products. None of the doubled peaks coincide with resonances from the apo or holo forms of mACP 9, indicating that modification with ATSL creates two significantly different conformations. All resolvable doublets comprise a minor peak with chemical shifts close to the corresponding holo signal (D) and a major peak showing a larger shift perturbation (E), with an intensity ratio of approximately 1:1.4. In zz-HSQC experiments, no chemical exchange cross-peaks between resolved doublets were detected (results not shown), implying that if the two states are capable of interconverting, this must occur on a timescale slower than 0.5 s. As I shows, sites that give rise to doublets include those displaying the strongest PRE effects (H), the largest chemical shift differences between the apo and holo forms (G), and the most significant perturbations in 2-butenoyl- and butyryl-loaded samples, but affect fewer sites than those changed in an octanoyl-loaded sample.
Amide signals from the prosthetic group were also doubled and displayed relaxation properties ( τ eff values of 6.5 ns and 5.4 ns for the major and minor forms, respectively; ) intermediate between those of the protein backbone (∼10.5 ns) and the more dynamic arm of the holo species (∼3.9 ns). Consistent with these results, the H N4 and H N8 sites in both forms exhibited more intense 1H- 1H NOEs than the holo state, although once again only intra-Ppant connections were observed.Aiming to mimic the length of a butyryl substrate chain, we modified a sample of the holo domain using PMTS. The 1H, 15N-HSQC spectrum of PMTS-modified mACP 9 showed a single set of resonances with chemical shift changes (F) more extensive than those observed for butyryl-mACP 9 (F), but smaller than for either of the ATSL-modified forms (D and E). Together with an absence of peak doubling for the reduced MTSL sample, these observations suggest that multiple ATSL-mACP 9 states are a property of the altered nitroxide moiety rather than a consequence of the linkage mode, such as conformational isomerism of the disulfide bond.
The most likely explanation is that the ATSL-modified prosthetic group populates two distinct conformers, both of which dock against the surface of the ACP domain, differing perhaps in the orientation of the acetoxy moiety (Supplementary Figure S16). Within the Ppant group, motions are restricted compared with the freedom of movement experienced in the holo state, but a significant degree of flexibility is retained. Although ATSL is not a natural substrate for mACP 9, these results provide a further demonstration that longer substrates can interact with the surface of a type I PKS ACP domain. Evidence that the prosthetic group possesses moderately restricted dynamics in both ATSL-loaded conformers is consistent with the picture conveyed by the solution structure of octanoyl-mACP 9, in which the Ppant arm shows a degree of conformational heterogeneity that decreases as the acyl group approaches the protein surface. ACPacyl carrier proteinATacyltransferaseATSL(1-acetoxy-2,2,5,5-tetramethyl-∆3-pyrroline-3-methyl) methanethiosulfonatecryo-EMcryo-electron microscopyDHdehydrataseERenoyl reductaseESI MSelectrospray injection mass spectrometryFASfatty acid synthaseHSQCheteronuclear single quantum coherenceKRketoreductaseKSketosynthaseMTSL(1-oxyl-2,2,5,5-tetramethyl-∆3-pyrroline-3-methyl) methanethiosulfonateNRPSnon-ribosomal peptide synthetasePKSpolyketide synthasePMTSn-propyl methanethiosufonatePpant4′-phosphopantetheinePREparamagnetic relaxation enhancementTEthioesterase. AUTHOR CONTRIBUTIONWilliam Broadhurst designed the research. Steven Vance, Olga Tkachenko and William Broadhurst conceived and designed the experiments.
Steven Vance, Olga Tkachenko, Ben Thomas, Mona Bassuni, Daniel Nietlispach and William Broadhurst performed the experiments. Steven Vance, Olga Tkachenko and William Broadhurst analysed the data. Steven Vance, Mona Bassuni, Hui Hong and William Broadhurst contributed reagents/materials/analysis tools. Steven Vance and William Broadhurst wrote the paper.
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