Serge X. Cohen

Developments in the CCP4 molecular-graphics project

Progress towards structure determination that is both high-throughput and high-value is dependent on the development of integrated and automatic tools for electron-density map interpretation and for the analysis of the resulting atomic models. Advances in map-interpretation algorithms are extending the resolution regime in which fully automatic tools can work reliably, but at present human intervention is required to interpret poor regions of macromolecular electron density, particularly where crystallographic data is only available to modest resolution [for example, I/sigma(I) < 2.0 for minimum resolution 2.5 A]. In such cases, a set of manual and semi-manual model-building molecular-graphics tools is needed. At the same time, converting the knowledge encapsulated in a molecular structure into understanding is dependent upon visualization tools, which must be able to communicate that understanding to others by means of both static and dynamic representations. CCP4 mg is a program designed to meet these needs in a way that is closely integrated with the ongoing development of CCP4 as a program suite suitable for both low- and high-intervention computational structural biology. As well as providing a carefully designed user interface to advanced algorithms of model building and analysis, CCP4 mg is intended to present a graphical toolkit to developers of novel algorithms in these fields.

Towards complete validated models in the next generation of ARP/wARP

The design of a new versatile control system that will underlie future releases of the automated model-building package ARP/wARP is presented. A sophisticated expert system is under development that will transform ARP/wARP from a very useful model-building aid to a truly automated package capable of delivering complete, well refined and validated models comparable in quality to the result of intensive manual checking, rebuilding, hypothesis testing, refinement and validation cycles of an experienced crystallographer. In addition to the presentation of this control system, recent advances, ideas and future plans for improving the current model-building algorithms, especially for completing partially built models, are presented. Furthermore, a concept for integrating validation routines into the iterative model-building process is also presented.

Breaking good resolutions with ARP/wARP.

New procedures are outlined that enable ARP/wARP to automatically build protein models with diffraction data extending to about 2.5 A. An overview of ongoing research is given and possible future advances are discussed.

ARP/wARP and molecular replacement: the next generation

A systematic test shows how ARP/wARP deals with automated model building for structures that have been solved by molecular replacement. A description of protocols in the flex-wARP control system and studies of two specific cases are also presented.

Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies

Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells.

Structure of the GCM domain-DNA complex: a DNA-binding domain with a novel fold and mode of target site recognition

Glia cell missing (GCM) transcription factors form a small family of transcriptional regulators in metazoans. The prototypical Drosophila GCM protein directs the differentiation of neuron precursor cells into glia cells, whereas mammalian GCM proteins are involved in placenta and parathyroid development. GCM proteins share a highly conserved 150 amino acid residue region responsible for DNA binding, known as the GCM domain. Here we present the crystal structure of the GCM domain from murine GCMa bound to its octameric DNA target site at 2.85 Å resolution. The GCM domain exhibits a novel fold consisting of two domains tethered together by one of two structural Zn ions. We observe the novel use of a β‐sheet in DNA recognition, whereby a five‐ stranded β‐sheet protrudes into the major groove perpendicular to the DNA axis. The structure combined with mutational analysis of the target site and of DNA‐contacting residues provides insight into DNA recognition by this new type of Zn‐containing DNA‐binding domain.

The GCM domain is a Zn-coordinating DNA-binding domain

Glial cells missing (GCM) proteins form a small family of transcriptional regulators involved in different developmental processes. They contain a DNA‐binding domain that is highly conserved from flies to mice and humans and consists of approximately 150 residues. The GCM domain of the mouse GCM homolog a was expressed in bacteria. Extended X‐ray absorption fine structure and particle‐induced X‐ray emission analysis techniques showed the presence of two Zn atoms with four‐fold coordination and cysteine/histidine residues as ligands. Zn atoms can be removed from the GCM domain by the Zn chelator phenanthroline only under denaturating conditions. This suggests that the Zn ions are buried in the interior of the GCM domain and that their removal abolishes DNA‐binding because it impairs the structure of the GCM domain. Our results define the GCM domain as a new type of Zn‐coordinating, sequence‐specific DNA‐binding domain.

Native mass spectrometry provides direct evidence for DNA mismatch-induced regulation of asymmetric nucleotide binding in mismatch repair protein MutS

The DNA mismatch repair protein MutS recognizes mispaired bases in DNA and initiates repair in an ATP-dependent manner. Understanding of the allosteric coupling between DNA mismatch recognition and two asymmetric nucleotide binding sites at opposing sides of the MutS dimer requires identification of the relevant MutS.mmDNA.nucleotide species. Here, we use native mass spectrometry to detect simultaneous DNA mismatch binding and asymmetric nucleotide binding to Escherichia coli MutS. To resolve the small differences between macromolecular species bound to different nucleotides, we developed a likelihood based algorithm capable to deconvolute the observed spectra into individual peaks. The obtained mass resolution resolves simultaneous binding of ADP and AMP.PNP to this ABC ATPase in the absence of DNA. Mismatched DNA regulates the asymmetry in the ATPase sites; we observe a stable DNA-bound state containing a single AMP.PNP cofactor. This is the first direct evidence for such a postulated mismatch repair intermediate, and showcases the potential of native MS analysis in detecting mechanistically relevant reaction intermediates.

A knowledge-driven approach for crystallographic protein model completion

A novel method that uses the conformational distribution of Cα atoms in known structures is used to build short missing regions (‘loops’) in protein models. An initial tree of possible loop paths is pruned according to structural and electron-density criteria and the most likely loop conformation(s) are selected and built.

“Conditional Restraints”: Restraining the Free Atoms in ARP/wARP

Summary The automated building of a protein model into an electron density map remains a challenging problem. In the ARP/wARP approach, model building is facilitated by initially interpreting a density map with free atoms of unknown chemical identity; all structural information for such chemically unassigned atoms is discarded. Here, this is remedied by applying restraints between free atoms, and between free atoms and a partial protein model. These are based on geometric considerations of protein structure and tentative (conditional) assignments for the free atoms. Restraints are applied in the REFMAC5 refinement program and are generated on an ad hoc basis, allowing them to fluctuate from step to step. A large set of experimentally phased and molecular replacement structures showcases individual structures where automated building is improved drastically by the conditional restraints. The concept and implementation we present can also find application in restraining geometries, such as hydrogen bonds, in low-resolution refinement.