You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


1.. Introduction {#sec1}
================

1.1.. SSGCID {#sec1.1}
------------

The Seattle Structural Genomics Center for Infectious Disease (SSGCID) is one of two consortia funded by NIAID to apply genome-scale approaches to the solution of protein structures from bio­defense organisms, as well as those causing emerging and re-emerging diseases. In its first three and a half years, the SSGCID has submitted ∼350 protein structures to the Protein Data Bank (PDB) and is on track to solve a further 100 per year going forward. For several organisms, this represents the majority of PDB submissions during this time, including 100% of the structures for *Ehrlichia*, *Anaplasma* and *Burkholderia*. SSGCID's target-selection strategy has focused on drug targets, essential enzymes, virulence factors and vaccine candidates from a number of bacterial (*Bartonella*, *Brucella*, *Ehrlichia*, *Anaplasma*, *Rickettsia*, *Burkholderia*, *Borrelia* and *Mycobacterium*) and eukaryotic (*Babesia*, *Cryptosporidium*, *Toxoplasma*, *Giardia*, *Entamoeba*, *Coccidioides* and *Encephalitozoon*) pathogens, as well as ssDNA and negative-strand ssRNA viruses. More than 3000 targets have been selected to date, with \>700 proteins being purified for crystallization trials. Crystallization screening and analysis of X-ray diffraction data sets for structure solution are performed at Emerald BioStructures.

1.2.. Aldolase {#sec1.2}
--------------

Aldolases (EC 4.1.2) are enzymes that cleave aldols and have been the subject of crystallographic study since 1971 (Heidner *et al.*, 1971[@bb6]). To date, more than 70 unique aldolase structures have been deposited in the PDB. The glycolytic enzyme fructose bisphosphate aldolase (EC 4.1.2.13) catalyzes the aldol cleavage of fructose 1,6-bisphos­phate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. It also catalyzes the reverse reaction. The hallmark of a class I aldolase is the presence of an active-site lysine residue which forms a Schiff base with the substrate.

*Bartonella henselae* is best known for causing cat scratch disease (CSD). It is a Gram-negative aerobic rod-shaped proteobacterium. Immunocompromised patients can develop severe complications from *B. henselae*. Here, we present the crystal structure of the class I fructose bisphosphate aldolase from *B. henselae* with the reactant bound at the active site.

2.. Materials and methods {#sec2}
=========================

All expression clones, purified proteins and protein structures produced by SSGCID are available to the scientific community.

2.1.. Protein purification and crystallization {#sec2.1}
----------------------------------------------

### 2.1.1.. Purification of *B. henselae* aldolase {#sec2.1.1}

The protein was expressed in *Escherichia coli* using BL21 (DE3) R3 Rosetta cells and autoinduction medium in a LEX Bioreactor. Starter cultures of LB broth with appropriate antibiotics were grown for ∼18 h at 310 K as described by Choi *et al.* (2011[@bb1]). ZYP-5052 auto-induction medium with tryptone, yeast extract, glucose and α-lactose supplemented with MgSO~4~ and trace metals was freshly prepared as per Studier's published protocol (Studier, 2005[@bb14]). Antibiotics (carbenicillin/ampicillin) were added to 2 l bottles of sterile auto-induction medium. The medium was inoculated with all of the overnight culture. Inoculated bottles were then placed into a LEX bioreactor. Cultures were grown for ∼24 h at 298 K; the temperature was then reduced to 288 K and growth continued for a further ∼72 h. To harvest, the medium was centrifuged at 4000*g* for 20 min at 277 K. The cell paste was flash-frozen in liquid nitrogen and stored at 193 K. The frozen cells were resuspended in lysis buffer \[25 m*M* HEPES pH 7.0, 500 m*M* NaCl, 5% glycerol, 30 m*M* imidazole, 0.025% sodium azide, 0.5% CHAPS, 10 m*M* MgCl~2~, 1 m*M* tris(2-carboxyethyl)phosphine (TCEP), 250 ng ml^−1^ 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and 0.05 µg ml^−1^ lysozyme\]. The resuspended cell pellet was disrupted on ice for 30 min with a Virtis sonicator (408912; 100 W power, with alternating cycles of 15 s pulse-on and 15 s pulse-off). The cell debris was incubated with 20 µl Benzonase nuclease (25 units ml^−1^) at room temperature for 45 min and clarified by centrifugation on a Sorvall SLA-1500 at 14 000 rev min^−1^ for 75 min at 277 K. The protein was purified from the clarified cell lysate by immobilized metal-affinity chromatography on a HisTrap FF 5 ml column (GE Healthcare) equilibrated with binding buffer (25 m*M* HEPES pH 7.0, 500 m*M* NaCl, 5% glycerol, 30 m*M* imidazole, 0.025% sodium azide, 1 m*M* TCEP). The recombinant protein was eluted with 250 m*M* imidazole. The purification tag (MAHHHHH­HMGTLEAQTQGPGS) was cleaved with 3C protease, leaving a GPGS remnant. The sample was further polished using size-exclusion chromatography (SEC) on a HiLoad 26/60 Superdex 75 column (GE Healthcare) in SEC buffer (25 m*M* HEPES pH 7.0, 500 m*M* NaCl, 2 m*M* DTT, 0.025% sodium azide, 5% glycerol). Fractions from a single peak were pooled. The protein was concentrated to 21 mg ml^−1^ using 10K Amicon Ultra centrifugal filters (Millipore). The concentration was determined by measuring the OD~280~ of the protein (extinction coefficient 29 450 *M* ^−1^ cm^−1^; molecular weight 39 495 Da) with a Nanodrop 1000 instrument. The concentrated sample was flash-frozen in liquid nitrogen and stored at 193 K. This purification yielded 17 mg protein per litre of cell culture with \>95% purity.

### 2.1.2.. Crystallization of *B. henselae* aldolase with the native substrate fructose 1,6-bisphosphate {#sec2.1.2}

*B. henselae* fructose bisphos­phate aldolase (BhFBPA) was initially crystallized in the apo form, but these crystals did not diffract. Crystallization screening was repeated in the presence of the natural substrate fructose 1,6-­bis­phosphate (FBP), which yielded crystals that diffracted to 2.35 Å resolution at a synchrotron-radiation light source. BhFBPA was cocrystallized *via* vapor diffusion at 290 K in drops consisting of 0.4 µl protein solution and 0.4 µl precipitant solution, with 80 µl precipitant solution in the reservoir. Protein at 21 mg ml^−1^ in SEC buffer was supplemented with fructose 1,6-bisphosphate (FBP) from a 100 m*M* aqueous stock to give a final FBP concentration of 10 m*M* and a final protein concentration of ∼19 mg ml^−1^. A number of crystals grew, ranging in size from approximately 50 to 200 µm. The precipitant consisted of 0.2 *M* sodium acetate, 0.1 *M* Tris pH 8.5, 30% PEG 4000 (condition B10 of Crystal Screen HT from Hampton Research). The cryoprotection solution consisted of 80% precipitant solution and 20% ethylene glycol. The crystal used for data collection was soaked in this cryoprotection solution for ∼15 s before being flash-cooled in liquid nitrogen.

### 2.1.3.. Data collection, processing, structure solution and refinement {#sec2.1.3}

Data were collected on ALS beamline 5.0.1 as part of the Collaborative Crystallography Program over a 35 min period. The wavelength was 0.97740 Å and the detector was an ADSC Q315 CCD. The crystal-to-detector distance was 350 mm. 220 frames were collected with a width of 1° in ϕ at 100 K.

All data were indexed, integrated and scaled with the *XDS* suite (Kabsch, 2010[@bb7]). Data-collection and processing statistics are presented in Table 1[▶](#table1){ref-type="table"}. Refinement and validation parameters are presented in Table 2[▶](#table2){ref-type="table"}.

The structure was solved by molecular replacement with *Phaser* (McCoy *et al.*, 2007[@bb10]) using a search model prepared from residues 11--­343 of *Babesia bovis* aldolase (PDB entry [3kx6](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=pdb&pdbId=3kx6); SSGCID, unpublished work) with the *CCP*4*i* (Winn *et al.*, 2011[@bb2]) interface to *CHAINSAW* (Stein, 2008[@bb12]), which uses an alignment between the target sequence and that of a homologous protein of known crystal structure to create an improved molecular-replacement model by truncation of non-identical side chains. The structure was rebuilt in *Coot* (Emsley & Cowtan, 2004[@bb5]) and refined with *REFMAC*5 (Murshudov *et al.*, 2011[@bb11]) using two TLS groups per monomer. Figures were prepared with *PyMOL* (<http://www.pymol.org>).

3.. Results and discussion {#sec3}
==========================

3.1.. Overall structure {#sec3.1}
-----------------------

BhFBPA crystallized with four monomers in the asymmetric unit (50% solvent content; Matthews coefficient *V* ~M~ = 2.45 Å^3^ Da^−1^; Matthews, 1968[@bb9]). BhFBPA adopts the typical TIM-barrel fold tertiary structure of aldolases as well as the typical homotetrameric assembly quaternary structure, with an r.m.s. difference for monomer *A* of 0.94 Å from the [3kx6](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=pdb&pdbId=3kx6) starting model. It has ∼50% sequence identity to many aldolases with unsolved structures, such as those from *Toxoplasma gondii*, *Arabidopsis thaliana* and *Crypto­sporidium parvum*. The final FBP-bound model contains four copies of FBPA including three residues of the four-residue N-terminal purification tag and residues 1--340 (of 343) of the protein; the FBP ligand was modelled in each monomer. 498 water molecules were modelled.

The final model showed good geometry (Table 2[▶](#table2){ref-type="table"}) as determined using the program *MolProbity* (Chen *et al.*, 2010[@bb4]).

3.2.. Reactant state {#sec3.2}
--------------------

As part of glycolysis, FBPA catalyzes the cleavage of fructose 1,6-­bisphosphate (FBP) into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). Here, we determined the 2.35 Å resolution crystal structure of BhFBPA (Tables 1[▶](#table1){ref-type="table"} and 2[▶](#table2){ref-type="table"}). This structure has clear electron density for FBP bound at the active site (Fig. 1[▶](#fig1){ref-type="fig"}). There is a covalent bond between C atom C2 of the linear FBP molecule and the N^ζ^ atom of Lys223 of the protein. Furthermore, phosphate group 1 of FBP makes hydrogen bonds to three amide N atoms as well as the side chains of Arg295 and Ser264 (Fig. 2[▶](#fig2){ref-type="fig"}). The alcohol groups along the linear carbon backbone of FBP also make hydrogen bonds to the side chains of the protein, as well as to nearby water molecules. Finally, the O atoms of phosphate group 2 are hydrogen bonded to side chains and to nearby water molecules.

SSGCID's interest in BhFBPA is as a potential drug target. We compared the active site of BhFBPA with those of homologous mammalian enzymes. The orientation of the FBP molecule in the active site differs from that observed in the structure of human muscle aldolase (PDB entry [4ald](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=pdb&pdbId=4ald); Dalby *et al.*, 1999[@bb3]; 54% sequence identity; r.m.s.d. of non-H atoms in the FBP ligand of 2.2 Å; r.m.s.d. for C^α^ atoms over the whole protein of 0.663 Å). However, the complex observed in [4ald](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=pdb&pdbId=4ald) does not show full formation of the Schiff base, suggesting that the state in [4ald](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=pdb&pdbId=4ald) precedes the formation of the covalent complex (Dalby *et al.*, 1999[@bb3]). The FBP conformation in BhFBPA is essentially identical to that of rabbit muscle aldolase A (PDB entry [1zai](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=pdb&pdbId=1zai); St-Jean *et al.*, 2005; r.m.s.d. of non-H atoms in the FBP ligand of 0.29 Å; r.m.s.d. for C^α^ atoms over a full monomer of 0.489).

4.. Conclusion {#sec4}
==============

The crystal structure of fructose bisphosphate aldolase from *B. henselae* was determined in complex with its reactant at 2.35 Å resolution. The structure obtained of the reactant Schiff-base state is similar to those of other aldolases in the reactant state \[PDB entries [1zai](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=pdb&pdbId=1zai) (St-Jean *et al.*, 2005[@bb13]), [3mbf](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=pdb&pdbId=3mbf) (Gardberg *et al.*, 2011[@bb15]) and [2qdg](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=pdb&pdbId=2qdg) (Lafrance-Vanasse & Sygusch, 2007[@bb8])\], especially rabbit muscle FBPA A (PDB entry [1zai](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=pdb&pdbId=1zai)). The similarity to the active site of mammalian FBPA A suggests that the design of a specific inhibitor for BhFBPA (that does not inhibit the human enzyme) would be exceptionally challenging and would have to rely on exploiting very minor differences in the chemical environment in and near the active site.

Supplementary Material
======================

PDB reference: [fructose bisphosphate aldolase, 3mmt](3mmt)

The authors thank the SSGCID teams at SBRI, UW and Emerald BioStructures. This research was funded under Federal Contract No. HHSN272200700057C from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services. The Berkeley Center for Structural Biology is supported in part by the National Institutes of Health, National Institute of General Medical Sciences and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences of the US Department of Energy under Contract No. DE-AC02-05CH11231.

![Weighted 2*F* ~o~ − *F* ~c~ electron-density map at 1.5σ for FBP and nearby residues at the active site of fructose bisphosphate aldolase from *B. henselae*. There is clear electron density for a Schiff base formed between Lys223 and the FBP molecule.](f-67-01051-fig1){#fig1}

![Bonding environment for FBP bound at the active site of fructose bisphosphate aldolase from *B. henselae*.](f-67-01051-fig2){#fig2}

###### Data-collection statistics

Values in parentheses are for the highest of 20 resolution shells.

  ----------------------------------------------- -----------------------------------------
  Space group                                     *P*2~1~2~1~2~1~
  Unit-cell parameters (Å)                        *a* = 72.39, *b* = 127.71, *c* = 157.63
  Resolution range                                50--2.35 (2.41--2.35)
  Unique reflections                              61670 (4530)
  Multiplicity                                    8.9 (8.4)
  Completeness (%)                                100 (99.0)
  *R*~merge~[†](#tfn1){ref-type="table-fn"} (%)   9.9 (55.3)
  Mean *I*/σ(*I*)                                 19.5 (4.2)
  ----------------------------------------------- -----------------------------------------

*R* ~merge~ = .

###### Refinement and model statistics

Values in parentheses are for the highest of 20 resolution shells.

  ------------------------------------------- ------------------------------------------------------------------
  Resolution range (Å)                        50--2.35 (2.41--2.35)
  *R*~cryst~[†](#tfn2){ref-type="table-fn"}   0.177 (0.233)
  *R*~free~[†](#tfn2){ref-type="table-fn"}    0.222 (0.272)
  R.m.s.d. bonds (Å)                          0.010
  R.m.s.d. angles (°)                         1.180
  Protein atoms                               10241
  Nonprotein atoms                            574
  Mean *B* factor (Å^2^)                      26.95
  Ligand *B* factor (Å^2^)                    33.2
  Residues in favored region (%)              97.56
  Residues in allowed region (%)              99.7
  *MolProbity*score \[percentile\]            1.50 \[99th\]
  PDB code                                    [3mmt](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=pdb&pdbId=3mmt)
  ------------------------------------------- ------------------------------------------------------------------

*R* ~cryst~ = . The free *R* factor was calculated using the 5% of the reflections that were omitted from the refinement.