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A standalone system, called PDB-Dev, has been developed for archiving integrative structures and making them publicly available. The paper describes the data standards, the software tools and the various components of the PDB-Dev data-collection, processing and archiving infrastructure., Structures of many complex biological assemblies are increasingly determined using integrative approaches, in which data from multiple experimental methods are combined. A standalone system, called PDB-Dev, has been developed for archiving integrative structures and making them publicly available. Here, the data standards and software tools that support PDB-Dev are described along with the new and updated components of the PDB-Dev data-collection, processing and archiving infrastructure. Following the FAIR (Findable, Accessible, Interoperable and Reusable) principles, PDB-Dev ensures that the results of integrative structure determinations are freely accessible to everyone.

Acta crystallographica / F 77(7), 215 - 225 (2021). doi:10.1107/S2053230X21006142, The crystal structures of domain-swapped tryptophan repressor (TrpR) variant Val58Ile before and after soaking with the physiological ligand L-tryptophan (L-Trp) indicate that L-Trp occupies the same location in the domain-swapped form as in native dimeric TrpR and makes equivalent residue contacts. This result is unexpected because the ligand binding-site residues arise from three separate polypeptide chains in the domain-swapped form. This work represents the first published structure of a domain-swapped form of TrpR with L-Trp bound. The presented structures also show that the protein amino-terminus, whether or not it bears a disordered extension of about 20 residues, is accessible in the large solvent channels of the domain-swapped crystal form, as in the structures reported previously in this form for TrpR without N-terminal extensions. These findings inspire the exploration of L-Trp analogs and N-terminal modifications as labels to orient guest proteins that cannot otherwise be crystallized in the solvent channels of crystalline domain-swapped TrpR hosts for potential diffraction analysis., Published by Blackwell, Oxford [u.a.]

As a discipline, structural biology has been transformed by the three-dimensional electron microscopy (3DEM) “Resolution Revolution” made possible by convergence of robust cryo-preservation of vitrified biological materials, sample handling systems, and measurement stages operating a liquid nitrogen temperature, improvements in electron optics that preserve phase information at the atomic level, direct electron detectors (DEDs), high-speed computing with graphics processing units, and rapid advances in data acquisition and processing software. 3DEM structure information (atomic coordinates and related metadata) are archived in the open-access Protein Data Bank (PDB), which currently holds more than 11,000 3DEM structures of proteins and nucleic acids, and their complexes with one another and small-molecule ligands (~ 6% of the archive). Underlying experimental data (3DEM density maps and related metadata) are stored in the Electron Microscopy Data Bank (EMDB), which currently holds more than 21,000 3DEM density maps. After describing the history of the PDB and the Worldwide Protein Data Bank (wwPDB) partnership, which jointly manages both the PDB and EMDB archives, this review examines the origins of the resolution revolution and analyzes its impact on structural biology viewed through the lens of PDB holdings. Six areas of focus exemplifying the impact of 3DEM across the biosciences are discussed in detail (icosahedral viruses, ribosomes, integral membrane proteins, SARS-CoV-2 spike proteins, cryogenic electron tomography, and integrative structure determination combining 3DEM with complementary biophysical measurement techniques), followed by a review of 3DEM structure validation by the wwPDB that underscores the importance of community engagement.

The history of the growth of the Protein Data Bank and the role that the community has played in developing standards and policies are described., The Protein Data Bank (PDB) has grown from a small data resource for crystallographers to a worldwide resource serving structural biology. The history of the growth of the PDB and the role that the community has played in developing standards and policies are described. This article also illustrates how other biophysics communities are collaborating with the worldwide PDB to create a network of interoperating data resources. This network will expand the capabilities of structural biology and enable the determination and archiving of increasingly complex structures.

PDBx/mmCIF, Protein Data Bank Exchange (PDBx) macromolecular Crystallographic Information Framework (mmCIF), has become the data standard for structural biology. With its early roots in the domain of small-molecule crystallography, PDBx/mmCIF provides an extensible data representation that is used for deposition, archiving, remediation, and public dissemination of experimentally determined three-dimensional (3D) structures of biological macromolecules by the Worldwide Protein Data Bank (wwPDB, wwpdb.org). Extensions of PDBx/mmCIF are similarly used for computed structure models by ModelArchive (modelarchive.org), integrative/hybrid structures by PDB-Dev (pdb-dev.wwpdb.org), small angle scattering data by Small Angle Scattering Biological Data Bank SASBDB (sasbdb.org), and for models computed generated with the AlphaFold 2.0 deep learning software suite (alphafold.ebi.ac.uk). Community-driven development of PDBx/mmCIF spans three decades, involving contributions from researchers, software and methods developers in structural sciences, data repository providers, scientific publishers, and professional societies. Having a semantically rich and extensible data framework for representing a wide range of structural biology experimental and computational results, combined with expertly curated 3D biostructure data sets in public repositories, accelerates the pace of scientific discovery. Herein, we describe the architecture of the PDBx/mmCIF data standard, tools used to maintain representations of the data standard, governance, and processes by which data content standards are extended, plus community tools/software libraries available for processing and checking the integrity of PDBx/mmCIF data. Use cases exemplify how the members of the Worldwide Protein Data Bank have used PDBx/mmCIF as the foundation for its pipeline for delivering Findable, Accessible, Interoperable, and Reusable (FAIR) data to many millions of users worldwide. published

Acta crystallographica / Section D 77(4), 471 - 485 (2021). doi:10.1107/S2059798321001078, Soaking small molecules into the solvent channels of protein crystals is the most common method of obtaining crystalline complexes with ligands such as substrates or inhibitors. The solvent channels of some protein crystals are large enough to allow the incorporation of macromolecules, but soaking of protein guests into protein crystals has not been reported. Such protein host crystals (here given the name hostals) incorporating guest proteins may be useful for a wide range of applications in biotechnology, for example as cargo systems or for diffraction studies analogous to the crystal sponge method. The present study takes advantage of crystals of the Escherichia coli tryptophan repressor protein (ds-TrpR) that are extensively domain-swapped and suitable for incorporating guest proteins by diffusion, as they are robust and have large solvent channels. Confocal fluorescence microscopy is used to follow the migration of cytochrome c and fluorophore-labeled calmodulin into the solvent channels of ds-TrpR crystals. The guest proteins become uniformly distributed in the crystal within weeks and enriched within the solvent channels. X-ray diffraction studies on host crystals with high concentrations of incorporated guests demonstrate that diffraction limits of ���2.5 �� can still be achieved. Weak electron density is observed in the solvent channels, but the guest-protein structures could not be determined by conventional crystallographic methods. Additional approaches that increase the ordering of guests in the host crystal are discussed that may support protein structure determination using the hostal system in the future. This host system may also be useful for biotechnological applications where crystallographic order of the guest is not required., Published by Wiley, Bognor Regis

Structures of biomolecular systems are increasingly computed by integrative modeling. In this approach, a structural model is constructed by combining information from multiple sources, including varied experimental methods and prior models. In 2019, a Workshop was held as a Biophysical Society Satellite Meeting to assess progress and discuss further requirements for archiving integrative structures. The primary goal of the Workshop was to build consensus for addressing the challenges involved in creating common data standards , building methods for federated data exchange, and developing mechanisms for validating integrative structures. The summary of the Workshop and the recommendations that emerged are presented here. Introduction When the Protein Data Bank (PDB) (Protein Data Bank, 1971) was first established in 1971, X-ray crystallography was the only method for determining three-dimensional structures of biological macromolecules at sufficient resolution to build atomic models. A decade later, structures of biomolecules in solution could also be determined by nuclear magnetic resonance (NMR) spectroscopy (Williamson et al., 1985). Recently, three-dimensional cryoelectron microscopy (3DEM) (Henderson et al., 1990) began to achieve unprecedented near-atomic resolution for large complex assemblies. Increasingly, investigators are also modeling structures based on data from more than one method (Rout and Sali, 2019). These integrative/hybrid approaches to structure determination consist of collecting information about a system using multiple experimental and computational methods, followed by integrative/hybrid modeling that converts this information into integrative/hybrid structure models. For succinctness, we will use the term integra-tive hereafter to refer to integrative/hybrid approaches, modeling, and models.

An evaluation system and a web infrastructure were developed for the second cryo-EM model challenge. The evaluation system includes tools to validate stereo-chemical plausibility of submitted models, check their fit to the corresponding density maps, estimate their overall and per-residue accuracy, and assess their similarity to reference cryo-EM or X-ray structures as well as other models submitted in this challenge. The web infrastructure provides a convenient interface for analyzing models at different levels of detail. It includes interactively sortable tables of evaluation scores for different subsets of models and different sublevels of structure organization, and a suite of visualization tools facilitating model analysis. The results are publicly accessible at http://model-compare.emdatabank.org.

OneDep, a unified system for deposition, biocuration, and validation of experimentally determined structures of biological macromolecules to the Protein Data Bank (PDB) archive, has been developed as a global collaboration by the Worldwide Protein Data Bank (wwPDB) partners. This new system was designed to ensure that the wwPDB could meet the evolving archiving requirements of the scientific community over the coming decades. OneDep unifies deposition, biocuration, and validation pipelines across all wwPDB, EMDB, and BMRB deposition sites with improved focus on data quality and completeness in these archives, while supporting growth in the number of depositions and increases in their average size and complexity. In this paper, we describe the design, functional operation, and supporting infrastructure of the OneDep system, and provide initial performance assessments.

The Protein Data Bank (PDB) is the single global repository for experimentally determined 3D structures of biological macromolecules and their complexes with ligands. The worldwide PDB (wwPDB) is the international collaboration that manages the PDB archive according to the FAIR principles: Findability, Accessibility, Interoperability and Reusability. The wwPDB recently developed OneDep, a unified tool for deposition, validation and biocuration of structures of biological macromolecules. All data deposited to the PDB undergo critical review by wwPDB Biocurators. This article outlines the importance of biocuration for structural biology data deposited to the PDB and describes wwPDB biocuration processes and the role of expert Biocurators in sustaining a high-quality archive. Structural data submitted to the PDB are examined for self-consistency, standardized using controlled vocabularies, cross-referenced with other biological data resources and validated for scientific/technical accuracy. We illustrate how biocuration is integral to PDB data archiving, as it facilitates accurate, consistent and comprehensive representation of biological structure data, allowing efficient and effective usage by research scientists, educators, students and the curious public worldwide. Database URL: https://www.wwpdb.org/

In this review, we describe how the interplay among science, technology and community interests contributed to the evolution of four structural biology data resources. We present the method by which data deposited by scientists are prepared for worldwide distribution, and argue that data archiving in a trusted repository must be an integral part of any scientific investigation.

The integration of cellular and molecular structural data is key to understanding the function of macromolecular assemblies and complexes in their in vivo context. Here we report on the outcomes of a workshop that discussed how to integrate structural data from a range of public archives. The workshop identified two main priorities: the development of tools and file formats to support segmentation (that is, the decomposition of a three-dimensional volume into regions that can be associated with defined objects), and the development of tools to support the annotation of biological structures. DOI: http://dx.doi.org/10.7554/eLife.25835.001

The crystal structure of E. coli catabolite activator protein with bound cobalt(II) and sulfate ions at 1.97 Å resolution is reported., The crystal structure of cyclic AMP–catabolite activator protein (CAP) from Escherichia coli containing cobalt(II) chloride and ammonium sulfate is reported at 1.97 Å resolution. Each of the two CAP subunits in the asymmetric unit binds one cobalt(II) ion, in each case coordinated by N-terminal domain residues His19, His21 and Glu96 plus an additional acidic residue contributed via a crystal contact. The three identified N-terminal domain cobalt-binding residues are part of a region of CAP that is important for transcription activation at class II CAP-dependent promoters. Sulfate anions mediate additional crystal lattice contacts and occupy sites corresponding to DNA backbone phosphate positions in CAP–DNA complex structures.

The α subunit C-terminal domain (αCTD) of RNA polymerase (RNAP) is a key element in transcription activation inEscherichia coli, possessing determinants responsible for the interaction of RNAP with DNA and with transcription factors. Here, the crystal structure ofE. coliαCTD (α subunit residues 245–329) determined to 2.0 Å resolution is reported. Crystals were obtained after reductive methylation of the recombinantly expressed domain. The crystals belonged to space groupP21and possessed both pseudo-translational symmetry and pseudo-merohedral twinning. The refined coordinate model (Rfactor = 0.193,Rfree= 0.236) has improved geometry compared with prior lower resolution determinations of the αCTD structure [Jeonet al.(1995),Science,270, 1495–1497; Benoffet al.(2002),Science,297, 1562–1566]. An extensive dimerization interface formed primarily by N- and C-terminal residues is also observed. The new coordinates will facilitate the improved modeling of αCTD-containing multi-component complexes visualized at lower resolution using X-ray crystallography and electron-microscopy reconstruction.

A new data model for PDB entries of viruses and other biological assemblies with regular noncrystallographic symmetry is described., A new scheme has been devised to represent viruses and other biological assemblies with regular noncrystallographic symmetry in the Protein Data Bank (PDB). The scheme describes existing and anticipated PDB entries of this type using generalized descriptions of deposited and experimental coordinate frames, symmetry and frame transformations. A simplified notation has been adopted to express the symmetry generation of assemblies from deposited coordinates and matrix operations describing the required point, helical or crystallographic symmetry. Complete correct information for building full assemblies, subassemblies and crystal asymmetric units of all virus entries is now available in the remediated PDB archive.

Vsp surface lipoproteins are serotype-defining antigens of relapsing fever spirochetes that undergo multiphasic antigenic variation to allow bacterial persistence in spite of an immune response. Two isogenic serotypes of Borrelia turicatae strain Oz1 differ in their Vsp sequences and in disease manifestations in infected mice: Vsp1 is associated with the selection of a neurological niche, while Vsp2 is associated with blood and skin infection. We report here crystal structures of the Vsp1 dimer at 2.7 and 2.2 Å. The structures confirm that relapsing fever Vsp proteins share a common helical fold with OspCs of Lyme disease-causing Borrelia . The fold features an inner stem formed by highly conserved N and C termini and an outer “dome” formed by the variable central residues. Both Vsp1 and OspC structures possess small water-filled cavities, or pockets, that are lined largely by variable residues and are thus highly variable in shape. These features appear to signify tolerance of the Vsp-OspC fold for imperfect packing of residues at its antigenic surface. Structural comparison of Vsp1 with a homology model for Vsp2 suggests that observed differences in disease manifestation may arise in part from distinct differences in electrostatic surface properties; additional predicted positively charged surface patches on Vsp2 compared to Vsp1 may be sufficient to explain the relative propensity of Vsp2 to bind to acidic glycosaminoglycans.

The catabolite activator protein (CAP) bends DNA in the CAP-DNA complex, typically introducing a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A1A2A3T4G5T6G7A8T9C10T11 -3' ("primary kink"). In previous work, we showed that CAP recognizes the nucleotide immediately 5' to the primary-kink site, T6, through an "indirect-readout" mechanism involving sequence effects on energetics of primary-kink formation. Here, to understand further this example of indirect readout, we have determined crystal structures of CAP-DNA complexes containing each possible nucleotide at position 6. The structures show that CAP can introduce a DNA kink at the primary-kink site with any nucleotide at position 6. The DNA kink is sharp with the consensus pyrimidine-purine step T6G7 and the non-consensus pyrimidine-purine step C6G7 (roll angles of approximately 42 degrees, twist angles of approximately 16 degrees ), but is much less sharp with the non-consensus purine-purine steps A6G7 and G6G7 (roll angles of approximately 20 degrees, twist angles of approximately 17 degrees). We infer that CAP discriminates between consensus and non-consensus pyrimidine-purine steps at positions 6-7 solely based on differences in the energetics of DNA deformation, but that CAP discriminates between the consensus pyrimidine-purine step and non-consensus purine-purine steps at positions 6-7 both based on differences in the energetics of DNA deformation and based on qualitative differences in DNA deformation. The structures further show that CAP can achieve a similar, approximately 46 degrees per DNA half-site, overall DNA bend through a sharp DNA kink, a less sharp DNA kink, or a smooth DNA bend. Analysis of these and other crystal structures of CAP-DNA complexes indicates that there is a large, approximately 28 degrees per DNA half-site, out-of-plane component of CAP-induced DNA bending in structures not constrained by end-to-end DNA lattice interactions and that lattice contacts involving CAP tend to involve residues in or near biologically functional surfaces.

A classification model of a DNA-binding protein chain was created based on identification of alpha helices within the chain likely to bind to DNA. Using the model, all chains in the Protein Data Bank were classified. For many of the chains classified with high confidence, previous documentation for DNA-binding was found, yet no sequence homology to the structures used to train the model was detected. The result indicates that the chain model can be used to supplement sequence based methods for annotating the function of DNA-binding. Four new candidates for DNA-binding were found, including two structures solved through structural genomics efforts. For each of the candidate structures, possible sites of DNA-binding are indicated by listing the residue ranges of alpha helices likely to interact with DNA.

The E. coli trp repressor (trpR) homodimer recognizes its palindromic DNA binding site through a pair of flexible helix-turn-helix (HTH) motifs displayed on an intertwined helical core. Flexible N-terminal arms mediate association between dimers bound to tandem DNA sites. The 2.5 Å X-ray structure of trpR crystallized in 30% (v/v) isopropanol reveals a substantial conformational rearrangement of HTH motifs and N-terminal arms, with the protein appearing in the unusual form of an ordered 3D domain-swapped supramolecular array. Small angle X-ray scattering measurements show that the self-association properties of trpR in solution are fundamentally altered by isopropanol.

Recently determined structures of the Escherichia coli catabolite activator protein (CAP) in complex with DNA, and in complex with the RNA polymerase alpha subunit C-terminal domain (alphaCTD) and DNA, have yielded insights into how CAP binds DNA and activates transcription. Comparison of multiple structures of CAP-DNA complexes has revealed the contributions of direct and indirect readout to DNA binding by CAP. The structure of the CAP-alphaCTD-DNA complex has provided the first structural description of interactions between a transcription activator and its functional target within the general transcription machinery. Using the structure of the CAP-alphaCTD-DNA complex, the structure of an RNA polymerase-DNA complex, and restraints from biophysical, biochemical and genetic experiments, it has been possible to construct detailed three-dimensional models of intact class I and class II transcription activation complexes.

We report the outcomes of the discussion initiated at the workshop entitled A 3D Cellular Context for the Macromolecular World and propose how data from emerging three-dimensional (3D) cellular imaging techniques—such as electron tomography, 3D scanning electron microscopy and soft X-ray tomography—should be archived, curated, validated and disseminated, to enable their interpretation and reuse by the biomedical community.

Outer surface protein A (OspA) is a major lipoprotein of the Borrelia burgdorferi spirochete, the causative agent of Lyme disease. Vaccination with OspA generates an immune response that can prevent bacterial transmission to a mammalian host during the attachment of an infected tick. However, the protective capacity of immune sera cannot be predicted by measuring total anti-OspA antibody. The murine monoclonal antibody LA-2 defines an important protective B-cell epitope of OspA against which protective sera have strong levels of reactivity. We have now mapped the LA-2 epitope of OspA using both NMR chemical-shift perturbation measurements in solution and X-ray crystal structure determination. LA-2 recognizes the three surface-exposed loops of the C-terminal domain of OspA that are on the tip of the elongated molecule most distant from the lipid-modified N terminus. The structure suggests that the natural variation at OspA sequence position 208 in the first loop is a major limiting factor for antibody cross-reactivity between different Lyme disease-causing Borrelia strains. The unusual Fab-dominated lattice of the crystal also permits a rare view of antigen flexibility within an antigen:antibody complex. These results provide a rationale for improvements in OspA-based vaccines and suggest possible designs for more direct tests of antibody protective levels in vaccinated individuals.

This report describes the outcomes of the Data Management Challenges in 3D Electron Microscopy workshop. Key topics discussed include data models, validation and raw-data archiving. The meeting participants agreed that the EMDataBank should take the lead in addressing these issues, and concrete action points were agreed upon that will have a substantial impact on the accessibility of three-dimensional EM data in biology and medicine.

The Protein Data Bank (PDB) was established in 1971 as a repository for the three dimensional structures of biological macromolecules. Since then, more than 85000 biological macromolecule structures have been determined and made available in the PDB archive. Through analysis of the corpus of data, it is possible to identify trends that can be used to inform us abou the future of structural biology and to plan the best ways to improve the management of the ever-growing amount of PDB data.

This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM (http://www.emdatabank.org) and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine.

Electron cryo-microscopy (cryoEM) is a rapidly maturing methodology in structural biology, which now enables the determination of 3D structures of molecules, macromolecular complexes and cellular components at resolutions as high as 3.5A, bridging the gap between light microscopy and X-ray crystallography/NMR. In recent years structures of many complex molecular machines have been visualized using this method. Single particle reconstruction, the most widely used technique in cryoEM, has recently demonstrated the capability of producing structures at resolutions approaching those of X-ray crystallography, with over a dozen structures at better than 5 A resolution published to date. This method represents a significant new source of experimental data for molecular modeling and simulation studies. CryoEM derived maps and models are archived through EMDataBank.org joint deposition services to the EM Data Bank (EMDB) and Protein Data Bank (PDB), respectively. CryoEM maps are now being routinely produced over the 3 - 30 A resolution range, and a number of computational groups are developing software for building coordinate models based on this data and developing validation techniques to better assess map and model accuracy. In this workshop we will present the results of the first cryoEM modeling challenge, in which computational groups were asked to apply their tools to a selected set of published cryoEM structures. We will also compare the results of the various applied methods, and discuss the current state of the art and how we can most productively move forward.

One obstacle to achieving complete understanding of the principles underlying sequence-dependent recognition of DNA is the paucity of structural data for DNA recognition sequences in their free (unbound) state. Here, we carried out crystallization screening of 50 DNA duplexes containing cognate protein binding sites and obtained new crystal structures of free DNA binding sites for three distinct modes of DNA recognition: anti-parallel β strands (MetR), helix-turn-helix motif + hinge helices (PurR), and zinc fingers (Zif268). Structural changes between free and protein-bound DNA are manifested differently in each case. The new DNA structures reveal that distinctive sequence-dependent DNA geometry dominates recognition by MetR, protein-induced bending of DNA dictates recognition by PurR, and deformability of DNA along the A - B continuum is important in recognition by Zif268. Together, our findings show that crystal structures of free DNA binding sites provide new information about the nature of protein–DNA interactions and thus lend insights towards a structural code for DNA recognition.

Structures of biomolecular systems are increasingly computed by integrative modeling that relies on varied types of experimental data and theoretical information. We describe here the proceedings and conclusions from the first wwPDB Hybrid/Integrative Methods Task Force Workshop held at the European Bioinformatics Institute in Hinxton, UK, on October 6 and 7, 2014. At the workshop, experts in various experimental fields of structural biology, experts in integrative modeling and visualization, and experts in data archiving addressed a series of questions central to the future of structural biology. How should integrative models be represented? How should the data and integrative models be validated? What data should be archived? How should the data and models be archived? What information should accompany the publication of integrative models? The workshop was supported by funding to PDBe by Wellcome Trust 088944; RCSB PDB by NSF DBI 1338415; PDBj by JST-NBDC; BMRB by NLM P41 LM05799; EMDataBank by NIH GM079429; and tax-deductible donations made to the wwPDB Foundation in support of wwPDB outreach activities.

Data management has emerged as one of the central issues in the high-throughput processes of taking a protein target sequence through to a protein sample. To simplify this task, and following extensive consultation with the international structural genomics community, we describe here a model of the data related to protein production. The model is suitable for both large and small facilities for use in tracking samples, experiments, and results through the many procedures involved. The model is described in Unified Modeling Language (UML). In addition, we present relational database schemas derived from the UML. These relational schemas are already in use in a number of data management projects.

Crystallization and preliminary neutron-diffraction measurements of wild-type variant Val58--Ile of the Escherichia coli trp repressor are reported. A vapor-diffusion chamber suitable for initial protein-solution Volumes in the range 0.2-0.5 ml was used to grow cube-shaped crystals with edge dimensions in the range 0.8-1.4 mm. Neutron Laue measurements to a nominal resolution of 2.1 A were recorded from a D(2)O-exchanged crystal using the LADI instrument at ILL. These results demonstrate that it will be possible for the first time to obtain a full-atom neutron structural model of a DNA-binding protein plus its associated solvent. Direct observation of hydrogen bonding between protein and solvent should enhance understanding of the role of solvent in protein-DNA recognition.

Cocrystals of a 2:1 complex of trp repressor dimers with a DNA duplex containing a single, central operator half-site sequence are described. Crystals with different, morphologies grew under diverse crystallization conditions within days to weeks by hanging drop vapor-diffusion. Twinned rods split along their longitudinal cleft produce single crystals with space group C 2 and unit cell dimensions a = 112·34 A b =90·16 A c =58·65 A and β = 113·92°. The crystals diffract to 2·4 A and are thus suitable for structural analysis by X-ray diffraction. Several heavy-atom derivative cocrystals have been obtained with iodouridine·substituted DNAs.

Two crystal forms of recombinant p26 capsid protein (CA) from the equine infectious anemia virus (EIAV) have in common an antiparallel four-helix bundle dimer interface between N-terminal domains (NTDs). The dimer interface provides a lenient scaffold to accommodate the wide sequence variation in these helices within lentivirus CA. Pairs of dimers weakly associate to form exact or approximate D2 symmetry tetramers. In one of the two crystal forms, the tetramers are linked via dimerization of C-terminal domains (CTDs). We propose that the observed NTD and CTD homodimer interactions are involved in the assembly of the lentivirus capsid. The NTD homodimer shape readily suggests a model for the mature capsid core, based on hexagonal packing with dimensions and surface topology resembling described EIAV capsid cores. Combining available data for human immunodeficiency virus and EIAV CA, we also propose an assembly pathway for maturation of the lentivirus capsid core following proteolytic cleavage of the gag polyprotein precursor.

The cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) gene from Bacillus circulans strain 251 was cloned and sequenced. It was found to code for a mature protein of 686 amino acid residues, showing 75% identity to the CGTase from B. circulans strain 8. The X-ray structure of the CGTase was elucidated in a maltodextrin-dependent crystal form and refined against X-ray diffraction data to 2.0 A resolution. The structure of the enzyme is nearly identical to the CGTase from B. circulans strain 8. Three maltose binding sites are observed at the protein surface, two in domain E and one in domain C. The maltose-dependence of CGTase crystallization can be ascribed to the proximity of two of the maltose binding sites to intermolecular crystal contacts. The maltose molecules bound in the E domain interact with several residues implicated in a raw starch binding motif conserved among a diverse group of starch converting enzymes.

Crystals of cyclomaltodextrin glucanotransferase from Bacillus circulans (EC 2.4.1.19) suitable for high-resolution X-ray analysis were obtained by vapor diffusion against 60% ( v v ) 2-methyl 2,4-pentanediol buffered with 100 m m -sodium Hepes, pH 7.55. The crystals have P212121 space group symmetry, with a = 120.4 A , b = 110.9 A and c = 66.4 A , and contain one molecule of 68,000 in the asymmetric unit. Growth of single enzyme crystals was found to require the presence of either α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or maltose in high molar excess, a requirement that could not be fulfilled by glucose, the basic building block of these compounds. Although the exact role of cyclic and linear maltodextrins in enzyme crystallization is not yet known, we have preliminary evidence that these compounds are degraded by the enzyme in the crystallization droplet.

Bactericidal Fab Michael Becker, Jonas Bunikis, Barbara D. Lade, John J. Dunn, Alan G. Barbour, Catherine L. Lawson, Biology Department, Brookhaven National Laboratory, Upton, NY. Department of Microbiology & Molecular Genetics and Department of Medicine, University of California at Irvine, College of Medicine, Irvine, CA. Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA. E-mail: mbecker@bnl.gov

Alcohol oxidase, purified from the yeast Hansenula polymorpha, was crystallized in vitro for the purpose of determining its structure at atomic resolution by X-ray diffraction methods. The crystals obtained yielded only extremely weak diffraction patterns: the maximal resolution observed was in the best case 6 Å. Electron microscopy of thin sections indicated that most crystals showed lattice defects which might explain the poor diffraction patterns: most surprising was the appearance of large holes interrupting an otherwise regular lattice in one of the crystal forms examined. Our results indicate that transmission electron microscopy is a suitable tool for the inspection of crystals to be used in X-ray crystallography. The method allows rapid determination of lattice defects and enables optimization of crystallization conditions.

Two novel colored fluorescent proteins were cloned and biophysically characterized from zooxanthellate corals (Anthozoa). A cyan fluorescent protein derived from the coral Montastrea cavernosa (mcCFP) is a trimeric complex with strong blue-shifted excitation and emission maxima at 432 and 477 nm, respectively. The native complex has a fluorescence lifetime of 2.66 % 0.01 ns and an inferred absolute quantum yield of 0.385. The spectroscopic properties of a green fluorescent protein cloned from Meandrina meandrites (mmGFP) resemble the commer- cially available GFP derived originally from the hydrozoan Aequorea victoria (avGFP). mmGFP is a monomeric protein with an excitation maximum at 398 nm and an emission maximum at 505 nm, a fluorescence lifetime of 3.10 % 0.01 ns and an absolute quantum yield of 0.645. Sequence homology with avGFP and the red fluorescent protein (DsRed) indicates that the proteins adopt the classic b-barrel configuration with 11 b-strands. The three amino acid residues that comprise the chromophore are QYG for mcCFP and TYG for mmGFP, compared with SYG for avGFP. A single point mutation, Ser-110 to Asn, was introduced into mmGFP by random mutagenesis. Denaturation and refolding experiments showed that the mutant has reduced aggregation, increased solubility and more efficient refolding relative to the wild type. Time-resolved emission lifetimes and anisotropies suggest that the electronic structure of the chromophore is highly dependent on the protonation state of adjoining residues. 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

An orthorhombic crystal form of trp repressor (aporepressor plus L-tryptophan ligand) was solved by molecular replacement, refined to 1.65 A resolution, and compared to the structure of the repressor in trigonal crystals. Even though these two crystal forms of repressor were grown under identical conditions, the refined structures have distinctly different conformations of the DNA-binding domains. Unlike the repressor/aporepressor structural transition, the conformational shift is not caused by the binding or loss of the L-tryptophan ligand. We conclude that while L-tryptophan binding is essential for forming a specific complex with trp operator DNA, the corepressor ligand does not lock the repressor into a single conformation that is complementary to the operator. This flexibility may be required by the various binding modes proposed for trp repressor in its search for and adherence to its three different operator sites.