IHMCIF: An Extension of the PDBx/mmCIF Data Standard for Integrative Structure Determination Methods.

IHMCIF (github.com/ihmwg/IHMCIF) is a data information framework that supports archiving and disseminating macromolecular structures determined by integrative or hybrid modeling (IHM), and making them Findable, Accessible, Interoperable, and Reusable (FAIR). IHMCIF is an extension of the Protein Data Bank Exchange/macromolecular Crystallographic Information Framework (PDBx/mmCIF) that serves as the framework for the Protein Data Bank (PDB) to archive experimentally determined atomic structures of biological macromolecules and their complexes with one another and small molecule ligands (e.g., enzyme cofactors and drugs). IHMCIF serves as the foundational data standard for the PDB-Dev prototype system, developed for archiving and disseminating integrative structures. It utilizes a flexible data representation to describe integrative structures that span multiple spatiotemporal scales and structural states with definitions for restraints from a variety of experimental methods contributing to integrative structural biology. The IHMCIF extension was created with the benefit of considerable community input and recommendations gathered by the Worldwide Protein Data Bank (wwPDB) Task Force for Integrative or Hybrid Methods (wwpdb.org/task/hybrid). Herein, we describe the development of IHMCIF to support evolving methodologies and ongoing advancements in integrative structural biology. Ultimately, IHMCIF will facilitate the unification of PDB-Dev data and tools with the PDB archive so that integrative structures can be archived and disseminated through PDB.

Form factor determination of biological molecules with X-ray free electron laser small-angle scattering (XFEL-SAS).

Free-electron lasers (FEL) are revolutionizing X-ray-based structural biology methods. While protein crystallography is already routinely performed at FELs, Small Angle X-ray Scattering (SAXS) studies of biological macromolecules are not as prevalent. SAXS allows the study of the shape and overall structure of proteins and nucleic acids in solution, in a quasi-native environment. In solution, chemical and biophysical parameters that have an influence on the structure and dynamics of molecules can be varied and their effect on conformational changes can be monitored in time-resolved XFEL and SAXS experiments. We report here the collection of scattering form factors of proteins in solution using FEL X-rays. The form factors correspond to the scattering signal of the protein ensemble alone; the scattering contributions from the solvent and the instrument are separately measured and accurately subtracted. The experiment was done using a liquid jet for sample delivery. These results pave the way for time-resolved studies and measurements from dilute samples, capitalizing on the intense and short FEL X-ray pulses.

Small conformational changes in IgG1 detected as acidic charge variants by cation exchange chromatography.

Fully characterizing the post-translational modifications present in charge variants of therapeutic monoclonal antibodies (mAbs), particularly acidic variants, is challenging and remains an open area of investigation. In this study, to test the possibility that chromatographically separated acidic fractions of therapeutic mAbs contain conformational variants, we undertook a mAb refolding approach using as a case study an IgG1 that contains many unidentified acidic peaks with few post-translational modifications, and examined whether different acidic peak fractions could be generated corresponding to these variants. The IgG1 drug substance was denatured by guanidine hydrochloride, without a reducing agent present, and gradually refolded by stepwise dialysis against arginine hydrochloride used as an aggregation suppressor. Each acidic chromatographic peak originally contained in the IgG1 drug substance was markedly increased by this stepwise refolding process, indicating that these acidic variants are conformational variants. However, no conformational changes were detected by small-angle X-ray scattering experiments for the whole IgG1, indicating that the conformational changes are minor. Chromatographic, thermal and fluorescence analyses suggested that the conformational changes are a localized denaturation effect centred around the aromatic amino acid regions. This study provides new insights into the characterization of acidic variants that are currently not fully understood.

Minimum information guidelines for experiments structurally characterizing intrinsically disordered protein regions.

An unambiguous description of an experiment, and the subsequent biological observation, is vital for accurate data interpretation. Minimum information guidelines define the fundamental complement of data that can support an unambiguous conclusion based on experimental observations. We present the Minimum Information About Disorder Experiments (MIADE) guidelines to define the parameters required for the wider scientific community to understand the findings of an experiment studying the structural properties of intrinsically disordered regions (IDRs). MIADE guidelines provide recommendations for data producers to describe the results of their experiments at source, for curators to annotate experimental data to community resources and for database developers maintaining community resources to disseminate the data. The MIADE guidelines will improve the interpretability of experimental results for data consumers, facilitate direct data submission, simplify data curation, improve data exchange among repositories and standardize the dissemination of the key metadata on an IDR experiment by IDR data sources.

Crosstalk between regulatory elements in disordered TRPV4 N-terminus modulates lipid-dependent channel activity.

Intrinsically disordered regions (IDRs) are essential for membrane receptor regulation but often remain unresolved in structural studies. TRPV4, a member of the TRP vanilloid channel family involved in thermo- and osmosensation, has a large N-terminal IDR of approximately 150 amino acids. With an integrated structural biology approach, we analyze the structural ensemble of the TRPV4 IDR and the network of antagonistic regulatory elements it encodes. These modulate channel activity in a hierarchical lipid-dependent manner through transient long-range interactions. A highly conserved autoinhibitory patch acts as a master regulator by competing with PIP2 binding to attenuate channel activity. Molecular dynamics simulations show that loss of the interaction between the PIP2-binding site and the membrane reduces the force exerted by the IDR on the structured core of TRPV4. This work demonstrates that IDR structural dynamics are coupled to TRPV4 activity and highlights the importance of IDRs for TRP channel function and regulation.

The preference signature of the SARS-CoV-2 Nucleocapsid NTD for its 5'-genomic RNA elements.

The nucleocapsid protein (N) of SARS-CoV-2 plays a pivotal role during the viral life cycle. It is involved in RNA transcription and accounts for packaging of the large genome into virus particles. N manages the enigmatic balance of bulk RNA-coating versus precise RNA-binding to designated cis-regulatory elements. Numerous studies report the involvement of its disordered segments in non-selective RNA-recognition, but how N organizes the inevitable recognition of specific motifs remains unanswered. We here use NMR spectroscopy to systematically analyze the interactions of N's N-terminal RNA-binding domain (NTD) with individual cis RNA elements clustering in the SARS-CoV-2 regulatory 5'-genomic end. Supported by broad solution-based biophysical data, we unravel the NTD RNA-binding preferences in the natural genome context. We show that the domain's flexible regions read the intrinsic signature of preferred RNA elements for selective and stable complex formation within the large pool of available motifs.

Conformational multiplicity of bacterial ferric binding protein revealed by small angle x-ray scattering and molecular dynamics calculations.

This study combines molecular dynamics (MD) simulations with small angle x-ray scattering (SAXS) measurements to investigate the range of conformations that can be adopted by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations in solution. To explore how the conformational distribution of proteins may be modified in the environmental niches of biological media, we focus on the periplasmic ferric binding protein A (FbpA) from Haemophilus influenzae involved in the mechanism by which bacteria capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its biological environment. While we show that these changes fall within the detectable range for SAXS as evidenced by differences observed in the theoretical scattering patterns calculated from the crystal structure models of apo and holo forms, detection of conformational changes due to the point mutation D52A and changes in ionic strength (IS) from SAXS scattering profiles have been challenging. Here, to reach conclusions, statistical analyses with SAXS profiles and results from different techniques were combined in a complementary fashion. The SAXS data complemented by size exclusion chromatography point to multiple and/or alternative conformations at physiological IS, whereas they are well-explained by single crystallographic structures in low IS buffers. By fitting the SAXS data with unique conformations sampled by a series of MD simulations under conditions mimicking the buffers, we quantify the populations of the occupied substates. We also find that the D52A mutant that we predicted by coarse-grained computational modeling to allosterically control the iron binding site in FbpA, responds to the environmental changes in our experiments with conformational selection scenarios that differ from those of the wild type.

2023 update of template tables for reporting biomolecular structural modelling of small-angle scattering data.

In 2017, guidelines were published for reporting structural modelling of small-angle scattering (SAS) data from biomolecules in solution that exemplified best-practice documentation of experiments and analysis. Since then, there has been significant progress in SAS data and model archiving, and the IUCr journal editors announced that the IUCr biology journals will require the deposition of SAS data used in biomolecular structure solution into a public archive, as well as adherence to the 2017 reporting guidelines. In this context, the reporting template tables accompanying the 2017 publication guidelines have been reviewed with a focus on making them both easier to use and more general. With input from the SAS community via the IUCr Commission on SAS and attendees of the triennial 2022 SAS meeting (SAS2022, Campinas, Brazil), an updated reporting template table has been developed that includes standard descriptions for proteins, glycosylated proteins, DNA and RNA, with some reorganization of the data to improve readability and interpretation. In addition, a specialized template has been developed for reporting SAS contrast-variation (SAS-cv) data and models that incorporates the additional reporting requirements from the 2017 guidelines for these more complicated experiments. To demonstrate their utility, examples of reporting with these new templates are provided for a SAS study of a DNA-protein complex and a SAS-cv experiment on a protein complex. The examples demonstrate how the tabulated information promotes transparent reporting that, in combination with the recommended figures and additional information best presented in the main text, enables the reader of the work to readily draw their own conclusions regarding the quality of the data and the validity of the models presented.

X-ray structure and function of fibronectin domains two and three of the neural cell adhesion molecule L1.

The cell adhesion molecule L1 (L1CAM, L1 in short) plays crucial roles during neural development, regeneration after injury, synapse formation, synaptic plasticity and tumor cell migration. L1 belongs to the immunoglobulin superfamily and comprises in its extracellular part six immunoglobulin (Ig)-like domains and five fibronectin type III homologous repeats (FNs). The second Ig-like domain has been validated for self- (so-called homophilic) binding between cells. Antibodies against this domain inhibit neuronal migration in vitro and in vivo. The fibronectin type III homologous repeats FN2 and FN3 bind small molecule agonistic L1 mimetics and contribute to signal transduction. FN3 has a stretch of 25 amino acids that can be triggered with a monoclonal antibody, or the L1 mimetics, to enhance neurite outgrowth and neuronal cell migration in vitro and in vivo. To correlate the structural features of these FNs with function, we determined a high-resolution crystal structure of a FN2FN3 fragment, which is functionally active in cerebellar granule cells and binds several mimetics. The structure illustrates that both domains are connected by a short linker sequence allowing a flexible and largely independent organization of both domains. This becomes further evident by comparing the X-ray crystal structure with models derived from Small-Angle X-ray Scattering (SAXS) data for FN2FN3 in solution. Based on the X-ray crystal structure, we identified five glycosylation sites which we believe are crucial for folding and stability of these domains. Our study signifies an advance in the understanding of structure-functional relationships of L1.

Data analysis and modeling of small-angle neutron scattering data with contrast variation from bio-macromolecular complexes.

Small-angle neutron scattering (SANS) with contrast variation (CV) is a valuable technique in the structural biology toolchest. Accurate structural parameters-e.g., radii of gyration, volumes, dimensions, and distance distribution(s)-can be derived from the SANS-CV data to yield the shape and disposition of the individual components within stable complexes. Contrast variation is achieved through the substitution of hydrogen isotopes (1H for 2H) in molecules and solvents to alter the neutron scattering properties of each component of a complex. While SANS-CV can be used a stand-alone technique for interrogating the overall structure of biomacromolecules in solution, it also complements other methods such as small-angle X-ray scattering, crystallography, nuclear magnetic resonance, and cryo-electron microscopy. Undertaking a SANS-CV experiment is challenging, due in part to the preparation of significant quantities of monodisperse samples that may require deuterium (2H) labeling. Nevertheless, SANS-CV can be used to study a diverse range biomacromolecular complexes including protein-protein and protein-nucleic acid systems, membrane proteins, and flexible systems resistant to crystallization. This chapter describes how to approach the data analysis and modeling of SANS data, including: (1) Analysis of the forward scattering (I(0)) and calculation of theoretical estimates of contrast; (2) Analysis of the contrast dependence of the radius of gyration using the Stuhrmann plot and parallel axis theorem; (3) Calculation of composite scattering functions to evaluate the size, shape, and dispositions of individual components within a complex, and; (4) Development of real-space models to fit the SANS-CV data using volume-element bead modeling or atomistic rigid body modeling.

Asymmetric horseshoe-like assembly of peroxisomal yeast oxalyl-CoA synthetase.

Oxalyl-CoA synthetase from Saccharomyces cerevisiae is one of the most abundant peroxisomal proteins in yeast and hence has become a model to study peroxisomal translocation. It contains a C-terminal Peroxisome Targeting Signal 1, which however is partly dispensable, suggesting additional receptor bindings sites. To unravel any additional features that may contribute to its capacity to be recognized as peroxisomal target, we determined its assembly and overall architecture by an integrated structural biology approach, including X-ray crystallography, single particle cryo-electron microscopy and small angle X-ray scattering. Surprisingly, it assembles into mixture of concentration-dependent dimers, tetramers and hexamers by dimer self-association. Hexameric particles form an unprecedented asymmetric horseshoe-like arrangement, which considerably differs from symmetric hexameric assembly found in many other protein structures. A single mutation within the self-association interface is sufficient to abolish any higher-level oligomerization, resulting in a homogenous dimeric assembly. The small C-terminal domain of yeast Oxalyl-CoA synthetase is connected by a partly flexible hinge with the large N-terminal domain, which provides the sole basis for oligomeric assembly. Our data provide a basis to mechanistically study peroxisomal translocation of this target.

Determinants of receptor tyrosine phosphatase homophilic adhesion: Structural comparison of PTPRK and PTPRM extracellular domains.

Type IIB receptor protein tyrosine phosphatases are cell surface transmembrane proteins that engage in cell adhesion via their extracellular domains (ECDs) and cell signaling via their cytoplasmic phosphatase domains. The ECDs of type IIB receptor protein tyrosine phosphatases form stable, homophilic, and trans interactions between adjacent cell membranes. Previous work has demonstrated how one family member, PTPRM, forms head-to-tail homodimers. However, as the interface was composed of residues conserved across the family, the determinants of homophilic specificity remain unknown. Here, we have solved the X-ray crystal structure of the membrane-distal N-terminal domains of PTPRK that form a head-to-tail dimer consistent with intermembrane adhesion. Comparison with the PTPRM structure demonstrates interdomain conformational differences that may define homophilic specificity. Using small-angle X-ray scattering, we determined the solution structures of the full-length ECDs of PTPRM and PTPRK, identifying that both are rigid extended molecules that differ in their overall long-range conformation. Furthermore, we identified one residue, W351, within the interaction interface that differs between PTPRM and PTPRK and showed that mutation to glycine, the equivalent residue in PTPRM, abolishes PTPRK dimer formation in vitro. This comparison of two members of the receptor tyrosine phosphatase family suggests that homophilic specificity is driven by a combination of shape complementarity and specific but limited sequence differences.

Technical considerations for small-angle neutron scattering from biological macromolecules in solution: Cross sections, contrasts, instrument setup and measurement.

Small angle scattering affords an approach to evaluate the structure of dilute populations of macromolecules in solution where the measured scattering intensities relate to the distribution of scattering-pair distances within each macromolecule. When small angle neutron scattering (SANS) with contrast variation is employed, additional structural information can be obtained regarding the internal organization of biomacromolecule complexes and assemblies. The technique allows for the components of assemblies to be selectively 'matched in' and 'matched out' of the scattering profiles due to the different ways the isotopes of hydrogen-protium 1H, and deuterium 2H (or D)-scatter neutrons. The isotopic substitution of 1H for D in the sample enables the controlled variation of the scattering contrasts. A contrast variation experiment requires trade-offs between neutron beam intensity, q-range, wavelength and q-resolution, isotopic labelling levels, sample concentration and path-length, and measurement times. Navigating these competing aspects to find an optimal combination is a daunting task. Here we provide an overview of how to calculate the neutron scattering contrasts of dilute biological macromolecule samples prior to an experiment and how this then informs the approach to configuring SANS instruments and the measurement of a contrast variation series dataset.

Substrate Induced Movement of the Metal Cofactor between Active and Resting State.

Regulation of enzyme activity is vital for living organisms. In metalloenzymes, far-reaching rearrangements of the protein scaffold are generally required to tune the metal cofactor's properties by allosteric regulation. Here structural analysis of hydroxyketoacid aldolase from Sphingomonas wittichii RW1 (SwHKA) revealed a dynamic movement of the metal cofactor between two coordination spheres without protein scaffold rearrangements. In its resting state configuration (M2+ R ), the metal constitutes an integral part of the dimer interface within the overall hexameric assembly, but sterical constraints do not allow for substrate binding. Conversely, a second coordination sphere constitutes the catalytically active state (M2+ A ) at 2.4 Šdistance. Bidentate coordination of a ketoacid substrate to M2+ A affords the overall lowest energy complex, which drives the transition from M2+ R to M2+ A . While not described earlier, this type of regulation may be widespread and largely overlooked due to low occupancy of some of its states in protein crystal structures.

Production and characterisation of modularly deuterated UBE2D1-Ub conjugate by small angle neutron and X-ray scattering.

This structural study exploits the possibility to use modular protein deuteration to facilitate the study of ubiquitin signalling, transfer, and modification. A protein conjugation reaction is used to combine protonated E2 enzyme with deuterated ubiquitin for small angle X-ray and neutron scattering with neutron contrast variation. The combined biomolecules stay as a monodisperse system during data collection in both protonated and deuterated buffers indicating long stability of the E2-Ub conjugate. With multiphase ab initio shape restoration and rigid body modelling, we reconstructed the shape of a E2-Ub-conjugated complex of UBE2D1 linked to ubiquitin via an isopeptide bond. Solution X-ray and neutron scattering data for this E2-Ub conjugate in the absence of E3 jointly indicate an ensemble of open and backbent states, with a preference for the latter in solution. The approach of combining protonated and labelled proteins can be used for solution studies to assess localization and movement of ubiquitin and could be widely applied to modular Ub systems in general.

Intrinsically disordered plant protein PARCL colocalizes with RNA in phase-separated condensates whose formation can be regulated by mutating the PLD.

In higher plants, long-distance RNA transport via the phloem is crucial for communication between distant plant tissues to align development with stress responses and reproduction. Several recent studies suggest that specific RNAs are among the potential long-distance information transmitters. However, it is yet not well understood how these RNAs enter the phloem stream, how they are transported, and how they are released at their destination. It was proposed that phloem RNA-binding proteins facilitate RNA translocation. In the present study, we characterized two orthologs of the phloem-associated RNA chaperone-like (PARCL) protein from Arabidopsis thaliana and Brassica napus at functional and structural levels. Microscale thermophoresis showed that these phloem-abundant proteins can bind a broad spectrum of RNAs and show RNA chaperone activity in FRET-based in vitro assays. Our SAXS experiments revealed a high degree of disorder, typical for RNA-binding proteins. In agroinfiltrated tobacco plants, eYFP-PARCL proteins mainly accumulated in nuclei and nucleoli and formed cytosolic and nuclear condensates. We found that formation of these condensates was impaired by tyrosine-to-glutamate mutations in the predicted prion-like domain (PLD), while C-terminal serine-to-glutamate mutations did not affect condensation but reduced RNA binding and chaperone activity. Furthermore, our in vitro experiments confirmed phase separation of PARCL and colocalization of RNA with the condensates, while mutation as well as phosphorylation of the PLD reduced phase separation. Together, our results suggest that RNA binding and condensate formation of PARCL can be regulated independently by modification of the C-terminus and/or the PLD.

Herpes simplex virus 1 protein pUL21 alters ceramide metabolism by activating the interorganelle transport protein CERT.

Herpes simplex virus (HSV)-1 dramatically alters the architecture and protein composition of cellular membranes during infection, but its effects upon membrane lipid composition remain unclear. HSV-1 pUL21 is a virus-encoded protein phosphatase adaptor that promotes dephosphorylation of multiple cellular and virus proteins, including the cellular ceramide (Cer) transport protein CERT. CERT mediates nonvesicular Cer transport from the endoplasmic reticulum to the trans-Golgi network, whereupon Cer is converted to sphingomyelin (SM) and other sphingolipids that play important roles in cellular proliferation, signaling, and membrane trafficking. Here, we use click chemistry to profile the kinetics of sphingolipid metabolism, showing that pUL21-mediated dephosphorylation activates CERT and accelerates Cer-to-SM conversion. Purified pUL21 and full-length CERT interact with submicromolar affinity, and we solve the solution structure of the pUL21 C-terminal domain in complex with the CERT Pleckstrin homology and steroidogenic acute regulatory-related lipid transfer domains using small-angle X-ray scattering. We identify a single amino acid mutation on the surface of pUL21 that disrupts CERT binding in vitro and in cultured cells. This residue is highly conserved across the genus Simplexvirus. In addition, we identify a pUL21 residue essential for binding to HSV-1 pUL16. Sphingolipid profiling demonstrates that Cer-to-SM conversion is severely diminished in the context of HSV-1 infection, a defect that is compounded when infecting with a virus encoding the mutated form of pUL21 that lacks the ability to activate CERT. However, virus replication and spread in cultured keratinocytes or epithelial cells is not significantly altered when pUL21-mediated CERT dephosphorylation is abolished. Collectively, we demonstrate that HSV-1 modifies sphingolipid metabolism via specific protein-protein interactions.

Recombinant AcnB, NrdR and RibD of Acinetobacter baumannii and their potential interaction with DNA adenine methyltransferase AamA.

In the last decades Acinetobacter baumannii developed into an increasingly challenging nosocomial pathogen. A. baumannii ATCC 17978 harbors a DNA-(adenine N6)-methyltransferase termed AamA. Previous studies revealed a low specific activity of AamA in vitro despite proven folding, which led us to speculate about possible interaction partners assisting AamA in targeting methylation sites. Here, applying a pulldown assay with subsequent mass spectrometry we identified aconitate hydratase 2 (AcnB) as possible interaction partner. In addition, we considered the putative transcriptional regulator gene nrdR (A1S_0220) and the pyrimidine deaminase/reductase gene ribD (A1S_0221) of A. baumannii strain ATCC 17978 to encode additional potential interaction partners due to their vicinity to the aamA gene (A1S_0222). Proteins were recombinantly produced in the milligram scale, purified to near homogeneity, and interactions with AamA were studied applying blue native gel electrophoreses, electrophoretic mobility shift assay, chemical cross-linking and co-immunoprecipitation. These analyses did not provide evidence of interaction between AamA and purified proteins. Solution structures of RibD, NrdR and AcnB were studied by small-angle X-ray scattering (SAXS) alone and in combination with AamA. While in the case of RibD and AcnB no evidence of an interaction with AamA was produced, addition of AamA to NrdR resulted in dissociation of long and rod-shaped polymeric NrdR structures, implying a specific but transient interaction. Moreover, we identified a molecular crowding effect possibly impeding the DNA methyltransferase activity in vivo and a sequence-independent DNA binding activity of AamA calling for continued efforts to identify the interaction network of AamA.