Macromolecular Crystal Structures Research Articles

The Automatic Solution of Macromolecular Crystal Structures via Molecular Replacement Techniques: REMO22 and Its Pipeline.

A description of REMO22, a new molecular replacement program for proteins and nucleic acids, is provided. This program, as with REMO09, can use various types of prior information through appropriate conditional distribution functions. Its efficacy in model searching has been validated through several test cases involving proteins and nucleic acids. Although REMO22 can be configured with different protocols according to user directives, it has been developed primarily as an automated tool for determining the crystal structures of macromolecules. To evaluate REMO22's utility in the current crystallographic environment, its experimental results must be compared favorably with those of the most widely used Molecular Replacement (MR) programs. To accomplish this, we chose two leading tools in the field, PHASER and MOLREP. REMO22, along with MOLREP and PHASER, were included in pipelines that contain two additional steps: phase refinement (SYNERGY) and automated model building (CAB). To evaluate the effectiveness of REMO22, SYNERGY and CAB, we conducted experimental tests on numerous macromolecular structures. The results indicate that REMO22, along with its pipeline REMO22 + SYNERGY + CAB, presents a viable alternative to currently used phasing tools.

Assessing PDB macromolecular crystal structure confidence at the individual amino acid residue level

Approximately 87% of the more than 190,000 atomic-level three-dimensional (3D) biostructures in the PDB were determined using macromolecular crystallography (MX). Agreement between 3D atomic coordinates and experimental data for >100 million individual amino acid residues occurring within ∼150,000 PDB MX structures was analyzed in detail. The real-space correlation coefficient (RSCC) calculated using the 3D atomic coordinates for each residue and experimental-data-derived electron density enables outlier detection of unreliable atomic coordinates (particularly important for poorly resolved side-chain atoms) and ready evaluation of local structure quality by PDB users. For human protein MX structures in PDB, comparisons of the per-residue RSCC metric with AlphaFold2-computed structure model confidence (pLDDT-predicted local distance difference test) document (1) that RSCC values and pLDDT scores are correlated (median correlation coefficient ∼0.41), and (2) that experimentally determined MX structures (3.5Å resolution or better) are more reliable than AlphaFold2-computed structure models and should be used preferentially whenever possible.

Assessing macromolecular crystal structures for agreement with experimental data at the individual amino acid residue level

Assessing macromolecular crystal structures for agreement with experimental data at the individual amino acid residue level

Notes of a protein crystallographer: the advantages of combining new integrated methods of structure solution with traditional data visuals.

The suggestion is made that combining analysis using the most advanced crystallographic software with the integrated visual tools of the field will result in more knowledgeable and better trained future generations of structural biologists. The use of integrated visuals could also expedite the structure solution of some recalcitrant and complex macromolecular crystal structures that resist automatic workflows.

Can the results of quantum refinement be improved with a continuum-solvation model?

Quantum refinement has repeatedly been shown to be a powerful approach to interpret and improve macromolecular crystal structures, allowing for the discrimination between different interpretations of the structure, regarding the protonation states or the nature of bound ligands, for example. In this method, the empirical restraints, used to supplement the crystallographic raw data in standard crystallographic refinement, are replaced by more accurate quantum mechanical (QM) calculations for a small, but interesting, part of the structure. Previous studies have shown that the results of quantum refinement can be improved if the charge of the QM system is reduced by adding neutralizing groups. However, this significantly increases the computation time for the refinement. In this study, we show that a similar improvement can be obtained if the original highly charged QM system is instead immersed in a continuum solvent in the QM calculations. The best results are typically obtained with a high dielectric constant (ɛ). The continuum solvent improves real-space Z values, electron-density difference maps and strain energies, and it normally does not affect the discriminatory power of the calculations between different chemical interpretations of the structure. However, for structures with a low charge in the QM system or with a low crystallographic resolution (>2 Å), no improvement of the structures is seen.

PDBrenum: A webserver and program providing Protein Data Bank files renumbered according to their UniProt sequences.

The Protein Data Bank (PDB) was established at Brookhaven National Laboratories in 1971 as an archive for biological macromolecular crystal structures. In mid 2021, the database has almost 180,000 structures solved by X-ray crystallography, nuclear magnetic resonance, cryo-electron microscopy, and other methods. Many proteins have been studied under different conditions, including binding partners such as ligands, nucleic acids, or other proteins; mutations, and post-translational modifications, thus enabling extensive comparative structure-function studies. However, these studies are made more difficult because authors are allowed by the PDB to number the amino acids in each protein sequence in any manner they wish. This results in the same protein being numbered differently in the available PDB entries. For instance, some authors may include N-terminal signal peptides or the N-terminal methionine in the sequence numbering and others may not. In addition to the coordinates, there are many fields that contain structural and functional information regarding specific residues numbered according to the author. Here we provide a webserver and Python3 application that fixes the PDB sequence numbering problem by replacing the author numbering with numbering derived from the corresponding UniProt sequences. We obtain this correspondence from the SIFTS database from PDBe. The server and program can take a list of PDB entries or a list of UniProt identifiers (e.g., "P04637" or "P53_HUMAN") and provide renumbered files in mmCIF format and the legacy PDB format for both asymmetric unit files and biological assembly files provided by PDBe.

QM/ELMO: A Multi-Purpose Fully Quantum Mechanical Embedding Scheme Based on Extremely Localized Molecular Orbitals

The development of computationally advantageous methods for the study of large systems is a long-standing research topic in theoretical chemistry. Among these techniques, a prominent place is certainly occupied by the multiscale embedding strategies, from the well-known QM/MM (quantum mechanics/molecular mechanics) methods to the latest and promising fully quantum mechanical approaches. In this Feature Article, we will briefly review the recently proposed QM/ELMO (quantum mechanics/extremely localized molecular orbital) scheme, namely a new multiscale embedding strategy in which the most chemically relevant region of the investigated system is treated at fully quantum chemical level, while the remaining part (namely, the environment) is described by means of transferred extremely localized molecular orbitals that remain frozen throughout the computation. Other than highlighting the theoretical bases, here we will also review the main results obtained through all the currently available variants of the novel method. In particular, we will show how the QM/ELMO embedding scheme has been successfully exploited to perform both ground and excited state calculations, reproducing the results of corresponding fully quantum mechanical computations but with a much lower computational cost. A first application to crystallography will be also discussed, and we will describe how the QM/ELMO approach has been recently coupled with the Hirshfeld atom refinement technique to accurately determine the positions of hydrogen atoms from X-ray diffraction data. Given the reliability and quality of the obtained results, future applications of the current versions of the QM/ELMO embedding strategy to different types of chemical problems are to be expected in the near future. Moreover, further algorithmic improvements and methodological developments are also envisaged, such as the development of a polarizable QM/ELMO scheme accounting for the effects of the QM region on the ELMO subsystem or the use of the new embedding approach in the context of quantum crystallography to perform unprecedented accurate refinements of macromolecular crystal structures.

Visualizing functional dynamicity in the DNA-dependent protein kinase holoenzyme DNA-PK complex by integrating SAXS with cryo-EM

Assembly of KU and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at DNA double strand breaks (DSBs) forms DNA-PK holoenzyme as a critical initiating step for non-homologous end joining (NHEJ) repair of DSBs produced by radiation and chemotherapies. Advanced cryo-electron microscopy (cryo-EM) imaging together with breakthrough macromolecular X-ray crystal (MX) structures of KU and DNA-PKcs recently enabled visualization of the ∼600 kDa DNA-PK assembly at near atomic resolution. These important static structures provide the foundation for definition and interpretation of functional movements crucial to mechanistic understanding that can be tested through solution state structure analysis. We herein therefore leverage Cryo-EM and MX structures for the interpretation of synchrotron small-angle X-ray scattering (SAXS) data on DNA-PK conformations in solution to inform the structural mechanism for NHEJ initiation. SAXS, which measures thermodynamic solution-state conformational states and assemblies outside of cryo- and solid-state conditions, unveils the inherent flexibility of KU, DNA-PKcs and DNA-PK. The combined structural measurements reveal mobility of KU80 C-terminal region (KU80CTR), motion/plasticity of HEAT (DNA-PKcs Huntingtin, Elongation Factor 3, PP2 A, and TOR1) regions, allosteric switching upon DNA-PKcs autophosphorylation, and dimeric arrangements of DNA-PK assembly. Importantly, the results uncover displacement of the N-terminal HEAT domain during autophosphorylation as suitable for a regulated release mechanism of DNA-PKcs from DNA-PK to control unproductive access to toxic and mutagenic DNA repair intermediates. These integrated analyses show that the marriage of SAXS with cryo-EM leverages the strengths of both techniques to enable assessment of functional conformations and flexibility defining atomic-resolution molecular mechanisms for DSB repair.

A new modulated crystal structure of the ANS complex of the St John's wort Hyp-1 protein with 36 protein molecules in the asymmetric unit of the supercell.

Superstructure modulation, with violation of the strict short-range periodic order of consecutive crystal unit cells, is well known in small-molecule crystallography but is rarely reported for macromolecular crystals. To date, one modulated macromolecular crystal structure has been successfully determined and refined for a pathogenesis-related class 10 protein from Hypericum perforatum (Hyp-1) crystallized as a complex with 8-anilinonaphthalene-1-sulfonate (ANS) [Sliwiak et al. (2015), Acta Cryst. D71, 829-843]. The commensurate modulation in that case was interpreted in a supercell with sevenfold expansion along c. When crystallized in the additional presence of melatonin, the Hyp-1-ANS complex formed crystals with a different pattern of structure modulation, in which the supercell shows a ninefold expansion of c, manifested in the diffraction pattern by a wave of reflection-intensity modulation with crests at l = 9n and l = 9n ± 4. Despite complicated tetartohedral twinning, the structure has been successfully determined and refined to 2.3 Å resolution using a description in a ninefold-expanded supercell, with 36 independent Hyp-1 chains and 156 ANS ligands populating the three internal (95 ligands) and five interstitial (61 ligands) binding sites. The commensurate superstructures and ligand-binding sites of the two crystal structures are compared, with a discussion of the effect of melatonin on the co-crystallization process.

Localized Molecular Orbital-Based Embedding Scheme for Correlated Methods.

Embedding strategies currently provide the best compromise between accuracy and computational cost in modeling chemical properties and processes of large and complex systems. In this framework, different methods have been proposed all over the years, from the very popular QM/MM approaches to the more recent and very promising density matrix and density functional embedding techniques. Here, we present a further development of the quantum mechanics/extremely localized molecular orbital technique (QM/ELMO) method, a recently proposed multiscale embedding strategy in which the chemically active region of the investigated system is treated at a fully quantum mechanical level, while the rest is described by frozen extremely localized molecular orbitals previously transferred from proper libraries or tailor-made model molecules. In particular, in this work we discuss and assess in detail the extension of the QM/ELMO approach to density functional theory and post-Hartree-Fock techniques by evaluating its performances when it is used to describe chemical reactions, bond dissociations, and intermolecular interactions. The preliminary test calculations have shown that, in the investigated cases, the new embedding strategy enables the results of the corresponding fully quantum mechanical computations to be reproduced within chemical accuracy in almost all the cases but with a significantly reduced computational cost, especially when correlated post-Hartree-Fock strategies are used to describe the quantum mechanical subsystem. In light of the obtained results, we already envisage the future application of the new correlated QM/ELMO techniques to the investigation of more challenging problems, such as the modeling of enzyme catalysis, the study of excited states of biomolecules, and the refinement of macromolecular X-ray crystal structures.

Prediction of models for ordered solvent in macromolecular structures by a classifier based upon resolution-independent projections of local feature data.

Current software tools for the automated building of models for macromolecular X-ray crystal structures are capable of assembling high-quality models for ordered macromolecule and small-molecule scattering components with minimal or no user supervision. Many of these tools also incorporate robust functionality for modelling the ordered water molecules that are found in nearly all macromolecular crystal structures. However, no current tools focus on differentiating these ubiquitous water molecules from other frequently occurring multi-atom solvent species, such as sulfate, or the automated building of models for such species. PeakProbe has been developed specifically to address the need for such a tool. PeakProbe predicts likely solvent models for a given point (termed a `peak') in a structure based on analysis (`probing') of its local electron density and chemical environment. PeakProbe maps a total of 19 resolution-dependent features associated with electron density and two associated with the local chemical environment to a two-dimensional score space that is independent of resolution. Peaks are classified based on the relative frequencies with which four different classes of solvent (including water) are observed within a given region of this score space as determined by large-scale sampling of solvent models in the Protein Data Bank. Designed to classify peaks generated from difference density maxima, PeakProbe also incorporates functionality for identifying peaks associated with model errors or clusters of peaks likely to correspond to multi-atom solvent, and for the validation of existing solvent models using solvent-omit electron-density maps. When tasked with classifying peaks into one of four distinct solvent classes, PeakProbe achieves greater than 99% accuracy for both peaks derived directly from the atomic coordinates of existing solvent models and those based on difference density maxima. While the program is still under development, a fully functional version is publicly available. PeakProbe makes extensive use of cctbx libraries, and requires a PHENIX licence and an up-to-date phenix.python environment for execution.

An enhanced set of displacement parameter restraints in CRYSTALS

Crystallographic restraints are widely used during refinement of small-molecule and macromolecular crystal structures. They can be especially useful for introducing additional observations and information into structure refinements against low-quality or low-resolution data (e.g. data obtained at high pressure) or to retain physically meaningful parameter values in disordered or unstable refinements. However, despite the fact that the anisotropic displacement parameters (ADPs) often constitute more than half of the total model parameters determined in a structure analysis, there are relatively few useful restraints for them, examples being Hirshfeld rigid-bond restraints, direct equivalence of parameters and SHELXL RIGU-type restraints. Conversely, geometric parameters can be subject to a multitude of restraints (e.g. absolute or relative distance, angle, planarity, chiral volume, and geometric similarity). This article presents a series of new ADP restraints implemented in CRYSTALS [Parois, Cooper & Thompson (2015), Chem. Cent. J. 9, 30] to give more control over ADPs by restraining, in a variety of ways, the directions and magnitudes of the principal axes of the ellipsoids in locally defined coordinate systems. The use of these new ADPs results in more realistic models, as well as a better user experience, through restraints that are more efficient and faster to set up. The use of these restraints is recommended to preserve physically meaningful relationships between displacement parameters in a structural model for rigid bodies, rotationally disordered groups and low-completeness data.

On the application of the expected log-likelihood gain to decision making in molecular replacement.

Molecular-replacement phasing of macromolecular crystal structures is often fast, but if a molecular-replacement solution is not immediately obtained the crystallographer must judge whether to pursue molecular replacement or to attempt experimental phasing as the quickest path to structure solution. The introduction of the expected log-likelihood gain [eLLG; McCoy et al. (2017), Proc. Natl Acad. Sci. USA, 114, 3637-3641] has given the crystallographer a powerful new tool to aid in making this decision. The eLLG is the log-likelihood gain on intensity [LLGI; Read & McCoy (2016), Acta Cryst. D72, 375-387] expected from a correctly placed model. It is calculated as a sum over the reflections of a function dependent on the fraction of the scattering for which the model accounts, the estimated model coordinate error and the measurement errors in the data. It is shown how the eLLG may be used to answer the question `can I solve my structure by molecular replacement?'. However, this is only the most obvious of the applications of the eLLG. It is also discussed how the eLLG may be used to determine the search order and minimal data requirements for obtaining a molecular-replacement solution using a given model, and for decision making in fragment-based molecular replacement, single-atom molecular replacement and likelihood-guided model pruning.

Mini-beam modes on standard MX beamline BL17U at SSRF.

The macromolecular crystallography beamlines at third-generation synchrotron facilities play a central role in solving macromolecular crystal structures and also in understanding the biological function at molecular levels. The MX beamline BL17U at Shanghai Synchrotron Radiation Facility is a typical standard MX beamline with a focused beam size (H × V) of FWHM around 80 μm × 45 μm. However the protein samples brought to the beamline are down to 5-10 m from the important and challenging science project now. These samples require smaller size beam. In order to achieve the mini-size beamline, two mini-beam modes have been developed on BL17U: the pinhole-based mini-beam and the focused mini-beam by compound refractive lens (CRL). Compared to the pinhole-based mode, three times increase in flux is obtained by the CRL mode at a similar beam size. The flux gain obtained by the CRL needs to be considered for data collection strategies. It takes few minutes to switch the beamline from the normal to CRL mini-beam mode.

Ab initio solution of macromolecular crystal structures without direct methods

The majority of macromolecular crystal structures are determined using the method of molecular replacement, in which known related structures are rotated and translated to provide an initial atomic model for the new structure. A theoretical understanding of the signal-to-noise ratio in likelihood-based molecular replacement searches has been developed to account for the influence of model quality and completeness, as well as the resolution of the diffraction data. Here we show that, contrary to current belief, molecular replacement need not be restricted to the use of models comprising a substantial fraction of the unknown structure. Instead, likelihood-based methods allow a continuum of applications depending predictably on the quality of the model and the resolution of the data. Unexpectedly, our understanding of the signal-to-noise ratio in molecular replacement leads to the finding that, with data to sufficiently high resolution, fragments as small as single atoms of elements usually found in proteins can yield ab initio solutions of macromolecular structures, including some that elude traditional direct methods.

Embarras de richesses - It is not good to be too anomalous: Accurate structure of selenourea, a chiral crystal of planar molecules.

Selenourea, SeC(NH2)2, recently found an application as a derivatization reagent providing a significant anomalous diffraction signal used for phasing macromolecular crystal structures. The crystal structure of selenourea itself was solved about 50 years ago, from data recorded on films and evaluated by eye and refined to R = 0.15 with errors of bond lengths and angles about 0.1 Å and 6°. In the current work this structure is re-evaluated on the basis of synchrotron data and refined to R1 = 0.021 with bond and angle errors about 0.007 Å and 0.5°. The nine planar molecules of selenourea pack either in the P31 or in the P32 unit cell. All unique molecules are connected by a complex network of Se•••H-N hydrogen bonds and Se•••Se contacts. The packing of selenourea molecules is highly pseudosymmetric, approximating either of the P31(2)12, R3, and R32 space groups. Because the overwhelming majority of diffracted X-ray intensity originates form the anomalously scattering selenium atoms, the measurable anomalous Bijvoet differences are diminished and it was not possible to solve this crystal structure based on the anomalous signal alone.

Long-wavelength macromolecular crystallography - First successful native SAD experiment close to the sulfur edge.

Phasing of novel macromolecular crystal structures has been challenging since the start of structural biology. Making use of anomalous diffraction of natively present elements, such as sulfur and phosphorus, for phasing has been possible for some systems, but hindered by the necessity to access longer X-ray wavelengths in order to make most use of the anomalous scattering contributions of these elements. Presented here are the results from a first successful experimental phasing study of a macromolecular crystal structure at a wavelength close to the sulfur K edge. This has been made possible by the in-vacuum setup and the long-wavelength optimised experimental setup at the I23 beamline at Diamond Light Source. In these early commissioning experiments only standard data collection and processing procedures have been applied, in particular no dedicated absorption correction has been used. Nevertheless the success of the experiment demonstrates that the capability to extract phase information can be even further improved once data collection protocols and data processing have been optimised.

The young person's guide to the PDB.

The Protein Data Bank (PDB), created in 1971 when merely seven protein crystal structures were known, today holds over 120, 000 experimentally-determined three-dimensional models of macromolecules, including gigantic structures comprised of hundreds of thousands of atoms, such as ribosomes and viruses. Most of the deposits come from X-ray crystallography experiments, with important contributions also made by NMR spectroscopy and, recently, by the fast growing Cryo-Electron Microscopy. Although the determination of a macromolecular crystal structure is now facilitated by advanced experimental tools and by sophisticated software, it is still a highly complicated research process requiring specialized training, skill, experience and a bit of luck. Understanding the plethora of structural information provided by the PDB requires that its users (consumers) have at least a rudimentary initiation. This is the purpose of this educational overview.

Selenourea: a convenient phasing vehicle for macromolecular X-ray crystal structures

Majority of novel X-ray crystal structures of proteins are currently solved using the anomalous diffraction signal provided by selenium after incorporation of selenomethionine instead of natural methionine by genetic engineering methods. However, selenium can be inserted into protein crystals in the form of selenourea (SeC(NH2)2), by adding the crystalline powder of selenourea into mother liquor or cryo-solution with native crystals, in analogy to the classic procedure of heavy-atom derivatization. Selenourea is able to bind to reactive groups at the surface of macromolecules primarily through hydrogen bonds, where the selenium atom may serve as acceptor and amide groups as donors. Selenourea has different chemical properties than heavy-atom reagents and halide ions and provides a convenient way of phasing crystal structures of macromolecules.

Septin crystallization for structural analysis.

Septins are filament-forming proteins found in many eukaryotes. Despite being important components of the cytoskeleton, only recently details of their macromolecular assemblies and crystal structures have started to appear in the literature. These are of fundamental importance to the understanding of cytoskeleton dynamics, membrane barrier formation, and bacterial caging, as well as essential cellular processes such as cell division, exocytosis, and vesicle trafficking. However, obtaining this data is frequently hindered by several experimental difficulties common to the majority of septin samples. Here we provide an overview of the current approaches to circumvent or minimize the experimental complications observed in septin crystallography focusing mainly, but not exclusively, on the choice of the septin construct and how to best prepare the sample itself.