Recombinant Chromobacterium violaceum Probable GTP-binding protein EngB (engB)

What is Chromobacterium violaceum and why is it significant for EngB research?

Chromobacterium violaceum is a Gram-negative betaproteobacterium found in soil and aquatic ecosystems in tropical and subtropical regions, including the Amazon basin. It is a versatile environmental organism that, while typically saprophytic, can cause rare but potentially fatal infections in humans and animals . The significance of C. violaceum for EngB research stems from its unique genomic properties revealed through complete genome sequencing, which has provided valuable insights into its molecular mechanisms and biotechnological potential . The EngB protein from C. violaceum represents an important model for understanding bacterial GTP-binding proteins involved in essential cellular processes, particularly in the context of organisms that transition between environmental and pathogenic lifestyles.

What is the general function of bacterial GTP-binding proteins like EngB?

Bacterial GTP-binding proteins, including EngB, function as molecular switches that regulate various cellular processes through GTP hydrolysis. These proteins typically cycle between GTP-bound (active) and GDP-bound (inactive) states, enabling them to control processes such as protein synthesis, cell division, and signal transduction. In C. violaceum, EngB is classified as a probable GTP-binding protein based on sequence homology with other bacterial GTPases. The protein likely plays a role in ribosome assembly or biogenesis, cell cycle regulation, or stress response mechanisms that may contribute to the bacterium's environmental adaptability and potential virulence .

How does the genetic context of engB in C. violaceum compare to other bacterial species?

The engB gene in C. violaceum is part of the bacterium's 4.75 Mb genome, which contains approximately 4,431 predicted protein-coding sequences . While specific operon organization for engB in C. violaceum was not detailed in the search results, bacterial engB genes are typically found in conserved genomic contexts across different species. In C. violaceum, genome analysis has revealed sophisticated regulatory networks, including the CviI/R quorum sensing system that regulates various phenotypes including violacein production . Understanding the genetic context of engB requires comparative genomic analysis with other bacterial species to identify conserved patterns in gene arrangement and potential co-regulation with genes involved in related cellular processes.

What are the most effective methods for expressing and purifying recombinant C. violaceum EngB protein?

For expressing recombinant C. violaceum EngB, researchers should consider the following methodology:

• Expression System Selection: Escherichia coli is an effective heterologous host for C. violaceum proteins, as demonstrated in previous studies with other C. violaceum proteins . BL21(DE3) or Rosetta strains are particularly suitable for proteins with potential rare codon usage.

• Vector Construction:

  • Amplify the engB gene using PCR with primers containing appropriate restriction sites (similar to approaches used for other C. violaceum genes )

  • Clone into expression vectors with strong inducible promoters (e.g., pET series) and fusion tags for purification

  • Common fusion tags include His6, GST, or MBP to enhance solubility and facilitate purification

• Expression Optimization:

  • Test multiple induction conditions (temperature, IPTG concentration, induction time)

  • Consider expressing at reduced temperatures (16-25°C) to enhance protein solubility

  • Supplement with rare tRNAs if codon optimization is necessary

• Purification Protocol:

  • Lyse cells using sonication or French press in buffer containing appropriate protease inhibitors

  • Perform initial purification using affinity chromatography based on the fusion tag

  • Further purify using size exclusion and/or ion exchange chromatography

  • Remove fusion tags if necessary using specific proteases

  • Verify protein purity using SDS-PAGE and identity using mass spectrometry

This methodology incorporates approaches similar to those used for molecular studies of other C. violaceum proteins, adapted specifically for GTP-binding proteins .

What challenges might researchers encounter when working with recombinant C. violaceum proteins and how can these be addressed?

Researchers working with recombinant C. violaceum proteins, including EngB, commonly encounter several challenges that require specific troubleshooting approaches:

• Protein Solubility Issues:

  • Challenge: C. violaceum proteins may form inclusion bodies in heterologous expression systems

  • Solution: Optimize expression conditions by lowering temperature (16-20°C), reducing inducer concentration, or using solubility-enhancing fusion partners (MBP, SUMO, or TrxA)

• Codon Usage Bias:

  • Challenge: Differences in codon usage between C. violaceum and expression hosts like E. coli

  • Solution: Use Rosetta or CodonPlus strains that supply rare tRNAs, or synthesize codon-optimized genes

• Protein Stability:

  • Challenge: Some C. violaceum proteins may exhibit reduced stability during purification

  • Solution: Incorporate stabilizing agents (glycerol, specific ions) in purification buffers, and optimize storage conditions through stability screening

• Contamination with Endotoxins:

  • Challenge: Gram-negative bacterial expression systems can introduce endotoxin contamination

  • Solution: Use endotoxin removal steps during purification, especially if downstream applications involve immunological studies

• Host Toxicity:

  • Challenge: Expression of certain C. violaceum proteins may be toxic to host cells

  • Solution: Use tightly regulated expression systems, consider cell-free expression systems, or express toxic domains separately

Addressing these challenges requires iterative optimization of experimental conditions, similar to approaches used in previous C. violaceum protein studies where molecular tools were carefully adapted to overcome species-specific barriers .

How should researchers design nucleotide binding and GTPase activity assays for C. violaceum EngB?

When designing nucleotide binding and GTPase activity assays for C. violaceum EngB, researchers should implement the following methodological framework:

Nucleotide Binding Assays:

• Fluorescence-Based Methods:

  • Measure intrinsic tryptophan fluorescence changes upon nucleotide binding

  • Use fluorescent GTP analogs (MANT-GTP, TNP-GTP) to determine binding kinetics

  • Design titration experiments to establish binding affinities for GTP, GDP, and other nucleotides

• Equilibrium Dialysis or Filter Binding:

  • Utilize radiolabeled nucleotides ([³H]-GTP or [γ-³²P]-GTP) for quantitative binding measurements

  • Implement controls with non-hydrolyzable GTP analogs (GTPγS, GMPPNP) to distinguish binding from hydrolysis

GTPase Activity Assays:

• Colorimetric Phosphate Detection:

  • Malachite green assay to measure released inorganic phosphate

  • MESG/PNP-based continuous assays for real-time monitoring

• HPLC-Based Analysis:

  • Separate and quantify GTP and GDP to directly measure conversion rates

  • Include appropriate controls with known GTPases (e.g., Ras) for comparison

• Coupled Enzyme Assays:

  • Use pyruvate kinase/lactate dehydrogenase system to regenerate GTP and monitor NADH oxidation

Experimental Parameters to Optimize:

When analyzing the data, researchers should determine key enzymatic parameters including Km, kcat, and kcat/Km for GTP hydrolysis, and compare these values with those of EngB proteins from other bacterial species to identify any unique characteristics of the C. violaceum enzyme.

How does the predicted structure of C. violaceum EngB compare to characterized bacterial GTPases?

Based on comparative analysis with other bacterial GTPases, C. violaceum EngB likely possesses a structure with several characteristic domains that define its function:

• G-Domain Architecture: The core structure would include the G-domain containing five conserved G-motifs (G1-G5) characteristic of the GTPase superfamily. These motifs are involved in nucleotide binding and hydrolysis, particularly:

  • G1/P-loop (GXXXXGKS/T): Critical for phosphate binding

  • G2/Switch I: Undergoes conformational changes upon GTP binding

  • G3/Switch II: Contains the catalytic residue for GTP hydrolysis

  • G4 and G5: Involved in guanine base recognition and specificity

• Structural Comparisons: While specific structural data for C. violaceum EngB is not available in the search results, comparative analysis with EngB proteins from other bacteria would suggest:

  • A central β-sheet surrounded by α-helices in a Rossmann fold

  • Unique structural elements that distinguish EngB from other bacterial GTPases like Era, Obg, or FtsY

  • Potential membrane interaction domains, as some EngB proteins associate with the bacterial membrane

• Phylogenetic Positioning: C. violaceum belongs to the Betaproteobacteria class , which may result in EngB structural variations compared to more extensively studied Gammaproteobacteria (like E. coli) or Firmicutes. These variations could affect substrate specificity or interaction partners.

Understanding these structural features is essential for elucidating the specific functions of C. violaceum EngB and its potential roles in bacterial physiology and pathogenesis .

What protein-protein interactions are predicted for C. violaceum EngB and how can these be experimentally verified?

C. violaceum EngB, like other bacterial GTPases, likely participates in several protein-protein interactions that mediate its cellular functions. Predicted interactions and verification methodologies include:

Predicted Interaction Partners:

• Ribosomal Components: EngB proteins often interact with ribosomal proteins or assembly factors, suggesting a role in ribosome biogenesis

• Cell Division Proteins: Potential interactions with divisome components, particularly those involved in Z-ring formation or regulation

• Membrane Proteins: Interactions with membrane proteins that might localize EngB to specific cellular regions

• GTPase-Activating Proteins (GAPs): Proteins that might stimulate the intrinsic GTPase activity of EngB

• Guanine Nucleotide Exchange Factors (GEFs): Factors that facilitate GDP/GTP exchange

Experimental Verification Methods:

• Affinity Purification Coupled with Mass Spectrometry (AP-MS):

  • Express tagged EngB in C. violaceum or heterologous hosts

  • Purify protein complexes under native conditions

  • Identify interaction partners using LC-MS/MS

  • Validate with reciprocal pull-downs

• Bacterial Two-Hybrid System:

  • Construct fusion proteins with split reporter domains

  • Screen against C. violaceum genomic library

  • Quantify interaction strength through reporter activity measurements

• Surface Plasmon Resonance (SPR) or Microscale Thermophoresis (MST):

  • Measure direct binding between purified EngB and candidate partners

  • Determine binding affinities and kinetics in different nucleotide-bound states

  • Assess effects of mutations on binding interactions

• Co-localization Studies:

  • Generate fluorescently tagged EngB and potential partners

  • Observe co-localization patterns using fluorescence microscopy

  • Correlate localization with cell cycle stages or stress conditions

• Cross-linking Coupled with Mass Spectrometry:

  • Utilize in vivo or in vitro crosslinking to capture transient interactions

  • Identify crosslinked peptides to map interaction interfaces

These approaches should be implemented systematically, starting with unbiased screening methods (AP-MS) followed by validation of specific interactions using multiple orthogonal techniques, an approach similar to that used in other C. violaceum protein interaction studies .

How might point mutations in conserved G-motifs affect EngB function in C. violaceum?

Point mutations in the conserved G-motifs of C. violaceum EngB would likely produce specific functional consequences that could be exploited for understanding protein function:

G1/P-loop (GXXXXGKS/T) Mutations:

• K→A substitution in the conserved lysine: Would significantly reduce nucleotide binding affinity and eliminate GTP hydrolysis

• S/T→A substitution: Would impair coordination of the Mg²⁺ ion, reducing both binding and hydrolysis

• These mutations could create a dominant-negative variant that binds nucleotides weakly but cannot hydrolyze GTP

G2/Switch I and G3/Switch II Mutations:

• Mutations in the catalytic glutamine/histidine: Would create a GTP-binding protein with severely reduced GTPase activity

• Mutations in switch regions: Would alter the conformational changes that occur during GTP hydrolysis, potentially "locking" the protein in either the GTP- or GDP-bound state

• These variants would be valuable for crystallographic studies to capture different conformational states

G4 and G5 Mutations:

• Mutations in guanine-recognizing residues: Would alter nucleotide specificity, potentially creating variants that can bind XTP or ATP instead of GTP

• These mutations could help define the nucleotide specificity determinants unique to C. violaceum EngB

Experimental Approaches to Study Mutations:

The functional consequences of these mutations in C. violaceum should be assessed both biochemically (using the purified mutant proteins) and in vivo (by complementation studies in engB deletion strains if viable, or using conditional expression systems if essential). These approaches would provide insights into the specific roles of EngB in C. violaceum physiology and potential connections to its environmental adaptability or pathogenicity .

How does EngB potentially contribute to C. violaceum pathogenicity mechanisms?

The potential contributions of EngB to C. violaceum pathogenicity involve several interconnected mechanisms based on what is known about both the organism's virulence factors and the general functions of bacterial GTPases:

• Stress Response and Environmental Adaptation:

  • C. violaceum transitions from environmental reservoirs to causing opportunistic infections

  • EngB might function in stress response pathways that enable adaptation to host conditions

  • This adaptation is critical for the bacterium's ability to colonize and establish infection after entering through damaged skin or via ingestion

• Cell Division and Growth Regulation:

  • Rapid bacterial replication is essential for establishing infection

  • As a GTPase potentially involved in ribosome assembly or cell division, EngB could regulate growth rates in response to host environments

  • C. violaceum infections are characterized by a rapid clinical course and multiple abscess formation, suggesting efficient bacterial proliferation

• Potential Role in Virulence Factor Expression:

  • C. violaceum produces various virulence factors including cytolytic proteins and factors involved in host cell adhesion and invasion

  • EngB might indirectly regulate the expression or secretion of these virulence factors through its effects on protein synthesis or cellular physiology

  • The severe nature of C. violaceum infections, including their high mortality rate, suggests coordinated expression of multiple virulence factors

• Contribution to Antibiotic Resistance:

  • C. violaceum exhibits resistance to multiple antibiotics including ampicillin, penicillin, rifampicin, erythromycin, and vancomycin

  • EngB's potential role in ribosome assembly might influence sensitivity to ribosome-targeting antibiotics

  • Targeting EngB could potentially enhance the efficacy of certain antibiotics against C. violaceum infections

Understanding these potential contributions of EngB to pathogenicity could inform the development of novel therapeutic approaches targeting this essential bacterial GTPase .

What is known about the regulation of engB expression in C. violaceum under different environmental conditions?

While specific information about engB regulation in C. violaceum is not directly provided in the search results, we can infer potential regulatory mechanisms based on what is known about C. violaceum gene regulation and bacterial GTPases in general:

• Quorum Sensing Regulation:

  • C. violaceum utilizes the CviI/R quorum sensing system that produces and responds to N-acylhomoserine lactones (AHLs), particularly C10-HSL

  • This system regulates various phenotypes including violacein production and potentially other cellular processes

  • engB expression might be influenced by quorum sensing, especially if its function relates to population density-dependent processes like biofilm formation

• Iron-Dependent Regulation:

  • C. violaceum has iron-responsive regulators such as Fur that control gene expression according to iron availability

  • The chuPRSTUV operon involved in heme utilization is regulated by Fur based on iron levels

  • If engB is involved in adaptation to nutrient limitation, its expression might similarly respond to iron availability, which is often limited in host environments

• Environmental Stress Responses:

  • As an environmental bacterium that can cause opportunistic infections, C. violaceum must adapt to various stresses

  • engB expression might be regulated in response to temperature shifts, pH changes, or oxidative stress encountered during host invasion

  • Such regulation would be consistent with the roles of bacterial GTPases in stress adaptation

Methodological Approaches to Study engB Regulation:

To investigate engB regulation in C. violaceum, researchers could employ the following approaches:

• Transcriptional Analysis:

  • qRT-PCR to measure engB mRNA levels under various conditions

  • RNA-seq to identify global expression patterns and co-regulated genes

  • Similar approaches have been used to study other C. violaceum genes

• Promoter Analysis:

  • Construction of reporter fusions (similar to those used for violacein studies )

  • Identification of potential transcription factor binding sites

  • Mutational analysis of regulatory regions

• Condition-Specific Expression Profiling:

  • Examine engB expression during:

    • Growth in different media compositions

    • Exposure to host-relevant conditions (serum, antimicrobial peptides)

    • Biofilm formation versus planktonic growth

    • Iron limitation and other nutrient stresses

These approaches would provide insights into the regulatory networks controlling engB expression and its potential roles in C. violaceum environmental adaptation and pathogenicity .

How might EngB function differ between environmental and infectious contexts for C. violaceum?

The functional versatility of EngB in C. violaceum likely reflects the organism's ability to transition between environmental survival and pathogenic behavior:

Environmental Context Functions:

• Adaptation to Nutrient Fluctuations:

  • In soil and aquatic habitats, C. violaceum faces varying nutrient availability

  • EngB may regulate ribosome assembly and protein synthesis rates in response to environmental nutrient levels

  • This regulation could help conserve energy during nutrient limitation while allowing rapid growth when conditions improve

• Temperature Adaptation:

  • As a resident of tropical and subtropical regions, C. violaceum experiences temperature variations

  • EngB might play a role in adapting cellular processes to temperature shifts

  • This adaptation could involve modulating membrane fluidity or protein folding processes

• Competitive Fitness:

  • EngB's potential role in growth regulation could influence competitive fitness in environmental niches

  • Efficient resource utilization regulated by EngB could provide advantages in microbial communities

Infectious Context Functions:

• Host Temperature Adaptation:

  • During infection, C. violaceum must adapt to the consistent higher temperature of the human host

  • EngB may facilitate the shift from environmental to host temperature

  • This adaptation is critical for successful colonization and proliferation

• Response to Host Defenses:

  • Infections trigger host immune responses including oxidative stress and nutrient restriction

  • EngB might coordinate stress responses and metabolic adjustments to counter these defenses

  • Its role in ribosome function could help rapidly synthesize virulence factors

• Tissue Invasion and Dissemination:

  • C. violaceum infections are characterized by rapid dissemination and abscess formation

  • EngB could support the increased metabolic demands of active invasion

  • Its potential involvement in cell division would facilitate the rapid proliferation needed for dissemination

Key Functional Transitions:

Understanding these context-dependent functions of EngB could provide insights into C. violaceum's successful adaptation from an environmental bacterium to an opportunistic pathogen capable of causing severe infections .

How might high-throughput screening be designed to identify inhibitors of C. violaceum EngB?

A comprehensive high-throughput screening (HTS) approach for identifying C. violaceum EngB inhibitors would involve the following methodological framework:

Primary Screening Assays:

• GTPase Activity-Based Screening:

  • Implement a phosphate-release assay using malachite green detection

  • Adapt to 384-well format for throughput optimization

  • Screen compound libraries at single concentrations (10-20 μM)

  • Include controls: positive (known GTPase inhibitors), negative (DMSO), and enzyme-free

• Thermal Shift Assay (TSA) Screening:

  • Monitor protein thermal stability changes upon compound binding

  • Identify compounds that either stabilize or destabilize EngB

  • This orthogonal approach can identify compounds that bind but might not affect GTPase activity in vitro

Assay Development Parameters:

Hit Validation and Characterization:

• Dose-Response Determination:

  • Test hits in 8-12 point dose-response curves

  • Determine IC50 values and rank compounds by potency

  • Establish structure-activity relationships for hit series

• Mechanism of Action Studies:

  • Nucleotide competition assays to determine competitive vs. non-competitive inhibition

  • Surface plasmon resonance to measure binding kinetics

  • X-ray crystallography or HDX-MS to identify binding sites

• Selectivity Profiling:

  • Counter-screen against human GTPases to assess selectivity

  • Test against EngB proteins from other bacterial species

  • Evaluate activity against a panel of unrelated GTPases

Secondary Cellular Assays:

• Antimicrobial Activity Assessment:

  • Determine minimum inhibitory concentrations against C. violaceum

  • Compare activity against C. violaceum strains with different antibiotic resistance profiles

  • Evaluate activity against other bacterial pathogens

• Target Engagement Validation:

  • Cellular thermal shift assays (CETSA) to confirm EngB binding in bacterial cells

  • Evaluate phenotypic effects consistent with EngB inhibition

  • Generate resistant mutants and sequence to confirm target

This comprehensive screening approach would identify and characterize potential inhibitors of C. violaceum EngB that could serve as chemical probes for studying its function or as starting points for antimicrobial development against this opportunistic pathogen .

What experimental approaches would most effectively elucidate the role of EngB in C. violaceum ribosome assembly?

To comprehensively investigate EngB's role in C. violaceum ribosome assembly, researchers should implement a multi-faceted experimental approach:

Genetic Manipulation Strategies:

• Conditional Expression Systems:

  • Generate strains with engB under control of inducible/repressible promoters

  • Use systems like Tet-on/off to create tunable expression

  • Monitor growth and ribosome profiles at varying expression levels

• Site-Directed Mutagenesis:

  • Create point mutations in key functional domains (G-motifs, potential ribosome interaction sites)

  • Perform complementation assays with mutant variants

  • Assess effects on ribosome assembly and cellular growth

Ribosome Assembly Analysis:

• Polysome Profiling:

  • Fractionate ribosomes on sucrose gradients following EngB depletion/overexpression

  • Quantify shifts in 30S, 50S, 70S, and polysome peaks

  • Compare profiles under various stress conditions relevant to C. violaceum's lifecycle

• rRNA Processing Analysis:

  • Use Northern blotting to monitor precursor and mature rRNA species

  • Implement pulse-chase labeling to track rRNA processing kinetics

  • Correlate processing defects with EngB activity levels

• Ribosome Component Analysis by Mass Spectrometry:

  • Isolate ribosomal particles from EngB-depleted and control cells

  • Characterize protein composition by quantitative proteomics

  • Identify ribosomal proteins with altered incorporation rates

Interaction Studies:

• Ribosome Binding Assays:

  • Examine direct binding of purified EngB to ribosomal subunits or precursors

  • Determine binding affinities in different nucleotide-bound states

  • Map interaction sites using crosslinking and mass spectrometry

• Co-sedimentation Analysis:

  • Analyze co-sedimentation of EngB with various ribosomal particles

  • Compare wild-type EngB with GTPase-deficient mutants

  • Assess nucleotide-dependence of interactions

• Localization Studies:

  • Track EngB localization using fluorescent protein fusions

  • Correlate with ribosomal markers and assembly factors

  • Implement time-lapse microscopy to observe dynamics during stress response

Functional Impact Assessment:

• Translational Fidelity Assays:

  • Use reporter constructs to measure translation accuracy upon EngB depletion

  • Assess miscoding, frameshifting, and stop codon readthrough rates

  • Compare with effects of known ribosome assembly defects

• Antibiotic Sensitivity Profiling:

  • Test whether EngB depletion alters sensitivity to ribosome-targeting antibiotics

  • Connect to C. violaceum's natural antibiotic resistance profile

  • Identify synergistic antibiotic combinations targeting EngB and ribosomes

This systematic approach would elucidate EngB's specific contribution to ribosome assembly in C. violaceum, potentially revealing unique adaptations related to the organism's environmental versatility and pathogenic potential .

How might cryo-EM approaches be optimized for structural studies of C. violaceum EngB in complex with its binding partners?

Optimizing cryo-EM approaches for structural studies of C. violaceum EngB complexes requires careful consideration of sample preparation, data collection, and processing strategies:

Sample Preparation Optimization:

• Complex Formation and Stabilization:

  • Express and purify EngB with potential binding partners (ribosomal components, other interaction proteins)

  • Utilize GTP analogs (GMPPNP, GTPγS) to stabilize specific conformational states

  • Implement GraFix (gradient fixation) or mild crosslinking to stabilize transient complexes

  • Screen buffer conditions extensively to minimize aggregation and optimize particle distribution

• Grid Preparation Enhancement:

  • Test multiple grid types (Quantifoil, C-flat, UltrAuFoil) with varying hole sizes

  • Optimize blotting parameters and ice thickness systematically

  • Implement glow discharge or plasma cleaning with different parameters

  • Consider additives like detergents below CMC to improve particle orientation distribution

Data Collection Strategy:

• Microscope and Detector Parameters:

  • Collect data on high-end microscopes (300kV Titan Krios or equivalent)

  • Utilize energy filters to enhance contrast

  • Implement state-of-the-art direct electron detectors with high DQE

  • Optimize dose rate and total dose for smaller complexes (40-60 e-/Ų)

• Collection Automation:

  • Employ automated data collection software with on-the-fly quality filtering

  • Implement aberration correction and beam-tilt correction

  • Use beam-image shift for efficient multi-hole data collection

  • Consider collecting tilt series for complexes with preferred orientation

Data Processing Workflow:

• Preprocessing Optimization:

  • Implement motion correction with dose-weighting

  • Perform CTF estimation with high accuracy

  • Utilize automated particle picking with subsequent 2D classification

  • Implement extensive cleaning through multiple rounds of 2D and 3D classification

• Advanced Processing Techniques:

  • Apply Bayesian polishing for enhanced resolution

  • Implement CTF refinement including higher-order aberrations

  • Utilize 3D variability analysis to capture conformational heterogeneity

  • Consider multi-body refinement for complexes with flexible domains

• Validation Approaches:

  • Perform half-map FSC analysis with gold-standard refinement

  • Implement local resolution estimation

  • Perform model-map validation

  • Consider tilt-pair validation for smaller complexes

Integration with Complementary Methods:

• Hybrid Structural Approaches:

  • Integrate available crystal structures of homologous proteins

  • Incorporate crosslinking-mass spectrometry data to validate interfaces

  • Use molecular dynamics simulations to interpret flexible regions

  • Implement integrative modeling approaches for complex assembly

• Functional Validation of Structural Insights:

  • Design mutants based on structural information

  • Test functional consequences in biochemical assays

  • Validate in vivo using complementation studies in C. violaceum

These optimized cryo-EM approaches would facilitate the determination of EngB complexes in functionally relevant states, providing unprecedented insights into its molecular mechanisms in C. violaceum and potentially revealing unique features related to this organism's environmental adaptation and pathogenic capabilities .