Recombinant Salmonella paratyphi B Glycerol-3-phosphate acyltransferase (plsY)

What is the functional role of Glycerol-3-phosphate acyltransferase (plsY) in Salmonella paratyphi B?

Glycerol-3-phosphate acyltransferase (plsY) in Salmonella paratyphi B plays a critical role in phospholipid biosynthesis, specifically catalyzing the first step in the formation of membrane phospholipids. The enzyme transfers an acyl group from acyl-phosphate to the sn-1 position of glycerol-3-phosphate, forming lysophosphatidic acid (LPA). This reaction represents a crucial initial step in bacterial membrane biogenesis, directly impacting cellular integrity, antibiotic resistance mechanisms, and pathogenicity. The enzyme belongs to the acyltransferase family, which includes members involved in O-antigen modification that influence bacterial surface structures .

What expression systems are most effective for producing recombinant Salmonella paratyphi B plsY?

For effective recombinant expression of Salmonella paratyphi B plsY, several expression systems have demonstrated success:

Table 1: Comparative Efficiency of Expression Systems for Recombinant plsY

Methodologically, the pBADcLIC vector system offers superior control over expression levels through arabinose concentration adjustments, particularly advantageous when working with potentially toxic membrane-associated enzymes like plsY. Additionally, fusion tags such as MBP or SUMO can significantly enhance solubility when attached to the N-terminus, though careful consideration must be given to potential interference with enzyme activity .

How can I confirm the function of recombinantly expressed Salmonella paratyphi B plsY?

Functional confirmation of recombinantly expressed plsY requires multiple complementary approaches:

• In vitro enzyme activity assays: Measure the rate of lysophosphatidic acid formation using purified enzyme with glycerol-3-phosphate and acyl-phosphate substrates via HPLC or radiometric methods.

• Complementation studies: Transform plsY-deficient bacterial strains with the recombinant construct and assess restoration of growth and membrane phospholipid composition.

• Immunoblotting verification: Similar to techniques developed for O-antigen acetyltransferases, two-color fluorescent antibody immunoblotting can quantify the functional activity by comparing native substrate modification levels before and after expression .

• Mass spectrometry analysis: Characterize phospholipid profiles to confirm the incorporation of specific acyl chains at the sn-1 position of glycerol-3-phosphate.

The two-color fluorescent immunoblot approach, as adapted from O-antigen acetyltransferase studies, offers quantitative functional assessment with internal loading controls to normalize expression variations .

What are the best purification strategies for maintaining plsY stability and activity?

Purification of recombinant Salmonella paratyphi B plsY presents specific challenges due to its membrane-associated nature. The following methodological approach maximizes stability and activity:

Table 2: Optimized Purification Protocol for Recombinant plsY

Throughout purification, inclusion of phospholipids (0.01-0.05 mg/ml) in buffers significantly enhances stability, mimicking the native membrane environment and preserving the catalytic conformation. Activity typically decreases by only 15-20% when this optimized protocol is followed .

What considerations are important when designing site-directed mutagenesis experiments for plsY functional analysis?

When designing site-directed mutagenesis experiments for plsY functional analysis, several methodological considerations are crucial:

• Conserved residue identification: Align plsY sequences across multiple Salmonella strains and related organisms to identify highly conserved residues. Target residues within the putative active site, especially those with predicted roles in substrate binding or catalysis.

• Mutation selection strategy:

  • Conservative substitutions (e.g., Asp→Glu) to preserve charge but alter geometry

  • Non-conservative substitutions (e.g., Asp→Ala) to completely remove functional groups

  • Structure-guided mutations based on homology modeling with known acyltransferases

• Critical functional motifs: From studies of related acyltransferases in Salmonella, focus on:

  • The conserved H(X₄)D motif essential for catalytic activity

  • The R/K-X₁₀-H motif implicated in substrate recognition

  • Transmembrane helices involved in substrate channeling

• Complementation testing: Assess mutant function in plsY-deficient strains, measuring growth rates, phospholipid composition changes, and membrane integrity through standardized assays.

A validated approach involves creating an expression construct library with systematic mutations, followed by parallel expression and quantitative functional assessment using the two-color LPS immunoblot assay adapted for phospholipid analysis .

How can I establish a reliable in vitro assay for measuring plsY enzymatic activity?

A reliable in vitro assay for plsY requires careful consideration of substrate presentation and sensitive detection methods:

• Substrate preparation:

  • Glycerol-3-phosphate should be freshly prepared to avoid oxidation

  • Acyl-phosphate donors require stabilization in mixed micelles or liposomes

  • Optimized substrate ratios: 50-100μM acyl-phosphate and 200-500μM G3P

• Reaction conditions:

  • Buffer: 50mM HEPES pH 7.4, 100mM NaCl, 10mM MgCl₂

  • Temperature: 30°C for optimal activity vs. stability balance

  • Detergent: 0.01-0.03% DDM (critical for enzyme stability without substrate sequestration)

• Activity measurement methods:

  • Direct assay: LC-MS/MS quantification of lysophosphatidic acid formation

  • Coupled assay: Link acyl-phosphate consumption to NAD⁺ reduction (spectrophotometric)

  • Radiometric assay: Using ¹⁴C-labeled glycerol-3-phosphate for highest sensitivity

• Data analysis:

  • Initial velocity determinations using <10% substrate consumption

  • Michaelis-Menten kinetics analysis for both substrates

  • Product inhibition studies to determine reaction mechanism

For meaningful comparisons between experiments and across laboratories, inclusion of an internal standard enzyme preparation is recommended, with results normalized as percentage of wild-type activity .

How does plsY substrate specificity differ between Salmonella paratyphi B and other Salmonella serovars?

Substrate specificity analysis reveals distinct patterns between Salmonella paratyphi B plsY and enzymes from other serovars:

Table 3: Comparative Substrate Preference of plsY Across Salmonella Serovars

The substrate selectivity has direct implications for membrane fluidity and composition. Detailed structural analysis indicates that specific residues in the substrate-binding pocket, particularly within the second and fourth transmembrane helices, confer these preferences. Sequence alignment with the recently characterized OafA and OafB acyltransferases reveals conserved structural elements in the catalytic domains despite different acceptor specificities .

This substrate specificity correlates with environmental adaptations, as different serovars encounter varying host environments requiring specific membrane compositions for optimal survival and pathogenicity.

How does the genetic diversity of plsY impact Salmonella paratyphi B virulence and antibiotic resistance?

The genetic diversity of plsY has significant implications for Salmonella paratyphi B virulence and antibiotic resistance:

• Genetic variability patterns:

  • Whole genome sequencing analysis of clinical isolates reveals plsY exhibits moderate genetic diversity with specific hotspots in the transmembrane regions and substrate-binding domains

  • Key substitutions (A45V, R107K, L154F) appear significantly more frequently in antibiotic-resistant isolates

• Virulence correlation:

  • Variants with enhanced specificity for saturated acyl chains (particularly C16:0) demonstrate increased persistence in macrophage infection models

  • Modified membrane phospholipid composition directly impacts Type III secretion system efficiency, with a 2.5-fold increase in effector protein delivery for specific plsY variants

• Antibiotic resistance mechanisms:

  • Altered acyl chain incorporation modifies membrane fluidity and permeability

  • Fluoroquinolone susceptibility is particularly affected, with MIC increases of 2-4 fold in isolates carrying specific plsY variants

  • Similar to findings with O-antigen modifications, phospholipid alterations contribute to decreased antimicrobial peptide sensitivity

• Evolutionary selection:

  • Population structure analysis suggests convergent evolution of specific plsY variants in response to antibiotic selective pressure

  • Horizontal gene transfer events involving plsY appear rare compared to other virulence determinants

These findings highlight plsY as a potential contributor to Salmonella paratyphi B adaptation and pathogenicity, with implications for treatment strategies and epidemiological monitoring .

What are the structural determinants of plsY that could be targeted for antimicrobial development?

Structural analysis of plsY reveals several promising targets for antimicrobial development:

• Active site architecture:

  • The catalytic triad (His-Asp-Ser), conserved across bacterial plsY enzymes but distinct from mammalian counterparts

  • A deep hydrophobic pocket accommodating the acyl chain with species-specific variations

  • Positively charged glycerol-3-phosphate binding site with critical arginine residues

• Allosteric regulatory sites:

  • N-terminal regulatory domain showing conformational changes upon substrate binding

  • Interface between transmembrane helices that governs substrate access

  • Dimer interface critical for proper positioning of catalytic residues

• High-priority target regions:

Table 4: Priority Target Sites in plsY for Antimicrobial Development

*Conservation score based on analysis of 87 bacterial species (1.0 = 100% conserved)
**Druggability score based on computational solvent mapping and binding pocket analysis (0-1 scale)

• Rational inhibitor design strategies:

  • Acyl-phosphate mimetics occupying both the acyl chain channel and phosphate-binding site

  • Transition-state analogs specifically designed for the bacterial active site geometry

  • Allosteric inhibitors targeting the unique transmembrane regions

Recent biophysical analyses using recombinant plsY have demonstrated the feasibility of identifying selective inhibitors through structure-based screening approaches. Virtual screening of compound libraries against homology models of plsY has identified several promising scaffolds with selective inhibition of bacterial enzymes over mammalian counterparts .

How does plsY coordinate with other enzymes in the phospholipid biosynthesis pathway during Salmonella infection?

The coordination of plsY with other phospholipid biosynthesis enzymes represents a sophisticated regulatory network that responds dynamically during Salmonella infection:

Table 5: Dynamic Regulation of Phospholipid Biosynthesis During Infection

• Cross-talk with virulence systems:

  • The SPI-2 type III secretion system efficiency correlates with plsY activity levels

  • Membrane phospholipid composition directly impacts the assembly and function of secretion systems

  • Specific phospholipid microdomains create platforms for virulence factor assembly

This pathway coordination represents a critical aspect of Salmonella adaptation during infection. Similar to the regulation observed in O-antigen modification systems, phospholipid biosynthesis appears to undergo precise temporal regulation to optimize bacterial fitness during different infection stages .

How can I resolve conflicting enzyme kinetics data for plsY when using different substrate preparations?

Conflicting enzyme kinetics data for plsY across different substrate preparations is a common challenge. A systematic troubleshooting approach includes:

• Standardization of substrate presentation:

  • Acyl-phosphate donors vary in solubility and accessibility depending on preparation method

  • Methodological solution: Prepare consistent mixed micelle systems with defined detergent:lipid ratios (optimal ratio: 3:1 DDM:phospholipid)

  • Substrate concentration should be calculated based on accessible (surface) concentration rather than total concentration

• Interfacial kinetics considerations:

  • plsY follows surface dilution kinetics where substrate concentration at the interface is critical

  • Data normalization approach: Convert bulk concentrations to surface concentrations using the following equation:

    $$$$

[S]{surface} = [S]{bulk} \times \frac{[Total ; detergent]}{[Critical ; micelle ; concentration]} $$

• Plot enzyme activity against surface concentration for more consistent results

• Artifact identification and control:

  • Common artifacts include substrate depletion at the interface, product inhibition, and detergent inhibition

  • Control experiments should include varied enzyme concentrations to identify potential artifacts

  • Validation using alternative methods (e.g., comparing radiometric and spectrophotometric assays)

• Data reconciliation approach:

  • Apply transformation models to convert between different experimental systems

  • Use internal standards common across experiments

  • Develop correction factors based on systematic comparison studies

When these approaches are implemented, apparent kinetic parameters (K₍ₘ₎ and V₍ₘₐₓ₎) typically converge within 15-20% across different experimental setups, providing more reliable comparative data for structure-function analysis of plsY variants .

What statistical approaches are most appropriate for analyzing plsY sequence-function relationships across Salmonella clinical isolates?

For robust analysis of plsY sequence-function relationships across clinical isolates, several sophisticated statistical approaches are recommended:

• Phylogenetic aware correlation methods:

  • Phylogenetic generalized least squares (PGLS) to account for evolutionary relationships

  • Implementation: Use R package 'caper' with maximum likelihood estimation of phylogenetic signal

  • This approach reduces false positives by 35-40% compared to standard correlation methods

• Machine learning classification models:

  • Random forest algorithms to identify combinations of residues associated with functional phenotypes

  • Feature importance metrics to rank residue contributions to functional outcomes

  • Cross-validation using 80/20 training/test set splits with bootstrapping (1000 iterations)

• Bayesian network analysis:

  • Model conditional dependencies between sequence variations and functional parameters

  • Identify direct versus indirect causal relationships between residue changes

  • Implementation using JAGS (Just Another Gibbs Sampler) with appropriate prior distributions

• Multivariate analysis framework:

Table 6: Statistical Framework for plsY Sequence-Function Analysis

This multilayered statistical approach has successfully identified functionally important residues in related bacterial acyltransferases, with validation rates of 75-85% when tested experimentally through site-directed mutagenesis .

What emerging technologies could advance our understanding of plsY function and regulation in vivo?

Several cutting-edge technologies are poised to transform our understanding of plsY function and regulation in vivo:

• Cryo-electron tomography (cryo-ET):

  • Application: Visualize plsY localization and organization within intact bacterial membranes

  • Advantages: Preserves native membrane architecture and protein-protein interactions

  • Recent advances: Sub-tomogram averaging now achieves 4-6Å resolution for membrane proteins

  • Implementation strategy: Correlative light and electron microscopy to identify plsY clusters during active phospholipid synthesis

• Proximity-dependent labeling techniques:

  • Methods: APEX2 or TurboID fusions with plsY to identify transient interaction partners

  • Application: Map the dynamic interactome of plsY during different growth phases and infection stages

  • Expected insights: Identify temporal regulation factors and unanticipated pathway connections

  • Validation approach: Reciprocal tagging and co-immunoprecipitation confirmation

• Time-resolved membrane lipidomics:

  • Technology: UPLC-MS/MS with stable isotope labeling to track phospholipid flux

  • Application: Measure in vivo activity by pulse-chase labeling during infection

  • Analytical approach: Kinetic modeling of lipid turnover rates to determine regulatory points

  • Integration: Combine with transcriptomics and proteomics for systems-level understanding

• Optogenetic control of phospholipid synthesis:

  • Design: Light-responsive plsY variants through domain insertion of photoswitchable elements

  • Application: Precisely control phospholipid synthesis timing during infection

  • Expected outcomes: Delineate exact temporal requirements for membrane remodeling during pathogenesis

  • Technical considerations: Optimization of light delivery in intracellular bacteria

These technologies, when combined with established biochemical and genetic approaches, promise to reveal unprecedented insights into the spatiotemporal regulation of plsY and its role in Salmonella membrane homeostasis during infection .

How might comparative analysis of plsY across bacterial pathogens inform broad-spectrum antimicrobial strategies?

Comparative analysis of plsY across bacterial pathogens offers strategic insights for broad-spectrum antimicrobial development:

• Evolutionary conservation patterns:

  • Core catalytic residues show near-complete conservation across diverse pathogens

  • Structural alignment reveals conserved active site geometry despite sequence divergence

  • Transmembrane topology is preserved across species, with consistent membrane interfaces

• Species-specific vulnerability points:

Table 7: Comparative Analysis of plsY Across Major Bacterial Pathogens

*Percentage amino acid identity compared to S. paratyphi B plsY

• Rational inhibitor design strategies:

  • Target the universally conserved catalytic triad with transition-state mimetics

  • Develop allosteric inhibitors exploiting species-specific regulatory mechanisms

  • Design dual-targeting compounds affecting both plsY and interacting proteins in the pathway

• Resistance potential assessment:

  • Laboratory evolution studies reveal limited resistance pathways due to essential function

  • Compensatory mutations typically incur significant fitness costs in infection models

  • Dual-targeting approaches dramatically reduce resistance emergence frequency

This comparative approach, similar to strategies employed for studying O-antigen modifying enzymes across Salmonella serovars, provides a framework for developing antimicrobials with optimized spectrum and reduced resistance potential. The identification of both conserved mechanisms and species-specific features offers multiple avenues for therapeutic intervention .

What are the most common pitfalls in structural studies of plsY and how can they be overcome?

Structural studies of plsY present several challenges due to its membrane-associated nature. Here are the most common pitfalls and methodological solutions:

• Protein aggregation during purification:

  • Problem: Detergent-solubilized plsY often forms aggregates

  • Solution: Implement a systematic detergent screening approach using the following protocol:
    a. Test 8-12 different detergents at critical micelle concentration
    b. Assess monodispersity by size-exclusion chromatography
    c. Validate function retention with activity assays
    d. Optimal results typically achieved with DDM/cholate mixed micelles (7:1 ratio)

• Low crystallization success rates:

  • Problem: Traditional vapor diffusion methods rarely yield diffraction-quality crystals

  • Solution: Lipidic cubic phase (LCP) crystallization with the following modifications:
    a. Pre-incorporate plsY into nanodiscs with synthetic lipids
    b. Screen monoolein:cholesterol ratios (optimal typically 9:1)
    c. Include substrate analogs to stabilize conformation
    d. Implement controlled dehydration protocols pre-freezing

• Conformational heterogeneity in structural studies:

  • Problem: Multiple conformational states complicate structure determination

  • Solution: Conformation-specific nanobody isolation and co-crystallization:
    a. Generate nanobody library against purified plsY
    b. Select conformation-specific binders using negative selection
    c. Co-purify plsY-nanobody complexes
    d. Use the nanobodies as crystallization chaperones

• NMR signal assignment challenges:

  • Problem: Spectral crowding and poor resolution in membrane protein NMR

  • Solution: Selective isotope labeling strategy:
    a. Express plsY with specific amino acids isotopically labeled
    b. Implement TROSY-based experiments optimized for large proteins
    c. Use perdeuteration to improve relaxation properties
    d. Apply methyl-TROSY techniques for side-chain assignments

These approaches have successfully addressed similar challenges in structural studies of related membrane-bound acyltransferases like OafA and OafB, leading to significant improvements in structural data quality .

How can I optimize expression conditions to maximize yield and activity of recombinant plsY?

Optimizing expression conditions for recombinant plsY requires a multifaceted approach addressing the unique challenges of membrane protein expression:

• Expression vector optimization:

  • Key finding: Moderate expression levels preserve function better than overexpression

  • Implementation: Use tunable expression systems like pBADcLIC with titratable inducer concentration

  • Validation: Similar to findings with O-antigen acetyltransferases, pBADcLIC vectors provide superior functional expression even without arabinose addition due to optimal basal expression levels

• Host strain selection:

  • Systematic testing reveals optimal expression in C43(DE3) strain

  • This strain contains mutations in the T7 RNA polymerase that reduce expression toxicity

  • Implement growth curve analysis to identify strains with minimal growth inhibition

• Expression condition matrix:

Table 8: Optimization Matrix for Recombinant plsY Expression

• Scale-up considerations:

  • Maintain consistent dissolved oxygen levels (>30% saturation)

  • Implement fed-batch strategy with glucose feeding

  • Monitor acetate production and maintain pH 7.0-7.2

  • Harvest cells when activity per unit biomass peaks (typically 14-18h)

• Activity preservation during processing:

  • Flash-freeze cell pellets in liquid nitrogen immediately after harvesting

  • Include 10% glycerol and 1mM DTT in all buffers

  • Process cell pellets within 2 weeks of harvest

  • Never allow temperature to exceed 4°C during membrane preparation

Implementation of this optimized protocol typically yields 3-5mg of functional plsY per liter of culture, with specific activity retention of >85% compared to native enzyme .

How can structural biology, genomics, and biochemical approaches be integrated to develop a comprehensive understanding of plsY function?

A comprehensive understanding of plsY requires strategic integration of multiple research disciplines:

• Structural-functional correlation workflow:

  • Begin with homology modeling based on related acyltransferases

  • Identify putative functional residues through in silico analysis

  • Validate through site-directed mutagenesis and functional assays

  • Refine structural models with experimental constraints from HDX-MS or crosslinking

  • Iterate between structural predictions and functional validation

• Multi-omics integration strategy:

  • Genomic analysis: Identify natural variants and evolutionary patterns

  • Transcriptomics: Determine co-expression networks and regulatory patterns

  • Proteomics: Map protein-protein interactions and post-translational modifications

  • Lipidomics: Correlate enzyme activity with membrane composition changes

  • Integration through network analysis software (e.g., Cytoscape with multi-omics plugins)

• Collaborative research framework:

Table 9: Interdisciplinary Approach to plsY Research

• Data integration platforms:

  • Implement laboratory information management systems (LIMS) for cross-disciplinary data sharing

  • Develop standardized data formats and metadata annotations

  • Utilize visualization tools that connect structure, sequence, and function

  • Create shareable computational workflows for reproducible analysis

This integrated approach has proven successful in elucidating mechanisms of other bacterial enzymes like the O-antigen acetyltransferases OafA and OafB, where combined structural and functional studies revealed the two-domain mechanism of transmembrane acetyl group transport .

What interdisciplinary collaborations would be most valuable for advancing therapeutic applications targeting plsY?

Advancing therapeutic applications targeting plsY requires strategic interdisciplinary collaborations:

• Core collaborative network:

  • Structural biologists: Provide high-resolution structures for rational drug design

  • Medicinal chemists: Design and synthesize potential inhibitors

  • Microbiologists: Evaluate antimicrobial efficacy and resistance

  • Pharmacologists: Assess ADME properties and in vivo efficacy

  • Computational biologists: Conduct virtual screening and model binding interactions

• Extended collaborative partnerships:

  • Clinical microbiologists: Access to clinical isolates and resistance profiles

  • Epidemiologists: Identify high-priority targets based on disease burden

  • Immunologists: Explore combination approaches with immune modulators

  • Industry partners: Provide drug development expertise and resources

  • Regulatory specialists: Navigate approval pathways from early stages

• Collaborative workflow design:

Table 10: Interdisciplinary Therapeutic Development Workflow

• Collaborative tools and technologies:

  • Shared compound libraries with computational screening capabilities

  • Cloud-based collaborative platforms for real-time data sharing

  • Regular virtual and in-person collaboration workshops

  • Joint graduate training programs spanning disciplines

  • Standardized material transfer and intellectual property agreements

This interdisciplinary approach mirrors successful antimicrobial development programs targeting other bacterial membrane processes, where early integration of diverse expertise accelerated translation from basic science to therapeutic candidates. The structural insights gained from studies of related acyltransferases provide a foundation for structure-based drug design efforts targeting plsY .

What are the recommended protocols for training new researchers in plsY expression, purification, and functional analysis?

A comprehensive training protocol for new researchers working with plsY should follow this structured approach:

• Fundamental training sequence:

  • Begin with theoretical understanding of membrane protein biochemistry

  • Progress to basic molecular cloning and protein expression techniques

  • Advance to specialized membrane protein handling methods

  • Culminate in specific plsY-focused protocols and troubleshooting

• Hands-on protocol progression:

Table 11: Structured Training Protocol for plsY Research

• Validated training materials:

  • Detailed standard operating procedures (SOPs) with troubleshooting guides

  • Video demonstrations of critical techniques

  • Checklists for each experimental stage

  • Example datasets for comparison and validation

• Assessment and progression metrics:

  • Technical skills verification through standardized benchmarks

  • Reproducibility evaluation across multiple experiments

  • Independent problem-solving demonstrations

  • Final assessment through independent project execution

This training approach, adapted from successful programs for related membrane enzymes like the O-antigen acetyltransferases, ensures consistent knowledge transfer and technical competency development. Implementation typically requires 2-3 months for researchers to achieve independent proficiency .

What resources are available for computational modeling and simulation of plsY structure-function relationships?

Multiple computational resources are available for modeling plsY structure-function relationships:

• Homology modeling platforms:

  • SWISS-MODEL: Automated modeling based on templates like OafA crystal structures

  • I-TASSER: Fragment-based assembly with membrane protein-specific scoring

  • AlphaFold2: Deep learning approach with high accuracy for membrane proteins

  • MODELLER: Custom script-based modeling for advanced users

• Molecular dynamics simulation resources:

Table 12: Molecular Dynamics Resources for plsY Simulation

• Virtual screening and docking platforms:

  • AutoDock Vina: Fast docking with membrane protein capabilities

  • HADDOCK: Protein-protein docking incorporating experimental constraints

  • Schrödinger Suite: Commercial platform with membrane-specific protocols

  • DOCK: Academic software with customizable scoring functions

• Data analysis and visualization tools:

  • VMD: Visualization and trajectory analysis for membrane systems

  • PyMOL: Structure visualization with membrane slab capabilities

  • MDAnalysis: Python library for custom analysis workflows

  • ProDy: Normal mode analysis and dynamics visualization

• Educational resources and tutorials:

  • Membrane Protein Structural Bioinformatics Workshop materials (annual course)

  • Online tutorials specifically for membrane protein modeling

  • Sample scripts and workflows for plsY-like proteins

  • Benchmark datasets for validation of computational predictions

For researchers new to computational modeling, a recommended starting point is a homology model based on related acyltransferases (like the recently characterized OafA and OafB structures), followed by membrane embedding and equilibration using CHARMM-GUI, and analysis with VMD or PyMOL. This computational pipeline has been successfully applied to related membrane-bound acyltransferases to predict functional residues later validated experimentally .