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1.  Flexible torsion-angle noncrystallographic symmetry restraints for improved macromolecular structure refinement 
Flexible torsion angle-based NCS restraints have been implemented in phenix.refine, allowing improved model refinement at all resolutions. Rotamer correction and rotamer consistency checks between NCS-related amino-acid side chains further improve the final model quality.
One of the great challenges in refining macromolecular crystal structures is a low data-to-parameter ratio. Historically, knowledge from chemistry has been used to help to improve this ratio. When a macromolecule crystallizes with more than one copy in the asymmetric unit, the noncrystallographic symmetry relationships can be exploited to provide additional restraints when refining the working model. However, although globally similar, NCS-related chains often have local differences. To allow for local differences between NCS-related molecules, flexible torsion-based NCS restraints have been introduced, coupled with intelligent rotamer handling for protein chains, and are available in phenix.refine for refinement of models at all resolutions.
PMCID: PMC4014122  PMID: 24816103
macromolecular crystallography; noncrystallographic symmetry; NCS; refinement; automation
2.  Automating crystallographic structure solution and refinement of protein–ligand complexes 
A software system for automated protein–ligand crystallography has been implemented in the Phenix suite. This significantly reduces the manual effort required in high-throughput crystallographic studies.
High-throughput drug-discovery and mechanistic studies often require the determination of multiple related crystal structures that only differ in the bound ligands, point mutations in the protein sequence and minor conformational changes. If performed manually, solution and refinement requires extensive repetition of the same tasks for each structure. To accelerate this process and minimize manual effort, a pipeline encompassing all stages of ligand building and refinement, starting from integrated and scaled diffraction intensities, has been implemented in Phenix. The resulting system is able to successfully solve and refine large collections of structures in parallel without extensive user intervention prior to the final stages of model completion and validation.
PMCID: PMC3919266  PMID: 24419387
protein–ligand complexes; automation; crystallographic structure solution and refinement
3.  Towards automated crystallographic structure refinement with phenix.refine  
phenix.refine is a program within the PHENIX package that supports crystallographic structure refinement against experimental data with a wide range of upper resolution limits using a large repertoire of model parameterizations. This paper presents an overview of the major phenix.refine features, with extensive literature references for readers interested in more detailed discussions of the methods.
phenix.refine is a program within the PHENIX package that supports crystallographic structure refinement against experimental data with a wide range of upper resolution limits using a large repertoire of model parameterizations. It has several automation features and is also highly flexible. Several hundred parameters enable extensive customizations for complex use cases. Multiple user-defined refinement strategies can be applied to specific parts of the model in a single refinement run. An intuitive graphical user interface is available to guide novice users and to assist advanced users in managing refinement projects. X-ray or neutron diffraction data can be used separately or jointly in refinement. phenix.refine is tightly integrated into the PHENIX suite, where it serves as a critical component in automated model building, final structure refinement, structure validation and deposition to the wwPDB. This paper presents an overview of the major phenix.refine features, with extensive literature references for readers interested in more detailed discussions of the methods.
PMCID: PMC3322595  PMID: 22505256
structure refinement; PHENIX; joint X-ray/neutron refinement; maximum likelihood; TLS; simulated annealing; subatomic resolution; real-space refinement; twinning; NCS
4.  Use of knowledge-based restraints in phenix.refine to improve macromolecular refinement at low resolution 
Recent developments in PHENIX are reported that allow the use of reference-model torsion restraints, secondary-structure hydrogen-bond restraints and Ramachandran restraints for improved macromolecular refinement in phenix.refine at low resolution.
Traditional methods for macromolecular refinement often have limited success at low resolution (3.0–3.5 Å or worse), producing models that score poorly on crystallographic and geometric validation criteria. To improve low-resolution refinement, knowledge from macromolecular chemistry and homology was used to add three new coordinate-restraint functions to the refinement program phenix.refine. Firstly, a ‘reference-model’ method uses an identical or homologous higher resolution model to add restraints on torsion angles to the geometric target function. Secondly, automatic restraints for common secondary-structure elements in proteins and nucleic acids were implemented that can help to preserve the secondary-structure geometry, which is often distorted at low resolution. Lastly, we have implemented Ramachandran-based restraints on the backbone torsion angles. In this method, a ϕ,ψ term is added to the geometric target function to minimize a modified Ramachandran landscape that smoothly combines favorable peaks identified from non­redundant high-quality data with unfavorable peaks calculated using a clash-based pseudo-energy function. All three methods show improved MolProbity validation statistics, typically complemented by a lowered R free and a decreased gap between R work and R free.
PMCID: PMC3322597  PMID: 22505258
macromolecular crystallography; low resolution; refinement; automation
5.  PHENIX: a comprehensive Python-based system for macromolecular structure solution 
The PHENIX software for macromolecular structure determination is described.
Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. How­ever, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallo­graphic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
PMCID: PMC2815670  PMID: 20124702
PHENIX; Python; macromolecular crystallography; algorithms
6.  MolProbity: all-atom structure validation for macromolecular crystallography 
MolProbity structure validation will diagnose most local errors in macromolecular crystal structures and help to guide their correction.
MolProbity is a structure-validation web service that provides broad-spectrum solidly based evaluation of model quality at both the global and local levels for both proteins and nucleic acids. It relies heavily on the power and sensitivity provided by optimized hydrogen placement and all-atom contact analysis, complemented by updated versions of covalent-geometry and torsion-angle criteria. Some of the local corrections can be performed automatically in MolProbity and all of the diagnostics are presented in chart and graphical forms that help guide manual rebuilding. X-ray crystallography provides a wealth of biologically important molecular data in the form of atomic three-dimensional structures of proteins, nucleic acids and increasingly large complexes in multiple forms and states. Advances in automation, in everything from crystallization to data collection to phasing to model building to refinement, have made solving a structure using crystallo­graphy easier than ever. However, despite these improvements, local errors that can affect biological interpretation are widespread at low resolution and even high-resolution structures nearly all contain at least a few local errors such as Ramachandran outliers, flipped branched protein side chains and incorrect sugar puckers. It is critical both for the crystallographer and for the end user that there are easy and reliable methods to diagnose and correct these sorts of errors in structures. MolProbity is the authors’ contribution to helping solve this problem and this article reviews its general capabilities, reports on recent enhancements and usage, and presents evidence that the resulting improvements are now beneficially affecting the global database.
PMCID: PMC2803126  PMID: 20057044
all-atom contacts; clashscore; automated correction; KiNG; ribose pucker; Ramachandran plots; side-chain rotamers; model quality; systematic errors; database improvement

Results 1-6 (6)