Rapid Structure Determination Research Articles

Metallo-supramolecular branched polymer protects particles from air-water interface in single-particle cryo-electron microscopy

Recent technological breakthroughs in single-particle cryo-electron microscopy (cryo-EM) enable rapid atomic structure determination of biological macromolecules. A major bottleneck in the current single particle cryo-EM pipeline is the preparation of good quality frozen cryo-EM grids, which is mostly a trial-and-error process. Among many issues, preferred particle orientation and sample damage by air–water interface (AWI) are common practical problems. Here we report a method of applying metallo-supramolecular branched polymer (MSBP) in the cryo-sample preparation for high-resolution single-particle cryo-EM. Our data shows that MSBP keeps a majority of particles away from air–water interface and mitigates preferred orientation as verified by the analyses of apoferritin, hemagglutinin) trimer and various sample proteins. The use of MSBP is a simple method to improve particle distribution for high-resolution structure determination in single-particle cryo-EM.

DFT-based 11B solid-state NMR calculations for guiding fine local structure identification and phase-property modulation in ZnxY1-xBO3-0.5x borate oxide ion conductors.

Solid-state nuclear magnetic resonance (NMR) spectroscopy serves as a powerful technique for probing local structures. However, the interpretation of NMR signals mainly based on empirical knowledge could lead to imprecise local structural determinations. To address this, density functional theory (DFT)-based theoretical NMR calculations, aided by experimental three-dimensional continuous rotation electronic diffraction (3D cRED) technique, were performed for ZnxY1-xBO3-0.5x borate oxide ion conductors. The calculations provided fine local structure identification for the experimental 11B NMR spectra of ZnxY1-xBO3-0.5x, providing rich information on multiple experimental 11B NMR signals towards the complex boron oxide anions associated with bridging oxygen vacancies and the coexistence of the monoclinic (C2/c), hexagonal (P63/m), and trigonal (R32) phases in ZnxY1-xBO3-0.5x. Owing to the advantages of solid-state NMR in identifying closely related phases compared to X-ray/neutron diffraction technique, along with the advanced 3D cRED technique that allows for rapid phase identification and structure determination, we provide a fine local structure identification and a more inclusive insight into the coexistence of multiple phases in borate with the same composition. More importantly, this work provides guidance for phase and property modulation. Phase modulation in ZnxY1-xBO3-0.5x was carried out with thermodynamic and kinetic modulation and eventually realized the tuning of the local structures and the resultant oxide ion conductivity of ZnxY1-xBO3-0.5x. This work provides a theoretical and experimental platform to access the flexible structural assignment of boron oxide anions and therefore offers new guidance and insights into the defect structures and the phase-property modulation of inorganic solid functional materials beyond borate oxide ion conductors.

Rapid and efficient ambient temperature X-ray crystal structure determination at Turkish Light Source

High-resolution biomacromolecular structure determination is essential to better understand protein function and dynamics. Serial crystallography is an emerging structural biology technique which has fundamental limitations due to either sample volume requirements or immediate access to the competitive X-ray beamtime. Obtaining a high volume of well-diffracting, sufficient-size crystals while mitigating radiation damage remains a critical bottleneck of serial crystallography. As an alternative, we introduce the plate-reader module adapted for using a 72-well Terasaki plate for biomacromolecule structure determination at a convenience of a home X-ray source. We also present the first ambient temperature lysozyme structure determined at the Turkish light source (Turkish DeLight). The complete dataset was collected in 18.5 min with resolution extending to 2.39 Å and 100% completeness. Combined with our previous cryogenic structure (PDB ID: 7Y6A), the ambient temperature structure provides invaluable information about the structural dynamics of the lysozyme. Turkish DeLight provides robust and rapid ambient temperature biomacromolecular structure determination with limited radiation damage.

Protein-to-structure pipeline for ambient-temperature in situ crystallography at VMXi.

The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized. Such experiments can allow protein dynamics to be characterized and are particularly well suited to challenging protein targets that may form fragile crystals that are difficult to cryo-cool. Room-temperature data collection also enables time-resolved experiments. In contrast to the high-throughput highly automated pipelines for determination of structures at cryogenic temperatures widely available at synchrotron beamlines, room-temperature methodology is less mature. Here, the current status of the fully automated ambient-temperature beamline VMXi at Diamond Light Source is described, and a highly efficient pipeline from protein sample to final multi-crystal data analysis and structure determination is shown. The capability of the pipeline is illustrated using a range of user case studies representing different challenges, and from high and lower symmetry space groups and varied crystal sizes. It is also demonstrated that very rapid structure determination from crystals in situ within crystallization plates is now routine with minimal user intervention.

Cryo-EM structures of wild-type and E138K/M184I mutant HIV-1 RT/DNA complexed with inhibitors doravirine and rilpivirine

Structures trapping a variety of functional and conformational states of HIV-1 reverse transcriptase (RT) have been determined by X-ray crystallography. These structures have played important roles in explaining the mechanisms of catalysis, inhibition, and drug resistance and in driving drug design. However, structures of several desired complexes of RT could not be obtained even after many crystallization or crystal soaking experiments. The ternary complexes of doravirine and rilpivirine with RT/DNA are such examples. Structural study of HIV-1 RT by single-particle cryo-electron microscopy (cryo-EM) has been challenging due to the enzyme's relatively smaller size and higher flexibility. We optimized a protocol for rapid structure determination of RT complexes by cryo-EM and determined six structures of wild-type and E138K/M184I mutant RT/DNA in complexes with the nonnucleoside inhibitors rilpivirine, doravirine, and nevirapine. RT/DNA/rilpivirine and RT/DNA/doravirine complexes have structural differences between them and differ from the typical conformation of nonnucleoside RT inhibitor (NNRTI)-bound RT/double-stranded DNA (dsDNA), RT/RNA-DNA, and RT/dsRNA complexes; the primer grip in RT/DNA/doravirine and the YMDD motif in RT/DNA/rilpivirine have large shifts. The DNA primer 3'-end in the doravirine-bound structure is positioned at the active site, but the complex is in a nonproductive state. In the mutant RT/DNA/rilpivirine structure, I184 is stacked with the DNA such that their relative positioning can influence rilpivirine in the pocket. Simultaneously, E138K mutation opens the NNRTI-binding pocket entrance, potentially contributing to a faster rate of rilpivirine dissociation by E138K/M184I mutant RT, as reported by an earlier kinetic study. These structural differences have implications for understanding molecular mechanisms of drug resistance and for drug design.

Methyl probes in proteins for determining ligand binding mode in weak protein–ligand complexes

Structures of protein–ligand complexes provide critical information for drug design. Most protein–ligand complex structures are determined using X-ray crystallography, but where crystallography is not able to generate a structure for a complex, NMR is often the best alternative. However, the available tools to enable rapid and robust structure determination of protein–ligand complexes by NMR are currently limited. This leads to situations where projects are either discontinued or pursued without structural data, rendering the task more difficult. We previously reported the NMR Molecular Replacement (NMR2) approach that allows the structure of a protein–ligand complex to be determined without requiring the cumbersome task of protein resonance assignment. Herein, we describe the NMR2 approach to determine the binding pose of a small molecule in a weak protein–ligand complex by collecting sparse protein methyl-to-ligand NOEs from a selectively labeled protein sample and an unlabeled ligand. In the selective labeling scheme all methyl containing residues of the protein are protonated in an otherwise deuterated background. This allows measurement of intermolecular NOEs with greater sensitivity using standard NOESY pulse sequences instead of isotope-filtered NMR experiments. This labelling approach is well suited to the NMR2 approach and extends its utility to include larger protein–ligand complexes.

Rapid Structure Determination of Bioactive 4″-Tetrahydrofurfuryl Macrozone Reaction Mixture Components by LC-SPE/Cryo NMR and MS.

LC-SPE/cryo NMR and MS methodologies have been developed and employed for a rapid structure determination of 4″-tetrahydrofurfuryl macrozone reaction mixture components. Macrozones, novel conjugates of azithromycin, and thiosemicarbazones have shown very good in vitro antibacterial activities against susceptible and some resistant bacterial strains and are promising agents for further development. The post-column multiple trapping of the chromatographically separated reaction mixture components on the SPE cartridges increased the sensitivity and together with cryogenically cooled NMR probe made it possible to identify and structurally characterize main 4″-tetrahydrofurfuryl macrozone reaction mixture compounds including those present at very low concentration level. This approach has several advantages over a classical off-line procedure, efficiency and low solvent consumption being the two most important ones. All identified components were process-related. It has been demonstrated that two different kinds of compounds with respect to structure were identified, i.e., macrolide-related and thiosemicarbazone-related ones. This methodology can serve as a platform for reliable and effective macrolides reaction components structure profiling, serving as both isolation and identification tools.

An automated platform for structural analysis of membrane proteins through serial crystallography

SummaryMembrane proteins are central to many pathophysiological processes, yet remain very difficult to analyze structurally. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins because of lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperatures. We apply this approach to human integral membrane proteins, which allowed us to identify different conformational states of intramembrane enzyme-product complexes and analyze by molecular dynamics simulations the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting, and serial crystallography, enabling screening of small-molecule libraries with membrane protein crystals grown in meso. This approach brings needed automation to this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins.

An Ultrastable π–π Stacked Porous Organic Molecular Framework as a Crystalline Sponge for Rapid Molecular Structure Determination

The crystalline sponge method is a pragmatic and promising strategy for molecular structure determination. However, the dominant metal–organic framework crystal sponge platforms always face poor ch...

The copper(II)-binding tripeptide GHK, a valuable crystallization and phasing tag for macromolecular crystallography.

The growth of diffraction-quality crystals and experimental phasing remain two of the main bottlenecks in protein crystallography. Here, the high-affinity copper(II)-binding tripeptide GHK was fused to the N-terminus of a GFP variant and an MBP-FG peptide fusion. The GHK tag promoted crystallization, with various residues (His, Asp, His/Pro) from symmetry molecules completing the copper(II) square-pyramidal coordination sphere. Rapid structure determination by copper SAD phasing could be achieved, even at a very low Bijvoet ratio or after significant radiation damage. When collecting highly redundant data at a wavelength close to the copper absorption edge, residual S-atom positions could also be located in log-likelihood-gradient maps and used to improve the phases. The GHK copper SAD method provides a convenient way of both crystallizing and phasing macromolecular structures, and will complement the current trend towards native sulfur SAD and MR-SAD phasing.

Experimental observation of the liquid-liquid transition in bulk supercooled water under pressure.

We prepared bulk samples of supercooled liquid water under pressure by isochoric heating of high-density amorphous ice to temperatures of 205 ± 10 kelvin, using an infrared femtosecond laser. Because the sample density is preserved during the ultrafast heating, we could estimate an initial internal pressure of 2.5 to 3.5 kilobar in the high-density liquid phase. After heating, the sample expanded rapidly, and we captured the resulting decompression process with femtosecond x-ray laser pulses at different pump-probe delay times. A discontinuous structural change occurred in which low-density liquid domains appeared and grew on time scales between 20 nanoseconds to 3 microseconds, whereas crystallization occurs on time scales of 3 to 50 microseconds. The dynamics of the two processes being separated by more than one order of magnitude provides support for a liquid-liquid transition in bulk supercooled water.

Liquid-liquid transitions under pressure

Water Phases Theoretical simulations suggest that deeply supercooled water undergoes a transition between high- and low-density forms, but this transition is difficult to study experimentally because it occurs under conditions in which ice crystallization is extremely rapid. Kim et al. combined x-ray lasers for rapid structure determination with infrared femtosecond pulses for rapid heating of amorphous ice layers formed at about 200 kelvin. The heating process created high-density liquid water at increased pressures. As the layer expanded and decompressed, low-density liquid domains appeared and grew on time scales between 20 nanoseconds and 3 microseconds, which was much faster than competing ice crystallization. Science , this issue p. [978][1] [1]: /lookup/doi/10.1126/science.abb9385

Structure of the Pandemic

During global pandemics, the spread of information needs to be faster than the spread of the virus in order to ensure the health and safety of human populations worldwide. In our current crisis, the demand for SARS-CoV-2 drugs and vaccines highlights the importance of biological targets and their three-dimensional shape. In particular, structural biology as a field was poised to quickly respond to crises due to previous experience and expertise and because of its early adoption of open access practices.

Serial protein crystallography in an electron microscope

Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nano-crystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 Å and 1.80 Å, respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation.

Development of “Plug and Play” Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM

"Universal" synthetic antibody (sAB)-based fiducial marks have been generated by customized phage display selections to facilitate the rapid structure determination of G protein-coupled receptor (GPCR) signaling complexes by single-particle cryo-electron microscopy (SP cryo-EM). sABs were generated to the two major G protein subclasses: trimeric Gi and Gs, as well as mini-Gs, and were tested to ensure binding in the context of their cognate GPCRs. Epitope binning revealed that multiple distinct epitopes exist for each G(αβγ) protein. Several Gβγ-specific sABs, cross-reactive between trimeric Gi and Gs, were identified suggesting they could be used across all subclasses in a "plug and play" fashion. sABs were also generated to a representative of another class of GPCR signaling partner, G protein receptor kinase 1 (GRK1) and evaluated further, supporting the generalizability of the approach. EM data suggested that the subclass-specific sABs provide effective single and dual fiducials for multiple GPCR signaling complexes.

Discovery of complex metal oxide materials by rapid phase identification and structure determination

Discovery of complex metal oxide materials by rapid phase identification and structure determination

Rapid Structure Determination of Molecular Solids Using Chemical Shifts Directed by Unambiguous Prior Constraints.

NMR-based crystallography approaches involving the combination of crystal structure prediction methods, ab initio calculated chemical shifts and solid-state NMR experiments are powerful methods for crystal structure determination of microcrystalline powders. However, currently structural information obtained from solid-state NMR is usually included only after a set of candidate crystal structures has already been independently generated, starting from a set of single-molecule conformations. Here, we show with the case of ampicillin that this can lead to failure of structure determination. We propose a crystal structure determination method that includes experimental constraints during conformer selection. In order to overcome the problem that experimental measurements on the crystalline samples are not obviously translatable to restrict the single-molecule conformational space, we propose constraints based on the analysis of absent cross-peaks in solid-state NMR correlation experiments. We show that these absences provide unambiguous structural constraints on both the crystal structure and the gas-phase conformations, and therefore can be used for unambiguous selection. The approach is parametrized on the crystal structure determination of flutamide, flufenamic acid, and cocaine, where we reduce the computational cost by around 50%. Most importantly, the method is then shown to correctly determine the crystal structure of ampicillin, which would have failed using current methods because it adopts a high-energy conformer in its crystal structure. The average positional RMSE on the NMR powder structure is ⟨rav⟩ = 0.176 Å, which corresponds to an average equivalent displacement parameter Ueq = 0.0103 Å2.

Discovery of Complex Metal Oxide Materials by Rapid Phase Identification and Structure Determination.

The discovery of new inorganic functional materials is of fundamental importance in synthetic and materials science. In the past, the discovering new materials relied on a slow and serendipitous trial-and-error process, especially in the well-studied oxide systems. Here, we presented a strategy to shorten the period of discovery of new complex metal oxide materials by rapid phase identification and structure determination with 3D electron diffraction (ED) techniques, which do not require pure samples or single crystal growth. With such strategy, three new complex metal oxide materials (BiTi0.855Fe1.145O4.93, BiTi4FeO11 and BiTi2FeO7) were discovered in the simple ternary Bi2O3-Fe2O3-TiO2 system. To our best knowledge, it is the first time to discover three new complex metal oxide materials with new structure types in a single study of ternary metal oxide system. The structures of new materials were refined by combining powder X-ray diffraction (PXRD) with powder neutron diffraction (PND). The most striking feature in this system is that BiTi0.855Fe1.145O4.93 presents edge-shared five-coordinated iron/titanium polyhedra. In addition, another new phase BiTi4GaO11, which is isostructural with BiTi4FeO11, can be obtained when replacing Fe in BiTi4FeO11 with Ga. The band structure investigation of BiTi0.855Fe1.145O4.93, BiTi4FeO11, BiTi2FeO7 and BiTi4GaO11 shown that they were semiconductors with band gaps of 1.65, 2.0, 1.9, and 2.8 eV, respectively. Although this study focused on rapid developing of new inorganic functional materials, this method for developing new materials is available to all fields in chemistry and material chemistry where the limiting factors are impurity, submicrometer-sized crystals, etc.

CS-ROSETTA.

Chemical Shift-Rosetta (CS-Rosetta) is an automated method that employs NMR chemical shifts to model protein structures de novo. In this chapter, we introduce the terminology and central concepts of CS-Rosetta. We describe the architecture and functionality of automatic NOESY assignment (AutoNOE) and structure determination protocols (Abrelax and RASREC) within the CS-Rosetta framework. We further demonstrate how CS-Rosetta can discriminate near-native structures against a large conformational search space using restraints obtained from NMR data, and/or sequence and structure homology. We highlight how CS-Rosetta can be combined with alternative automated approaches to (i) model oligomeric systems and (ii) create NMR-based structure determination pipeline. To show its practical applicability, we emphasize on the computational requirements and performance of CS-Rosetta for protein targets of varying molecular weight and complexity. Finally, we discuss the current Python interface, which enables easy execution of protocols for rapid and accurate high-resolution structure determination.

Combinatorial use of disulfide bridges and native sulfur-SAD phasing for rapid structure determination of coiled-coils.

Coiled-coils are ubiquitous protein–protein interaction motifs found in many eukaryotic proteins. The elongated, flexible and often irregular nature of coiled-coils together with their tendency to form fibrous arrangements in crystals imposes challenges on solving the phase problem by molecular replacement. Here, we report the successful combinatorial use of native and rational engineered disulfide bridges together with sulfur-SAD phasing as a powerful tool to stabilize and solve the structure of coiled-coil domains in a straightforward manner. Our study is a key example of how modern sulfur SAD combined with mutagenesis can help to advance and simplify the structural study of challenging coiled-coil domains by X-ray crystallography.