Macromolecular Crystals Research Articles

Practical courses on advanced methods in macromolecular crystallization: 20 years of history and future perspectives.

The first Federation of European Biochemical Societies Advanced Course on macromolecular crystallization was launched in the Czech Republic in October 2004. Over the past two decades, the course has developed into a distinguished event, attracting students, early career postdoctoral researchers and lecturers. The course topics include protein purification, characterization and crystallization, covering the latest advances in the field of structural biology. The many hands-on practical exercises enable a close interaction between students and teachers and offer the opportunity for students to crystallize their own proteins. The course has a broad and lasting impact on the scientific community as participants return to their home laboratories and act as nuclei by communicating and implementing their newly acquired knowledge and skills.

Lattice-Cluster-Theory-Informed Cross-Fractionation Chromatography Revealing Degree of Crystallinity of Single Macromolecular Species.

The relationship between macromolecular architecture and crystallization properties is a relevant research topic in polymer science and technology. The average degree of crystallinity of disperse polymers is a well-studied quantity and is accessible by various experimental methods. However, how the different macromolecular species contribute to the degree of crystallinity and, in particular, the relationship between a certain macromolecular architecture and the degree of crystallinity are not accessible today, neither experimentally nor theoretically. Therefore, in this work, a lattice cluster theory (LCT)-informed cross-fractionation chromatography (CFC) approach is developed to access the degree of crystallinity of single and nonlinear macromolecular species crystallizing from solution. The method entangles high-throughput experimental data from CFC with the LCT for semicrystalline polymers to predict the degree of crystallinity of polymer species with different molecular weights and branching. The approach is applied to a linear low-density polyethylene (ethylene/1-octene copolymer) and a high-density polyethylene, which have specific and different bivariate distributions. The degree of crystallinity of individual macromolecular species of these polymer samples is calculated, and the predicted average degree of crystallinity is compared with experimental measurements, thus successfully validating the approach. Furthermore, the average segment length between branches is introduced as a characteristic molecular feature of branched polyethylene, and its relationship with the degree of crystallinity of certain species is established.

On-Surface Fabrication toward Polar 2D Macromolecular Crystals.

Polar 2D macromolecular structures have attracted significant attention because of their ferroelectricity and ferro-magnetism. However, it is challenging to synthesize them experimentally because dipoles or spins of these macromolecules tend to cancel each other. So far, there has been no successful strategy for assembling macromolecules in a unidirectional manner, achieving stereoregular polymerization on metal surfaces, and creating polar 2D polymer crystals. Recent progress in molecular assembly, on-surface polymer synthesis, and direct control of molecules using electric field applications provides an opportunity to develop such strategies. In this regard, we first review past studies on chiral and achiral molecular assembly, on-surface polymer synthesis, and orientation control of polar molecules. Then, we discuss our newly developed approach called "vectorial on-surface synthesis", which is based on "dynamic chirality" of compass precursors, stereoselective polymerization, and favorable interchain interactions originating from CH-π interactions. Finally, we conclude with a prospective outlook.

Triple-Mechanism Enhanced Flexible SiO2 Nanofiber Composite Hydrogel with High Stiffness and Toughness for Cartilaginous Ligaments.

Hydrogels are widely used in tissue engineering, soft robotics and wearable electronics. However, it is difficult to achieve both the required toughness and stiffness, which severely hampers their application as load-bearing materials. This study presents a strategy to develop a hard and tough composite hydrogel. Herein, flexible SiO2 nanofibers (SNF) are dispersed homogeneously in a polyvinyl alcohol (PVA) matrix using the synergistic effect of freeze-drying and annealing through the phase separation, the modulation of macromolecular chain movement and the promotion of macromolecular crystallization. When the stress is applied, the strong molecular interaction between PVA and SNF effectively disperses the load damage to the substrate. Freeze-dried and annealed-flexible SiO2 nanofibers/polyvinyl alcohol (FDA-SNF/PVA) reaches a preferred balance between enhanced stiffness (13.71 ± 0.28MPa) and toughness (9.9 ± 0.4MJ m-3). Besides, FDA-SNF/PVA hydrogel has a high tensile strength of 7.84 ± 0.10MPa, super elasticity (no plastic deformation under 100 cycles of stretching), fast deformation recovery ability and excellent mechanical properties that are superior to the other tough PVA hydrogels, providing an effective way to optimize the mechanical properties of hydrogels for potential applications in artificial tendons and ligaments.

Effect of ball burnishing on surface roughness, wettability, mechanical integrity and antifouling resistance of UHMWPE

The aim of this paper is to investigate the effect of plasticity burnishing on the surface properties and antifouling resistance of UHMWPE. Ball burnishing experiments at different pressures (44–131 bar) and path strategies (1: Single Parallel and 2: Cross Parallel) were conducted. The treated surface was characterised in terms of surface roughness, surface topography, and contact angle (CA) to evaluate its surface wettability. Microhardness, wear and impact resistance were measured to assess mechanical integrity. Results show that as the burnishing pressure increased, surface roughness decreased. Strategy 2 demonstrated the most significant reduction in surface roughness. No clear relationship between burnishing pressure and CA was found. However, Strategy 2 with relatively higher pressure generated specific surface micro-texture that was responsible for higher CA over Strategy 1. Marine salt spray tests demonstrated improved water adsorption stably, creating a uniform hydration layer on the surface treated with Strategy 2. Consequently, in-field marine fouling test results revealed an improved antifouling resistance of the burnished surface. The burnishing increased microhardness, wear and impact resistance. Improvement in surface integrity was attributed to stretching and plastic yielding of entangled macromolecular chains and crystals networks in UHMWPE. The findings indicate that ball burnishing with appropriate path pattern and pressure could be a viable route to improve surface wettability and antifouling resistance.

Electron counting with direct electron detectors in MicroED

The combination of high sensitivity and rapid readout makes it possible for electron-counting detectors to record cryogenic electron microscopy data faster and more accurately without increasing the number of electrons used for data collection. This is especially useful for MicroED of macromolecular crystals where the strength of the diffracted signal at high resolution is comparable to the surrounding background. The ability to decrease fluence also alleviates concerns about radiation damage which limits the information that can be recovered from a diffraction measurement. The major concern with electron-counting direct detectors lies at the low end of the resolution spectrum: their limited linear range makes strong low-resolution reflections susceptible to coincidence loss and careful data collection is required to avoid compromising data quality. Nevertheless, these cameras are increasingly deployed in cryo-EM facilities, and several have been successfully used for MicroED. Provided coincidence loss can be minimized, electron-counting detectors bring high potential rewards.

Excessive free radical grafting interferes with the macromolecular association and crystallization of brined porcine myofibrils during heat-set gelatinization

Free radical grafting and oxidative modification show superiority in myofibrillar protein (MP) aggregation patterns during salting process, but their consequent formation mechanisms of protein hydration network require further evaluation. Herein, we explored the effect of salt-curing (0, 1, 3 and 5 %) on MP protein polymer substrate, water-protein interaction, crystallization events and thermal stability under H2O2/ascorbate-based hydroxyl radical (•OH)-generating system (HRGS) (1, 10, 20 mM H2O2). Results showed that moderate salting (≤3%) favored the water binding of MP gels during the oxidation course. Accordingly, the maximum thermal stability (Tm) of MP gels was obtained at 3 % salting could be greatly attributed to the protein chain solubilization and refolding process. However, 5 % salt synergized with •OH oxidation intensified diffraction peak 2 (the most striking diffraction feature). Microstructural analysis validated a maximum compactness of MP gel following brining with 5 % salt at potent oxidation strength (20 mM H2O2). This study maybe promises efficient strategy to the myogenetic fibril products and biomaterials.

Macromolecular Architecture-Dependent Polymorphous Crystallization Behavior of PVDF in the PVDF/γ-BL System via Thermally Induced Phase Separation.

This study investigates the effect of the macromolecular architecture of poly(vinylidene fluoride) (PVDF) on its thermally induced phase separation (TIPS) behavior and polymorphic crystallization in the PVDF/γ-butyrolactone (PVDF/γ-BL) system. Preparative PVDF fractions with specific macromolecular architecture and phase constitution are generated. The results show that PVDF's macromolecular architecture, particularly the degree of branching and regio-defects, plays a significant role in its temperature-dependent crystallization and resulting polymorphic phases. While regio-defects dominate crystallization in the temperature range between 30 and 25 °C, the degree of branching becomes decisive in the 25-20 °C interval. The developed fractions of PVDF are further analyzed in terms of their molecular weight distribution, revealing that the PVDF fractions crystallized out of solution have similar molecular weight distributions with lower dispersity compared with the feed polymer. These findings are crucial for macromolecular separation and adjustment of PVDF polymorphic properties and hence for the development of tailor-made PVDF matrix materials for composites and membranes. The findings suggest the possibility of polymorphous phase tailoring of PVDF based on macromolecular architecture due to temperature-controlled crystallization out of solution and strongly motivate further research to reveal deeper knowledge of regio-defect and branching influence of PVDF solution crystallization.

Morphological Investigation of Protein Crystals by Atomic Force Microscopy

In this review, we discuss the progress in the investigation of macromolecular crystals obtained through the use of atomic force microscopy (AFM), a powerful tool for imaging surfaces and specimens at high resolution. AFM enables the visualization of soft samples at the nanoscale and can provide precise visual details over a wide size range, from the molecular level up to hundreds of micrometers. The nonperturbative nature, the ability to scan in a liquid environment, and the lack of need for freezing, fixing, or staining make AFM a well-suited tool for studying fragile samples such as macromolecular crystals. Starting from the first morphological investigations revealing the surface morphology of protein crystals, this review discusses the achievements of AFM in understanding the crystal growth processes, both at the micro- and nanoscale. The capability of AFM to investigate the sample structure at the single molecular level is analyzed considering in-depth the structure of S-layers. Lastly, high-speed atomic force microscopy (HS-AFM) is discussed as the evolution to overcome the limitations of low imaging speed, allowing for the observation of molecular dynamics and weakly adsorbed, diffusing molecules. HS-AFM has provided intuitive views and directly visualized phenomena that were previously described indirectly, answering questions that were challenging to address using other characterization methods.

Processing macromolecular diffuse scattering data.

Diffuse scattering is a powerful technique to study disorder and dynamics of macromolecules at atomic resolution. Although diffuse scattering is always present in diffraction images from macromolecular crystals, the signal is weak compared with Bragg peaks and background, making it a challenge to visualize and measure accurately. Recently, this challenge has been addressed using the reciprocal space mapping technique, which leverages ideal properties of modern X-ray detectors to reconstruct the complete three-dimensional volume of continuous diffraction from diffraction images of a crystal (or crystals) in many different orientations. This chapter will review recent progress in reciprocal space mapping with a particular focus on the strategy implemented in the mdx-lib and mdx2 software packages. The chapter concludes with an introductory data processing tutorial using Python packages DIALS, NeXpy, and mdx2.

Molecular-dynamics simulations of macromolecular diffraction, part I: Preparation of protein crystal simulations.

Molecular-dynamics (MD) simulations of protein crystals enable the prediction of structural and dynamical features of both the protein and the solvent components of macromolecular crystals, which can be validated against diffraction data from X-ray crystallographic experiments. The simulations have been useful for studying and predicting both Bragg and diffuse scattering in protein crystallography; however, the preparation is not yet automated and includes choices and tradeoffs that can impact the results. Here we examine some of the intricacies and consequences of the choices involved in setting up MD simulations of protein crystals for the study of diffraction data, and provide a recipe for preparing the simulations, packaged in an accompanying Jupyter notebook. This article and the accompanying notebook are intended to serve as practical resources for researchers wishing to put these models to work.

MD simulations of macromolecular crystals: Implications for the analysis of Bragg and diffuse scattering.

Some of our most detailed information about structure and dynamics of macromolecules comes from X-ray-diffraction studies in crystalline environments. More than 170,000atomic models have been deposited in the Protein Data Bank, and the number of observations (typically of intensities of Bragg diffraction peaks) is generally quite large, when compared to other experimental methods. Nevertheless, the general agreement between calculated and observed intensities is far outside the experimental precision, and the majority of scattered photons fall between the sharp Bragg peaks, and are rarely taken into account. This chapter considers how molecular dynamics simulations can be used to explore the connections between microscopic behavior in a crystalline lattice and observed scattering intensities, and point the way to new atomic models that could more faithfully recapitulate Bragg intensities and extract useful information from the diffuse scattering that lies between those peaks.

Gentle equilibration of macromolecular crystals to new solutions with minimal handling

Gentle equilibration of macromolecular crystals to new solutions with minimal handling

EWALD: A macromolecular diffractometer for the second target station.

Revealing the positions of all the atoms in large macromolecules is powerful but only possible with neutron macromolecular crystallography (NMC). Neutrons provide a sensitive and gentle probe for the direct detection of protonation states at near-physiological temperatures and clean of artifacts caused by x rays or electrons. Currently, NMC use is restricted by the requirement for large crystal volumes even at state-of-the-art instruments such as the macromolecular neutron diffractometer at the Spallation Neutron Source. EWALD's design will break the crystal volume barrier and, thus, open the door for new types of experiments, the study of grand challenge systems, and the more routine use of NMC in biology. EWALD is a single crystal diffractometer capable of collecting data from macromolecular crystals on orders of magnitude smaller than what is currently feasible. The construction of EWALD at the Second Target Station will cause a revolution in NMC by enabling key discoveries in the biological, biomedical, and bioenergy sciences.

Characterization of Putative Transcriptional Regulator Orf90

The rapid emergence of antibiotic resistant bacteria is a pressing concern, threatening public health and food security worldwide. Antibiotic resistance commonly originates through conjugative plasmid transfer (CPT), where plasmids are transferred and replicated between bacteria. Although the overall mechanism of CPT is known, many plasmid‐encoded proteins essential for CPT remain unstudied. One such unstudied plasmid encoded protein is orf90, a putative transcriptional regulator located on the multiresistant plasmid pSK41 from Staphylococcus aureus. Methicillin resistant S. aureus(MRSA) is considered a serious threat by the Centers for Disease Control and Prevention (CDC), and how it acquires resistance plasmids is still an open area of study. In this investigation, we sought to gain valuable insight into the structure and character of orf90. We successfully expressed and purified two orf90 constructs, an untagged version (His‐orf90) and a maltose binding protein tagged version (His‐MBP‐orf90). We hypothesize that orf90 will bind the pSK41 origin of transfer and that one of our constructs will yield usable crystals for structure solution. Crystallography trials for His‐Orf90 did not yield macromolecular crystals, possibly due to the sample purity. Binding of His‐Orf90 to a 205 bp region of pSK41 containing the conserved origin of transfer (oriT) was confirmed via EMSA (Electrophoretic Mobility Shift Assay). Additionally, crystallization screens with His‐MBP‐orf90 identified three favorable conditions for crystallization. We have shown that orf90 binds to the pSK41 oriT and that His‐MBP‐orf90 is a promising crystallization construct. Characterizing pSK41 Orf90’s structure and function will further enhance our understanding of CPT, potentially yielding useful data for research aimed at combating antibiotic resistance.

Detecting ice artefacts in processed macromolecular diffraction data with machine learning.

Contamination with diffraction from ice crystals can negatively affect, or even impede, macromolecular structure determination, and therefore detecting the resulting artefacts in diffraction data is crucial. However, once the data have been processed it can be very difficult to automatically recognize this problem. To address this, a set of convolutional neural networks named Helcaraxe has been developed which can detect ice-diffraction artefacts in processed diffraction data from macromolecular crystals. The networks outperform previous algorithms and will be available as part of the AUSPEX web server and the CCP4-distributed software.

Integral equation models for solvent in macromolecular crystals.

The solvent can occupy up to ∼70% of macromolecular crystals, and hence, having models that predict solvent distributions in periodic systems could improve the interpretation of crystallographic data. Yet, there are few implicit solvent models applicable to periodic solutes, and crystallographic structures are commonly solved assuming a flat solvent model. Here, we present a newly developed periodic version of the 3D-reference interaction site model (RISM) integral equation method that is able to solve efficiently and describe accurately water and ion distributions in periodic systems; the code can compute accurate gradients that can be used in minimizations or molecular dynamics simulations. The new method includes an extension of the Ornstein–Zernike equation needed to yield charge neutrality for charged solutes, which requires an additional contribution to the excess chemical potential that has not been previously identified; this is an important consideration for nucleic acids or any other charged system where most or all the counter- and co-ions are part of the “disordered” solvent. We present several calculations of proteins, RNAs, and small molecule crystals to show that x-ray scattering intensities and the solvent structure predicted by the periodic 3D-RISM solvent model are in closer agreement with the experiment than are intensities computed using the default flat solvent model in the refmac5 or phenix refinement programs, with the greatest improvement in the 2 to 4 Å range. Prospects for incorporating integral equation models into crystallographic refinement are discussed.

Determination of the critical chain length for macromolecular crystallization using structurally flexible polyketones.

Critical chain length that divides small molecule crystallization from macromolecular crystallization is an important index in macro-organic chemistry to predict chain-length dependent properties of oligomers and polymers. However, extensive research on crystallization behavior of individual oligomers has been inhibited by difficulties in their synthesis and crystallization. Here, we report on the determination of critical chain length of macromolecular crystallization for structurally flexible polyketones consisting of 3,3-dimethylpentane-2,4-dione. Discrete polyketone oligomers were synthesized via stepwise elongation up to 20-mer. Powder and single crystal X-ray diffraction showed that the critical chain length for polyketones existed at an unexpectedly short chain length, 5-mer. While shorter oligomers adopted unique conformations and packing structures in the solid state, higher oligomers longer than 4-mer produced helical conformations and similar crystal packing. The critical chain length helped with understanding the inexplicable changes in melting point in the shorter chain length region resulting from chain conformations and packing styles.

Sectorization of Macromolecular Single Crystals Unveiled by Probing Shear Anisotropy.

Polymer single crystals continue to infiltrate emerging technologies such as flexible organic field-effect transistors because of their excellent translational symmetry and chemical purity. However, owing to the methodological challenges, direct imaging of the polymer chains folding direction resulting in sectorization of single crystals has rarely been investigated. Herein, we directly image the sectorization of polymer single crystals through anisotropic elastic deformation on the surface of macromolecular single crystals. A variant of friction force microscopy, in which the scanning direction of the probe tip is parallel with the cantilever axis, allows for high contrast imaging of the sectorization in polymer single crystals. The lateral deflection of the cantilever resulting from shear forces transverse to the scan direction shows a close connection with the in-plane components of the elastic tensor of the polymer single crystals, which is of a fundamentally different origin than the friction forces. This allows for fast, facile, and nondestructive characterization of the microstructure and in-plane elastic anisotropy of compliant crystalline materials such as polymers.

A Comparison of Structure Determination of Small Organic Molecules by 3D Electron Diffraction at Cryogenic and Room Temperature

3D electron diffraction (3D ED), also known as micro-crystal electron diffraction (MicroED), is a rapid, accurate, and robust method for structure determination of submicron-sized crystals. 3D ED has mainly been applied in material science until 2013, when MicroED was developed for studying macromolecular crystals. MicroED was considered as a cryo-electron microscopy method, as MicroED data collection is usually carried out in cryogenic conditions. As a result, some researchers may consider that 3D ED/MicroED data collection on crystals of small organic molecules can only be performed in cryogenic conditions. In this work, we determined the structure for sucrose and azobenzene tetracarboxylic acid (H4ABTC). The structure of H4ABTC is the first crystal structure ever reported for this molecule. We compared data quality and structure accuracy among datasets collected under cryogenic conditions and room temperature. With the improvement in data quality by data merging, it is possible to reveal hydrogen atom positions in small organic molecule structures under both temperature conditions. The experimental results showed that, if the sample is stable in the vacuum environment of a transmission electron microscope (TEM), the data quality of datasets collected under room temperature is at least as good as data collected under cryogenic conditions according to various indicators (resolution, I/σ(I), CC1/2 (%), R1, Rint, ADRA).