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1.  Low-resolution refinement tools in REFMAC5 
Low-resolution refinement tools implemented in REFMAC5 are described, including the use of external structural restraints, helical restraints and regularized anisotropic map sharpening.
Two aspects of low-resolution macromolecular crystal structure analysis are considered: (i) the use of reference structures and structural units for provision of structural prior information and (ii) map sharpening in the presence of noise and the effects of Fourier series termination. The generation of interatomic distance restraints by ProSMART and their subsequent application in REFMAC5 is described. It is shown that the use of such external structural information can enhance the reliability of derived atomic models and stabilize refinement. The problem of map sharpening is considered as an inverse deblurring problem and is solved using Tikhonov regularizers. It is demonstrated that this type of map sharpening can automatically produce a map with more structural features whilst maintaining connectivity. Tests show that both of these directions are promising, although more work needs to be performed in order to further exploit structural information and to address the problem of reliable electron-density calculation.
PMCID: PMC3322599  PMID: 22505260
low-resolution refinement; REFMAC5
2.  Evaluating the solution from MrBUMP and BALBES  
The automated pipelines for molecular replacement MrBUMP and BALBES are reviewed, with an emphasis on understanding their output. Conclusions are drawn from their performance in extensive trials.
Molecular replacement is one of the key methods used to solve the problem of determining the phases of structure factors in protein structure solution from X-ray image diffraction data. Its success rate has been steadily improving with the development of improved software methods and the increasing number of structures available in the PDB for use as search models. Despite this, in cases where there is low sequence identity between the target-structure sequence and that of its set of possible homologues it can be a difficult and time-consuming chore to isolate and prepare the best search model for molecular replacement. MrBUMP and BALBES are two recent developments from CCP4 that have been designed to automate and speed up the process of determining and preparing the best search models and putting them through molecular replacement. Their intention is to provide the user with a broad set of results using many search models and to highlight the best of these for further processing. An overview of both programs is presented along with a description of how best to use them, citing case studies and the results of large-scale testing of the software.
PMCID: PMC3069746  PMID: 21460449
MrBUMP; BALBES; molecular replacement
3.  REFMAC5 for the refinement of macromolecular crystal structures 
The general principles behind the macromolecular crystal structure refinement program REFMAC5 are described.
This paper describes various components of the macromolecular crystallographic refinement program REFMAC5, which is distributed as part of the CCP4 suite. REFMAC5 utilizes different likelihood functions depending on the diffraction data employed (amplitudes or intensities), the presence of twinning and the availability of SAD/SIRAS experimental diffraction data. To ensure chemical and structural integrity of the refined model, REFMAC5 offers several classes of restraints and choices of model parameterization. Reliable models at resolutions at least as low as 4 Å can be achieved thanks to low-resolution refinement tools such as secondary-structure restraints, restraints to known homologous structures, automatic global and local NCS restraints, ‘jelly-body’ restraints and the use of novel long-range restraints on atomic displacement parameters (ADPs) based on the Kullback–Leibler divergence. REFMAC5 additionally offers TLS parameterization and, when high-resolution data are available, fast refinement of anisotropic ADPs. Refinement in the presence of twinning is performed in a fully automated fashion. REFMAC5 is a flexible and highly optimized refinement package that is ideally suited for refinement across the entire resolution spectrum encountered in macromolecular crystallography.
PMCID: PMC3069751  PMID: 21460454
REFMAC5; refinement
4.  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.
Automatic iterative model (re-)building, as implemented in ARP/wARP and its new control system flex-wARP, is particularly well suited to follow structure solution by molecular replacement. More than 100 molecular-replacement solutions automatically solved by the BALBES software were submitted to three standard protocols in flex-wARP and the results were compared with final models from the PDB. Standard metrics were gathered in a systematic way and enabled the drawing of statistical conclusions on the advantages of each protocol. Based on this analysis, an empirical estimator was proposed that predicts how good the final model produced by flex-wARP is likely to be based on the experimental data and the quality of the molecular-replacement solution. To introduce the differences between the three flex-wARP protocols (keeping the complete search model, converting it to atomic coordinates but ignoring atom identities or using the electron-density map calculated from the molecular-replacement solution), two examples are also discussed in detail, focusing on the evolution of the models during iterative rebuilding. This highlights the diversity of paths that the flex-wARP control system can employ to reach a nearly complete and accurate model while actually starting from the same initial information.
PMCID: PMC2394809  PMID: 18094467
model building; refinement; molecular replacement
5.  BALBES: a molecular-replacement pipeline 
The fully automated pipeline, BALBES, integrates a redesigned hierarchical database of protein structures with their domains and multimeric organization, and solves molecular-replacement problems using only input X-ray and sequence data.
The number of macromolecular structures solved and deposited in the Protein Data Bank (PDB) is higher than 40 000. Using this information in macromolecular crystallo­graphy (MX) should in principle increase the efficiency of MX structure solution. This paper describes a molecular-replacement pipeline, BALBES, that makes extensive use of this repository. It uses a reorganized database taken from the PDB with multimeric as well as domain organization. A system manager written in Python controls the workflow of the process. Testing the current version of the pipeline using entries from the PDB has shown that this approach has huge potential and that around 75% of structures can be solved automatically without user intervention.
PMCID: PMC2394813  PMID: 18094476
BALBES; molecular replacement

Results 1-5 (5)