Tom Terwilliger

Advances in machine learning have transformed structural biology, enabling swift and accurate prediction of protein structure from sequence. However, challenges persist in capturing sidechain packing, condition-dependent conformational dynamics, and biomolecular interactions, primarily due to scarcity of high-quality training data. Emerging techniq...

Giant tortoises exhibit exceptional longevity, often exceeding the human lifespan. To understand the genomic and epigenomic basis of their longevity, awe analyzed the DNA sequence and methylome of Jonathan, an Aldabra giant tortoise (Aldabrachelys gigantea), estimated to be 192 years old. Relative to other giant tortoises (Aldabrachelys gigantea an...

The interpretation of cryo-EM maps often includes the docking of known or predicted structures of the components, which is particularly useful when the map resolution is worse than 4 Å. Although it can be effective to search the entire map to find the best placement of a component, the process can be slow when the maps are large. However, frequentl...

The interpretation of cryo-EM maps often includes the docking of known or predicted structures of the components, which is particularly useful when the map resolution is worse than 4 Å. Although it can be effective to search the entire map to find the best placement of a component, the process can be slow when the maps are large. However, frequentl...

Advances in machine learning have enabled sufficiently accurate predictions of protein structure to be used in macromolecular structure determination with crystallography and cryo‐electron microscopy data. The Phenix software suite has AlphaFold predictions integrated into an automated pipeline that can start with an amino acid sequence and data, a...

Fluorescent proteins (FPs) are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered FPs such as superfolder green fluorescent protein (sfGFP) and superfolder Cherry (sfCherry) have exceptional refolding capability capable of delivering fluorescent readout in harsh environments...

Artificial intelligence-based protein structure prediction methods such as AlphaFold have revolutionized structural biology. The accuracies of these predictions vary, however, and they do not take into account ligands, covalent modifications or other environmental factors. Here, we evaluate how well AlphaFold predictions can be expected to describe...

Atomic model refinement at low resolution is often a challenging task. This is mostly because the experimental data are not sufficiently detailed to be described by atomic models. To make refinement practical and ensure that a refined atomic model is geometrically meaningful, additional information needs to be used such as restraints on Ramachandra...

BpeB and BpeF are multidrug efflux transporters from Burkholderia pseudomallei that enable multidrug resistance. Here, we report the crystal structures of BpeB and BpeF at 2.94 Å and 3.0 Å resolution, respectively. BpeB was found as an asymmetric trimer, consistent with the widely-accepted functional rotation mechanism for this type of transporter....

Fast, reliable docking of models into cryo-EM maps requires understanding of the errors in the maps and the models. Likelihood-based approaches to errors have proven to be powerful and adaptable in experimental structural biology, finding applications in both crystallography and cryo-EM. Indeed, previous crystallographic work on the errors in struc...

Optimized docking of models into cryo-EM maps requires exploiting an understanding of the signal expected in the data to minimize the calculation time while maintaining sufficient signal. The likelihood-based rotation function used in crystallography can be employed to establish plausible orientations in a docking search. A phased likelihood transl...

Experimental structure determination can be accelerated with artificial intelligence (AI)-based structure-prediction methods such as AlphaFold. Here, an automatic procedure requiring only sequence information and crystallo­graphic data is presented that uses AlphaFold predictions to produce an electron-density map and a structural model. Iterating...

Fast, reliable docking of models into cryo-EM maps requires understanding of the errors in the maps and the models. Likelihood-based approaches to errors have proven to be powerful and adaptable in experimental structural biology, finding applications in both crystallography and cryo-EM. Indeed, previous crystallographic work on the errors in struc...

Optimized docking of models into cryo-EM maps requires exploiting expected signal in the data to minimize the calculation time while maintaining sufficient signal. The likelihood-based rotation function used in crystallography can be employed to establish plausible orientations in a docking search. A phased likelihood translation function yields sc...

AI-based methods such as AlphaFold have raised the possibility of using predicted models in place of experimentally-determined structures. Here we assess the accuracy of AlphaFold predictions by comparing them to density maps obtained from automated redeterminations of recent crystal structures and to the corresponding deposited models. Some AlphaF...

Experimental structure determination can be accelerated with AI-based structure prediction methods such as AlphaFold. Here we present an automatic procedure requiring only sequence information and crystallographic data that uses AlphaFold predictions to produce an electron density map and a structural model. Iterating through cycles of structure pr...

The ability of Mycobacterium tuberculosis (Mtb) to persist in its host may enable an evolutionary advantage for drug resistant variants to emerge. A potential strategy to prevent persistence and gain drug efficacy is to directly target the activity of enzymes that are crucial for persistence. We present a method for expedited discovery and structur...

AlphaFold has recently become an important tool in providing models for experimental structure determination by X-ray crystallography and cryo-EM. Large parts of the predicted models typically approach the accuracy of experimentally determined structures, although there are frequently local errors and errors in the relative orientations of domains....

Machine-learning prediction algorithms such as AlphaFold and RoseTTAFold can create remarkably accurate protein models, but these models usually have some regions that are predicted with low confidence or poor accuracy. We hypothesized that by implicitly including new experimental information such as a density map, a greater portion of a model coul...

Regulation of bacteriophage gene expression involves repressor proteins that bind and downregulate early lytic promoters. A large group of mycobacteriophages code for repressors that are unusual in also terminating transcription elongation at numerous binding sites (stoperators) distributed across the phage genome. Here we provide the X-ray crystal...

Machine learning prediction algorithms such as AlphaFold can create remarkably accurate protein models, but these models usually have some regions that are predicted with low confidence or poor accuracy. We hypothesized that by implicitly including experimental information, a greater portion of a model could be predicted accurately, and that this m...

Split green fluorescent protein (GFP) has been used in a panoply of cellular biology applications to study protein translocation, monitor protein solubility and aggregation, detect protein–protein interactions, enhance protein crystallization, and even map neuron contacts. Recent work shows the utility of split fluorescent proteins for large scale...

Using single-particle electron cryo-microscopy (cryo-EM), it is possible to obtain multiple reconstructions showing the 3D structures of proteins imaged as a mixture. Here, it is shown that automatic map interpretation based on such reconstructions can be used to create atomic models of proteins as well as to match the proteins to the correct seque...

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of cur...

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Density modification uses expectations about features of a map such as a flat solvent and expected distributions of density in the region of the macromolecule to improve individual Fourier terms representing the map. This process transfers information from one part of a map to another and can improve the accuracy of a map. Here, the assumptions beh...

A density-modification procedure for improving maps from single-particle electron cryogenic microscopy (cryo-EM) is presented. The theoretical basis of the method is identical to that of maximum-likelihood density modification, previously used to improve maps from macromolecular X-ray crystallography. Key differences from applications in crystallog...

This paper describes outcomes of the 2019 Cryo-EM Map-based Model Metrics Challenge sponsored by EMDataResource (www.emdataresource.org). The goals of this challenge were (1) to assess the quality of models that can be produced using current modeling software, (2) to check the reproducibility of modeling results from different software developers a...

Density modification uses expectations about features of a map such as a flat solvent and expected distributions of density in the region of the macromolecule to improve individual Fourier terms representing the map. This process transfers information from one part of a map to another and can improve the accuracy of a map. Here the assumptions behi...

X-ray crystallography often requires non-native constructs involving mutations or truncations, and is challenged by membrane proteins and large multicomponent complexes. We present here a bottom-up endogenous structural proteomics approach whereby near-atomic-resolution cryo electron microscopy (cryoEM) maps are reconstructed ab initio from unident...

Mycobacterium tuberculosis (Mtb) is the most effective pathogen of mankind and is second only to human immunodeficiency virus/AIDS as the greatest cause of death worldwide due to a single infectious agent. Antibiotic resistance is a major reason why Tuberculosis (TB) is so difficult to eradicate and has become a challenge to overcome. Besides drug...

An iterative density modification procedure for improving maps produced by single-particle electron cryo-microscopy is presented. The theoretical basis of the method is identical to that of maximum-likelihood density modification, previously used to improve maps from macromolecular X-ray crystallography. Two key differences from applications in cry...

A procedure for building protein chains into maps produced by single‐particle electron cryo‐microscopy (cryo‐EM) is described. The procedure is similar to the way an experienced structural biologist might analyze a map, focusing first on secondary structure elements such as helices and sheets, then varying the contour level to identify connections...

Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist becau...

We report a fully automated procedure for the optimization and interpretation of reconstructions from cryo-electron microscopy (cryo-EM) data, available in Phenix as phenix.map_to_model. We applied our approach to 476 datasets with resolution of 4.5 Å or better, including reconstructions of 47 ribosomes and 32 other protein–RNA complexes. The media...

Burkholderia cenocepacia is an opportunistic pathogen that infects cystic fibrosis patients, causing pneumonia and septicemia. B. cenocepacia has intrinsic antibiotic resistance against monobactams, aminoglycosides, chloramphenicol and fluoroquinolones that is contributed by a homologue of BpeB, which is a member of the resistance-nodulation-cell d...

Recent advances in the field of electron cryomicroscopy (cryo-EM) have resulted in a rapidly increasing number of atomic models of biomacromolecules that have been solved using this technique and deposited in the Protein Data Bank and the Electron Microscopy Data Bank. Similar to macromolecular crystallography, validation tools for these models and...

A recently-developed method for identifying a compact, contiguous region representing the unique part of a density map was applied to 218 cryo-EM maps with resolutions of 4.5 Å or better. The key elements of the segmentation procedure are (1) identification of all regions of density above a threshold and (2) choice of a unique set of these regions,...

This article describes the implementation of real-space refinement in the phenix.real_space_refine program from the Phenix suite. Use of a simplified refinement target function enables very fast calculation, which in turn makes it possible to identify optimal data-restraints weight as part of routine refinements with little runtime cost. Refinement...

This article describes the implementation of real-space refinement in the phenix.real_space_refine program from the PHENIX suite. The use of a simplified refinement target function enables very fast calculation, which in turn makes it possible to identify optimal data-restraint weights as part of routine refinements with little runtime cost. Refine...

An algorithm for automatic map sharpening is presented that is based on optimization of the detail and connectivity of the sharpened map. The detail in the map is reflected in the surface area of an iso-contour surface that contains a fixed fraction of the volume of the map, where a map with high level of detail has a high surface area. The connect...

A recently-developed method for identifying a compact, contiguous region representing the unique part of a density map was applied to 218 cryo-EM maps with resolutions of 4.5 Å or better. The key elements of the segmentation procedure are (1) identification of all regions of density above a threshold and (2) choice of a unique set of these regions,...

Recent advances in the field of electron cryo-microscopy (cryo-EM) have resulted in a rapidly increasing number of atomic models of bio-macromolecules solved using this technique and deposited in the Protein Data Bank and the Electron Microscopy Data Bank. Similar to macromolecular crystallography, validation tools for these models and maps are req...

A fully automated procedure for optimization and interpretation of reconstructions from cryo-EM is developed and applied to 476 datasets with resolution of 4.5 Å or better, including reconstructions of 47 ribosomes and 32 other protein-RNA complexes. The median fraction of residues in the deposited structures reproduced automatically was 71% for re...

This article describes the implementation of real-space refinement in the phenix.real_space_refine program from the Phenix suite. Use of a simplified refinement target function enables fast calculation, which in turn makes it possible to identify optimal data-restraints weight as part of routine refinements with little runtime cost. Refinement of a...

Synopsis A procedure for optimizing the sharpening of a map based on maximizing the level of detail and connectivity of the map is developed and applied to 361 pairs of deposited cryo-EM maps and associated models. Abstract We present an algorithm for automatic map sharpening that is based on optimization of detail and connectivity of the sharpene...

Nucleoside tri-phosphates (NTP) form an important class of small molecule ligands that participate in, and are essential to a large number of biological processes. Here, we seek to identify the NTP binding proteome (NTPome) in M. tuberculosis (M.tb), a deadly pathogen. Identifying the NTPome is useful not only for gaining functional insights of the...

Responses to the letter to the editor ‘Atomic resolution’: a badly abused term in structural biology [Wlodawer & Dauter (2017), Acta Cryst. D73, 379–380].

The crystallographic maps that are routinely used during the structure-solution workflow are almost always model-biased because model information is used for their calculation. As these maps are also used to validate the atomic models that result from model building and refinement, this constitutes an immediate problem: anything added to the model...

A topical review is presented of the rapidly developing interest in and storage options for the preservation and reuse of raw data within the scientific domain of the IUCr and its Commissions, each of which operates within a great diversity of instrumentation. A résumé is included of the case for raw diffraction data deposition. An overall context...

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 Prot...

A key challenge in the SAD phasing method is solving a structure when the anomalous signal-to-noise ratio is low. A simple theoretical framework for describing measurements of anomalous differences and the resulting useful anomalous correlation and anomalous signal in a SAD experiment is presented. Here, the useful anomalous correlation is defined...

A key challenge in the SAD phasing method is solving a structure when the anomalous signal-to-noise ratio is low. Here, algorithms and tools for evaluating and optimizing the useful anomalous correlation and the anomalous signal in a SAD experiment are described. A simple theoretical framework [Terwilliger et al. (2016), Acta Cryst. D72, 346–358] i...

Merging of data from multiple crystals has proven to be useful for determination of the anomalously scattering atomic substructure for crystals with weak anomalous scatterers (e.g. S and P) and/or poor diffraction. Strategies for merging data from many samples, which require assessment of sample isomorphism, rely on metrics of variability in unit-c...

Structures of multisubunit macromolecular machines are primarily determined either by electron microscopy (EM) or by X-ray crystallography. In many cases, a structure for a complex can be obtained at low resolution (at a coarse level of detail) with EM and at a higher resolution (with finer detail) by X-ray crystallography. The integration of these...

Applications ranging from synthetic biology to protein crystallization could be advanced by facile systems for connecting multiple proteins together in predefined spatial relationships. One approach to this goal is to engineer many distinct assembly forms of a single carrier protein or scaffold, to which other proteins of interest can then be readi...

The final step of RNA crystallography involves the fitting of coordinates into electron density maps. The large number of backbone atoms in RNA presents a difficult and tedious challenge, particularly when experimental density is poor. The ERRASER-Phenix pipeline can improve an initial set of RNA coordinates automatically based on a physically real...

Identifying the intramolecular motions of proteins and nucleic acids is a major challenge in macromolecular X-ray crystallography. Because Bragg diffraction describes the average positional distribution of crystalline atoms with imperfect precision, the resulting electron density can be compatible with multiple models of motion. Diffuse X-ray scatt...

A method is presented that modifies a 2mFobs − DFmodel σA-weighted map such that the resulting map can strengthen a weak signal, if present, and can reduce model bias and noise. The method consists of first randomizing the starting map and filling in missing reflections using multiple methods. This is followed by restricting the map to regions with...

Early on, crystallography was a domain of mineralogy and mathematics and dealt mostly with symmetry properties and imaginary crystal lattices. This changed when Wilhelm Conrad Röntgen discovered X-rays in 1895, and in 1912, Max von Laue and his associates discovered that X-ray irradiated salt crystals would produce diffraction patterns that could r...

Identifying the intramolecular motions of proteins and nucleic acids is a major challenge in macromolecular X-ray crystallography. While Bragg diffraction describes the average positional distribution of crystalline atoms, many different models can fit this distribution equally well. Diffuse X-ray scattering can reduce this degeneracy by directly r...

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two pr...

We describe a likelihood-based method for determining the substructure of anomalously scattering atoms in macromolecular crystals that allows successful structure determination by single-wavelength anomalous diffraction (SAD) X-ray analysis with weak anomalous signal. With the use of partial models and electron density maps in searches for anomalou...

Significance A major challenge of protein crystallography is to accurately describe protein structure variations. Whereas Bragg peaks only yield a picture in which the variations are superimposed, diffuse X-ray scattering (diffraction between the peaks) reports on the way different atoms move together and can be used to increase the accuracy of mod...

This article describes some of the activities of the IUCr Diffraction Data Deposition Working Group and introduces a collection of articles discussing the archiving of diffraction images.

Accurate crystal structures of macromolecules are of high importance in the biological and biomedical fields. Models of crystal structures in the Protein Data Bank (PDB) are in general of very high quality as deposited. However, methods for obtaining the best model of a macromolecular structure from a given set of experimental X-ray data continue t...

Numerical comparison of crystallographic contour maps is used extensively in structure solution and model refinement, analysis and validation. However, traditional metrics such as the map correlation coefficient (map CC, real-space CC or RSCC) sometimes contradict the results of visual assessment of the corresponding maps. This article explains suc...