Electron diffraction of 1,4-dichlorobenzene embedded in superfluid helium droplets.

We perform electron diffraction of 1,4-dichlorobenzene (C6H4Cl2, referred to as 2ClB) embedded in superfluid helium droplets to investigate the structure evolution of cluster growth. Multivariable linear regression fittings are used to determine the concentration and the best model structures of the clusters. At a droplet source temperature of 22 K with droplets containing on average 5000 He atoms, the fitting results agree with the doping statistics modeled using the Poisson distribution: the largest molecular clusters are tetramers, while the abundances of monomers and dimers are the highest and are similar. Molecular dimers of 2ClB are determined to have a parallel structure with a 60° rotation for the Cl-Cl molecular axes. However, a better agreement between experiment and fitting is obtained by reducing the interlayer distance that had been calculated using the density functional theory for dimers. Further calculations using the highest level quantum mechanical calculations prove that the reduction in interlayer distance does not significantly increase the energy of the dimer. Cluster trimers adopt a dimer structure with the additional monomer slanted against the dimer, and tetramers take on a stacked structure. The structure evolution with cluster size is extraordinary, because from trimer to tetramer, one monomer needs to be rearranged, and neither the trimer nor the tetramer adopts the corresponding global minimum structure obtained using high level coupled-cluster theory calculations. This phenomenon may be related to the fast cooling process in superfluid helium droplets during cluster formation.

Ten things I `hate' about refinement.

Macromolecular refinement is an optimization process that aims to produce the most likely macromolecular structural model in the light of experimental data. As such, macromolecular refinement is one of the most complex optimization problems in wide use. Macromolecular refinement programs have to deal with the complex relationship between the parameters of the atomic model and the experimental data, as well as a large number of types of prior knowledge about chemical structure. This paper draws attention to areas of unfinished business in the field of macromolecular refinement. In it, we describe ten refinement topics that we think deserve attention and discuss directions leading to macromolecular refinement software that would make the best use of modern computer resources to meet the needs of structural biologists of the twenty-first century.

A complete Fourier-synthesis-based backbone-conformation-dependent library for proteins.

While broadening the applicability of (φ/ψ)-dependent target values for the bond angles in the peptide backbone, sequence/conformation categories with too few residues to analyze via previous methods were encountered. Here, a method of describing a conformation-dependent library (CDL) using two-dimensional Fourier coefficients is reported where the number of coefficients for individual categories is determined via complete cross-validation. Sample sizes are increased further by selective blending of categories with similar patterns of conformational dependence. An additional advantage of the Fourier-synthesis-based CDL is that it uses continuous functions and has no artifactual steps near the edges of populated regions of φ/ψ space. A set of libraries for the seven main-chain bond angles, along with the ω and ζ angles, was created based on a set of Fourier analyses of 48 368 residues selected from high-resolution models in the wwPDB. This new library encompasses both trans- and cis-peptide bonds and outperforms currently used discrete CDLs.

Arginine off-kilter: guanidinium is not as planar as restraints denote.

Crystallographic refinement of macromolecular structures relies on stereochemical restraints to mitigate the typically poor data-to-parameter ratio. For proteins, each amino acid has a unique set of geometry restraints which represent stereochemical information such as bond lengths, valence angles, torsion angles, dihedrals and planes. It has been shown that the geometry in refined structures can differ significantly from that present in libraries; for example, it was recently reported that the guanidinium moiety in arginine is not symmetric. In this work, the asymmetry of the Nϵ-Cζ-Nη1 and Nϵ-Cζ-Nη2 valence angles in the guanidinium moiety is confirmed. In addition, it was found that the Cδ atom can deviate significantly (more than 20°) from the guanidinium plane. This requires the relaxation of the planar restraint for the Cδ atom, as it otherwise causes the other atoms in the group to compensate by distorting the guanidinium core plane. A new set of restraints for the arginine side chain have therefore been formulated, and are available in the software package Phenix, that take into account the asymmetry of the group and the planar deviation of the Cδ atom. This is an example of the need to regularly revisit the geometric restraint libraries used in macromolecular refinement so that they reflect the best knowledge of the structural chemistry of their components available at the time.

Making the invisible enemy visible.

During the COVID-19 pandemic, structural biologists rushed to solve the structures of the 28 proteins encoded by the SARS-CoV-2 genome in order to understand the viral life cycle and enable structure-based drug design. In addition to the 204 previously solved structures from SARS-CoV-1, 548 structures covering 16 of the SARS-CoV-2 viral proteins have been released in a span of only 6 months. These structural models serve as the basis for research to understand how the virus hijacks human cells, for structure-based drug design, and to aid in the development of vaccines. However, errors often occur in even the most careful structure determination - and may be even more common among these structures, which were solved quickly and under immense pressure. The Coronavirus Structural Task Force has responded to this challenge by rapidly categorizing, evaluating and reviewing all of these experimental protein structures in order to help downstream users and original authors. In addition, the Task Force provided improved models for key structures online, which have been used by Folding@Home, OpenPandemics, the EU JEDI COVID-19 challenge and others.

Electron Diffraction of Pyrene Nanoclusters Embedded in Superfluid Helium Droplets.

We report electron diffraction of pyrene nanoclusters embedded in superfluid helium droplets. Using a least-squares fitting procedure, we have been able to separate the contribution of helium from those of the pyrene nanoclusters and determine the most likely structures for dimers and trimers. We confirm that pyrene dimers form a parallel double-layer structure with an interlayer distance of 3.5 Šand suggest that pyrene trimers form a sandwich structure but that the molecular planes are not completely parallel. The relative contributions of the dimers and trimers are ∼6:1. This work is an extension of our effort of solving structures of biological molecules using serial single-molecule electron diffraction imaging. The success of electron diffraction from an all-light-atom sample embedded in helium droplets offers reassuring evidence of the feasibility of this approach.

Outcome of the First wwPDB/CCDC/D3R Ligand Validation Workshop.

Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent an important source of information concerning drug-target interactions, providing atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. Of the more than 115,000 entries extant in the Protein Data Bank (PDB) archive, ∼75% include at least one non-polymeric ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for in silico drug discovery and design, and the goodness-of-fit of ligand models to electron-density maps vary widely across the archive. We describe the proceedings and conclusions from the first Worldwide PDB/Cambridge Crystallographic Data Center/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30-31, 2015. Experts in protein crystallography from academe and industry came together with non-profit and for-profit software providers for crystallography and with experts in computational chemistry and data archiving to discuss and make recommendations on best practices, as framed by a series of questions central to structural studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived in the PDB? How should the ligands be best represented? How should structural models of macromolecule-ligand complexes be validated? What supplementary information should accompany publications of structural studies of biological macromolecules? Consensus recommendations on best practices developed in response to each of these questions are provided, together with some details regarding implementation. Important issues addressed but not resolved at the workshop are also enumerated.

A new default restraint library for the protein backbone in Phenix: a conformation-dependent geometry goes mainstream.

Chemical restraints are a fundamental part of crystallographic protein structure refinement. In response to mounting evidence that conventional restraints have shortcomings, it has previously been documented that using backbone restraints that depend on the protein backbone conformation helps to address these shortcomings and improves the performance of refinements [Moriarty et al. (2014), FEBS J. 281, 4061-4071]. It is important that these improvements be made available to all in the protein crystallography community. Toward this end, a change in the default geometry library used by Phenix is described here. Tests are presented showing that this change will not generate increased numbers of outliers during validation, or deposition in the Protein Data Bank, during the transition period in which some validation tools still use the conventional restraint libraries.

Residue-level global and local ensemble-ensemble comparisons of protein domains.

Many methods of protein structure generation such as NMR-based solution structure determination and template-based modeling do not produce a single model, but an ensemble of models consistent with the available information. Current strategies for comparing ensembles lose information because they use only a single representative structure. Here, we describe the ENSEMBLATOR and its novel strategy to directly compare two ensembles containing the same atoms to identify significant global and local backbone differences between them on per-atom and per-residue levels, respectively. The ENSEMBLATOR has four components: eePREP (ee for ensemble-ensemble), which selects atoms common to all models; eeCORE, which identifies atoms belonging to a cutoff-distance dependent common core; eeGLOBAL, which globally superimposes all models using the defined core atoms and calculates for each atom the two intraensemble variations, the interensemble variation, and the closest approach of members of the two ensembles; and eeLOCAL, which performs a local overlay of each dipeptide and, using a novel measure of local backbone similarity, reports the same four variations as eeGLOBAL. The combination of eeGLOBAL and eeLOCAL analyses identifies the most significant differences between ensembles. We illustrate the ENSEMBLATOR's capabilities by showing how using it to analyze NMR ensembles and to compare NMR ensembles with crystal structures provides novel insights compared to published studies. One of these studies leads us to suggest that a "consistency check" of NMR-derived ensembles may be a useful analysis step for NMR-based structure determinations in general. The ENSEMBLATOR 1.0 is available as a first generation tool to carry out ensemble-ensemble comparisons.

Conformation-dependent backbone geometry restraints set a new standard for protein crystallographic refinement.

Ideal values of bond angles and lengths used as external restraints are crucial for the successful refinement of protein crystal structures at all but the highest of resolutions. The restraints in common use today have been designed on the assumption that each type of bond or angle has a single ideal value that is independent of context. However, recent work has shown that the ideal values are, in fact, sensitive to local conformation, and, as a first step towards using such information to build more accurate models, ultra-high-resolution protein crystal structures have been used to derive a conformation-dependent library (CDL) of restraints for the protein backbone [Berkholz et al. (2009) Structure 17, 1316-1325]. Here, we report the introduction of this CDL into the phenix package and the results of test refinements of thousands of structures across a wide range of resolutions. These tests show that use of the CDL yields models that have substantially better agreement with ideal main-chain bond angles and lengths and, on average, a slightly enhanced fit to the X-ray data. No disadvantages of using the backbone CDL are apparent. In phenix, use of the CDL can be selected by simply specifying the cdl = True option. This successful implementation paves the way for further aspects of the context dependence of ideal geometry to be characterized and applied to improve experimental and predictive modeling accuracy.

Reinterpretation of the electron density at the site of the eighth bacteriochlorophyll in the FMO protein from Pelodictyon phaeum.

The Fenna-Matthews-Olson antenna protein from the green bacterium Pelodictyon phaeum mediates the energy transfer from a peripheral antenna complex to the membrane-bound reaction center. The three-dimensional structure of this protein has been previously modeled using X-ray diffraction to a resolution limit of 2.0 Å, with R (work) and R (free) values of 16.6 and 19.9%, respectively (Larson et al., Photosynth Res 107:139-150, 2011). This model shows the protein as consisting of ß-sheets surrounding several bacteriochlorophyll cofactors. While most of the model clearly matches the electron density maps, in this paper we re-examine the electron density for a specific feature, namely the eighth bacteriochlorophyll a cofactor. This electron density is now interpreted as arising primarily from the end of an otherwise disordered polyethylene glycol molecule. Additional electron density is present but the density is weak and cannot be unambiguously assigned. The new model has R (work) and R (free) values of 16.2 and 19.0%, respectively.

A conformation-dependent stereochemical library improves crystallographic refinement even at atomic resolution.

To utilize a new conformation-dependent backbone-geometry library (CDL) in protein refinements at atomic resolution, a script was written that creates a restraint file for the SHELXL refinement program. It was found that the use of this library allows models to be created that have a substantially better fit to main-chain bond angles and lengths without degrading their fit to the X-ray data even at resolutions near 1 Å. For models at much higher resolution (∼0.7 Å), the refined model for parts adopting single well occupied positions is largely independent of the restraints used, but these structures still showed much smaller r.m.s.d. residuals when assessed with the CDL. Examination of the refinement tests across a wide resolution range from 2.4 to 0.65 Šrevealed consistent behavior supporting the use of the CDL as a next-generation restraint library to improve refinement. CDL restraints can be generated using the service at http://pgd.science.oregonstate.edu/cdl_shelxl/.

Iron-containing urease in a pathogenic bacterium.

Helicobacter mustelae, a gastric pathogen of ferrets, synthesizes a distinct iron-dependent urease in addition to its archetypical nickel-containing enzyme. The iron-urease is oxygen-labile, with the inactive protein exhibiting a methemerythrin-like electronic spectrum. Significantly, incubation of the oxidized protein with dithionite under anaerobic conditions leads to restoration of activity and bleaching of the spectrum. Structural analysis of the oxidized species reveals a dinuclear iron metallocenter bridged by a lysine carbamate, closely resembling the traditional nickel-urease active site. Although the iron-urease is less active than the nickel-enzyme, its activity allows H. mustelae to survive the carnivore's low-nickel gastric environment.

Using a conformation-dependent stereochemical library improves crystallographic refinement of proteins.

The major macromolecular crystallographic refinement packages restrain models to ideal geometry targets defined as single values that are independent of molecular conformation. However, ultrahigh-resolution X-ray models of proteins are not consistent with this concept of ideality and have been used to develop a library of ideal main-chain bond lengths and angles that are parameterized by the phi/psi angle of the residue [Berkholz et al. (2009), Structure, 17, 1316-1325]. Here, it is first shown that the new conformation-dependent library does not suffer from poor agreement with ultrahigh-resolution structures, whereas current libraries have this problem. Using the TNT refinement package, it is then shown that protein structure refinement using this conformation-dependent library results in models that have much better agreement with library values of bond angles with little change in the R values. These tests support the value of revising refinement software to account for this new paradigm.

Lessons from the lysozyme of phage T4.

An overview is presented of some of the major insights that have come from studies of the structure, stability, and folding of T4 phage lysozyme. A major purpose of this review is to provide the reader with a complete tabulation of all of the variants that have been characterized, including melting temperatures, crystallographic data, Protein Data Bank access codes, and references to the original literature. The greatest increase in melting temperature (T(m)) for any point mutant is 5.1 degrees C for the mutant Ser 117 --> Val. This is achieved in part not only by hydrophobic stabilization but also by eliminating an unusually short hydrogen bond of 2.48 A that apparently has an unfavorable van der Waals contact. Increases in T(m) of more than 3-4 degrees C for point mutants are rare, whereas several different types of destabilizing substitutions decrease T(m) by 20 degrees C or thereabouts. The energetic cost of cavity creation and its relation to the hydrophobic effect, derived from early studies of "large-to-small" mutants in the core of T4 lysozyme, has recently been strongly supported by related studies of the intrinsic membrane protein bacteriorhodopsin. The L99A cavity in the C-terminal domain of the protein, which readily binds benzene and many other ligands, has been the subject of extensive study. Crystallographic evidence, together with recent NMR analysis, suggest that these ligands are admitted by a conformational change involving Helix F and its neighbors. A total of 43 nonisomorphous crystal forms of different monomeric lysozyme mutants were obtained plus three more for synthetically-engineered dimers. Among the 43 space groups, P2(1)2(1)2(1) and P2(1) were observed most frequently, consistent with the prediction of Wukovitz and Yeates.

The structural basis for the difference in absorbance spectra for the FMO antenna protein from various green sulfur bacteria.

The absorbance spectrum of the Fenna-Matthews-Olson protein--a component of the antenna system of Green Sulfur Bacteria--is always one of two types, depending on the species of the source organism. The FMO from Prosthecochloris aestuarii 2K has a spectrum of type 1 while that from Chlorobaculum tepidum is of type 2. The previously reported crystal structures for these two proteins did not disclose any rationale that would explain their spectral differences. We have collected a 1.3 A X-ray diffraction dataset of the FMO from Prosthecochloris aestuarii 2K, which has allowed us to identify an additional Bacteriochlorophyll-a molecule with chemical attachments to both sides of the central magnesium atom. A new analysis of the previously published X-ray data for the Chlorobaculum tepidum FMO shows the presence of a Bacteriochlorophyll-a molecule in an equivalent location but with a chemical attachment from only one side. This difference in binding is shown to be predictive of the spectral type of the FMO.

Evaluation at atomic resolution of the role of strain in destabilizing the temperature-sensitive T4 lysozyme mutant Arg 96 --> His.

Mutant R96H is a classic temperature-sensitive mutant of bacteriophage T4 lysozyme. It was in fact the first variant of the protein to be characterized structurally. Subsequently, it has been studied extensively by a variety of experimental and computational techniques, but the reasons for the loss of stability of the mutant protein remain controversial. In the crystallographic refinement of the mutant structure at 1.9 A resolution one of the bond angles at the site of substitution appeared to be distorted by about 11( degrees ), and it was suggested that this steric strain was one of the major factors in destabilizing the mutant. Different computationally-derived models of the mutant structure, however, did not show such distortion. To determine the geometry at the site of mutation more reliably, we have extended the resolution of the data and refined the wildtype (WT) and mutant structures to be better than 1.1 A resolution. The high-resolution refinement of the structure of R96H does not support the bond angle distortion seen in the 1.9 A structure determination. At the same time, it does confirm other manifestations of strain seen previously including an unusual rotameric state for His96 with distorted hydrogen bonding. The rotamer strain has been estimated as about 0.8 kcal/mol, which is about 25% of the overall reduction in stability of the mutant. Because of concern that contacts from a neighboring molecule in the crystal might influence the geometry at the site of mutation we also constructed and analyzed supplemental mutant structures in which this crystal contact was eliminated. High-resolution refinement shows that the crystal contacts have essentially no effect on the conformation of Arg96 in WT or on His96 in the R96H mutant.

Introduction to macromolecular refinement.

The process of refinement is such a large problem in function minimization that even the computers of today cannot perform the calculations to properly fit X-ray diffraction data. Each of the refinement packages currently under development reduces the difficulty of this problem by utilizing a unique combination of targets, assumptions, and optimization methods. This chapter summarizes the basic methods and underlying assumptions in the commonly used refinement packages. This information can guide the selection of a refinement package that is best suited for a particular refinement project.