Jeffrey C. Hoch

John L. Markley was an early pioneer of the application of nuclear magnetic resonance (NMR) spectroscopy to investigate the structure, dynamics, and function of biological macromolecules by NMR. A recurring theme of his career is the development of standards and shared resources that have enabled the biological NMR community to coherently and collectively advance the disciplines of structural biology and metabolomics. Spanning development standards for atom nomenclature, founding shared NMR resources at Purdue and UW-Madison, and founding the Biological Magnetic Resonance Data Bank (BMRB) and shepherding BMRB membership in the Worldwide Protein Data Bank, this appreciation revisits the many and lasting contributions by our friend and colleague John Markley in support of the global structural biology community.

With the continuous growth of the Protein Data Bank archive, the Worldwide Protein Data Bank (wwPDB) partnership anticipates that the entire complement of possible four-character PDB accession codes (for example 1ABC) will be exhausted by 2028. wwPDB is, therefore, revising the PDB accession code (PDB ID) to 12 characters by extending its length and prepending `pdb_' (for example pdb_1000axyz) in lower case. This change will enable the robust detection of references to PDB entries in published literature. On or about July 21st 2027, the PDB will convert to releasing entries with extended PDB IDs only, which will not be compatible with the legacy PDB format. A beta version of the PDB Archive (PDB Beta Archive) is now available to help communities adapt to and embrace the extended PDB IDs and PDBx/mmCIF format during a transition phase. All files in the current PDB archive are reorganized in the Beta Archive with extended PDB IDs (including file naming and directories) on an entry-level basis, mirroring the data organization of the PDB Versioned Archive. wwPDB encourages scientific journals, PDB community members and users to transition to the PDBx/mmCIF format and adopt the new PDB ID format as early as possible. The PDB Beta Archive will replace the current public archive, and 12-character PDB IDs will be solely assigned to all newly deposited PDB entries.

Positive-strand RNA viruses remodel host intracellular membranes to form replication organelles in the cytoplasm. SARS-CoV-2 and other coronaviruses, members of the Nidovirales order, replicate within double-membrane vesicles (DMVs) derived from the endoplasmic reticulum that protect newly synthesized viral RNA (vRNA). A recently discovered SARS-CoV-2 molecular pore complex composed of non-structural proteins 3 and 4 (nsp3 and nsp4) connects the DMV interior with the cytoplasm, enabling vRNA export for subsequent packaging into virions. The C-terminal region of nsp3, the Y domain, oligomerizes to form the base of the cytosolic crown of this pore, while the preceding transmembrane domain 2 (TM2) anchors the pore in the DMV membrane. However, the molecular mechanisms underlying pore formation and whether similar structures are conserved across other Nidovirales members remain unclear. Here, we combined AlphaFold structure prediction, structure-informed multiple sequence alignments, and molecular dynamics simulations to investigate the conservation and potential function of the C-terminal TM2-Y region of nsp3 across nidoviruses. We show that despite minimal sequence identity, all modeled TM2-Y regions adopt similar domain folds. In particular, the wedge-shaped TM2, the Zn-binding Y1 subdomain, and its adjacent Y2 subdomain constitute a conserved structural module present in most vertebrate-infecting Nidovirales families, including Coronaviridae , Tobaniviridae , Gresnaviridae , Olifoviridae, and Arteriviridae . Across these viruses, Zn(II) is coordinated with a distinctive octahedral geometry involving two water molecules. This conserved architecture likely represents an evolutionarily maintained mechanism for DMV pore formation, in which the Zn-binding region engages the membrane surface to induce local lipid bilayer destabilization that may precede DMV pore assembly. IMPORTANCE: Over the last 25 years, several coronaviruses, including highly pathogenic SARS-CoV, MERS-CoV, and SARS-CoV-2, have emerged in humans through zoonotic transmission. With nearly 100 coronaviruses known, further spillovers remain possible. Coronaviruses and other nidoviruses replicate within specialized replication organelles formed from host membranes; however, how they form remains poorly understood. This study reveals that coronaviruses and distantly related tobaniviruses and arteriviruses might share a conserved molecular framework for assembling membrane pores that connect the DMV interior, where vRNA is synthetized, and the cytoplasm, where virions are assembled. By identifying conserved structural elements in the C-terminal region of nsp3, including wedge-shaped TM2 domain and Zn-binding Y1/Y2 tandem, we show that vertebrate-infecting nidoviruses likely share structural mechanism for DMV pore formation. These findings provide insight into how diverse nidoviruses remodel host membranes and highlight the C-terminal region of nsp3 as a potential target for broad-spectrum antivirals.

The NMR Exchange Format (NEF) is a community-driven standard for representing NMR experimental data in a consistent, interoperable, and machine-readable form. Built on the STAR syntax, NEF provides a structured framework for storing and exchanging chemical shifts, peak lists, various types of structural restraints, and related metadata, thus allowing for data exchange across software platforms. By enabling direct, lossless transfer of information, NEF simplifies multi-software workflows, improves reproducibility, and supports FAIR (Findable, Accessible, Interoperable, Reusable) data principles. We describe the NEF specification, its current implementation across commonly used NMR software packages, and its application in areas including biomolecular structure determination, metabolomics, and ligand screening. Testing demonstrates that NEF can be used to exchange complete datasets between programs without loss of information or functionality. We also outline recent developments and future directions, such as inclusion of NMR relaxation data and support for non-standard residue topologies. NEF growing adoption highlights its potential as a unifying standard for NMR data, enabling more efficient, transparent and collaborative research.

The nutrient germinant receptors (GRs) in spores of Bacillus species consist of a cluster of three proteins– designated A, B, and C subunits– that play a critical role in initiating the germination of dormant spores in response to specific nutrient molecules. The Bacillus cereus GerI GR is essential for inosine-induced germination; however, the roles of the individual subunits and the mechanism by which germinant binding activates GR function remain unclear. In this study, we report the backbone chemical shift assignments of the N-terminal domain (NTD) of the A subunit of GerI (GerIA^NTD). Furthermore, we derive the secondary structure of GerIA^NTD in solution and compare it with the crystal structure of the NTD of the A subunit of a Bacillus megaterium GR. These findings lay the foundation for further NMR studies aimed at investigating the structure-function relationship of the GerI subunits, with a broader goal of understanding the molecular mechanism underlying germinant recognition and signal transduction in GRs across Bacillus species.

Background The National Cancer Institute issued a Request for Information (RFI; NOT-CA-23-007) in October 2022, soliciting input on using and reusing metabolomics data. This RFI aimed to gather input on best practices for metabolomics data storage, management, and use/reuse. Aim of review The nuclear magnetic resonance (NMR) Interest Group within the Metabolomics Association of North America (MANA) prepared a set of recommendations regarding the deposition, archiving, use, and reuse of NMR-based and, to a lesser extent, mass spectrometry (MS)-based metabolomics datasets. These recommendations were built on the collective experiences of metabolomics researchers within MANA who are generating, handling, and analyzing diverse metabolomics datasets spanning experimental (sample handling and preparation, NMR/MS metabolomics data acquisition, processing, and spectral analyses) to computational (automation of spectral processing, univariate and multivariate statistical analysis, metabolite prediction and identification, multi-omics data integration, etc.) studies. Key scientific concepts of review We provide a synopsis of our collective view regarding the use and reuse of metabolomics data and articulate several recommendations regarding best practices, which are aimed at encouraging researchers to strengthen efforts toward maximizing the utility of metabolomics data, multi-omics data integration, and enhancing the overall scientific impact of metabolomics studies.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Biomolecular structure analysis from experimental NMR studies generally relies on restraints derived from a combination of experimental and knowledge-based data. A challenge for the structural biology community has been a lack of standards for representing these restraints, preventing the establishment of uniform methods of model-vs-data structure validation against restraints and limiting interoperability between restraint-based structure modeling programs. The NMR exchange (NEF) and NMR-STAR formats provide a standardized approach for representing commonly used NMR restraints. Using these restraint formats, a standardized validation system for assessing structural models of biopolymers against restraints has been developed and implemented in the wwPDB OneDep data deposition-validation-biocuration system. The resulting wwPDB Restraint Violation Report provides a model vs. data assessment of biomolecule structures determined using distance and dihedral restraints, with extensions to other restraint types currently being implemented. These tools are useful for assessing NMR models, as well as for assessing biomolecular structure predictions based on distance restraints. Highlights PDB Structure Validation Report expanded to include Restraint Analysis NMR Exchange Format (NEF) and NMR-STAR for distance restraint representation Standard distance and dihedral restraint formats for model vs. restraint assessment Standardized restraint formats provide interoperability between modeling programs