Garib N. Murshudov

Overview of the CCP4 suite and current developments

An overview of the CCP4 software suite for macromolecular crystallography is given.

REFMAC5 for the refinement of macromolecular crystal structures

The general principles behind the macromolecular crystal structure refinement program REFMAC5 are described.

How good are my data and what is the resolution

The new scaling program AIMLESS is described and tests of refinements at different resolutions are compared with analyses from the scaling step.

Efficient anisotropic refinement of macromolecular structures using FFT

This paper gives the equations for the use of fast Fourier transformations in individual atomic anisotropic refinement. Restraints on bonded atoms, on the sphericity of each atom and between non-crystallographic symmetry related atoms are described. These have been implemented in the program REFMAC and its performance with several examples is analysed. All the tests show that anisotropic refinement not only reduces the R value and Rfree but also improves the fit to geometric targets, indicating that this parameterization is valuable for improving models derived from experimental data. The computer time taken is comparable to that for isotropic refinements.

REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use

One of the most important aspects of macromolecular structure refinement is the use of prior chemical knowledge. Bond lengths, bond angles and other chemical properties are used in restrained refinement as subsidiary conditions. This contribution describes the organization and some aspects of the use of the flexible and human/machine-readable dictionary of prior chemical knowledge used by the maximum-likelihood macromolecular-refinement program REFMAC5. The dictionary stores information about monomers which represent the constitutive building blocks of biological macromolecules (amino acids, nucleic acids and saccharides) and about numerous organic/inorganic compounds commonly found in macromolecular crystallography. It also describes the modifications the building blocks undergo as a result of chemical reactions and the links required for polymer formation. More than 2000 monomer entries, 100 modification entries and 200 link entries are currently available. Algorithms and tools for updating and adding new entries to the dictionary have also been developed and are presented here. In many cases, the REFMAC5 dictionary allows entirely automatic generation of restraints within REFMAC5 refinement runs.

Macromolecular TLS refinement in REFMAC at moderate resolutions.

Publisher Summary This chapter discusses the translation, rotation, and screw-rotation (TLS) parameterization of anisotropic displacement parameters (ADPs). In general, each atom can deviate anisotropically from its mean position, and six parameters are necessary to describe the mean square displacements fully. These parameters are referred to as the “ADPs,” are usually denoted U, and can be visualized as “thermal ellipsoids.” The addition of an extra six parameters per atom in macromolecular refinement is usually not justified by the data, except when atomic resolution data are available (

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.

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.

High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy

Three-dimensional (3D) structure determination by single particle electron cryomicroscopy (cryoEM) involves the calculation of an initial 3D model, followed by extensive iterative improvement of the orientation determination of the individual particle images and the resulting 3D map. Because there is much more noise than signal at high resolution in the images, this creates the possibility of noise reinforcement in the 3D map, which can give a false impression of the resolution attained. The balance between signal and noise in the final map at its limiting resolution depends on the image processing procedure and is not easily predicted. There is a growing awareness in the cryoEM community of how to avoid such over-fitting and over-estimation of resolution. Equally, there has been a reluctance to use the two principal methods of avoidance because they give lower resolution estimates, which some people believe are too pessimistic. Here we describe a simple test that is compatible with any image processing protocol. The test allows measurement of the amount of signal and the amount of noise from overfitting that is present in the final 3D map. We have applied the method to two different sets of cryoEM images of the enzyme beta-galactosidase using several image processing packages. Our procedure involves substituting the Fourier components of the initial particle image stack beyond a chosen resolution by either the Fourier components from an adjacent area of background, or by simple randomisation of the phases of the particle structure factors. This substituted noise thus has the same spectral power distribution as the original data. Comparison of the Fourier Shell Correlation (FSC) plots from the 3D map obtained using the experimental data with that from the same data with high-resolution noise (HR-noise) substituted allows an unambiguous measurement of the amount of overfitting and an accompanying resolution assessment. A simple formula can be used to calculate an unbiased FSC from the two curves, even when a substantial amount of overfitting is present. The approach is software independent. The user is therefore completely free to use any established method or novel combination of methods, provided the HR-noise test is carried out in parallel. Applying this procedure to cryoEM images of beta-galactosidase shows how overfitting varies greatly depending on the procedure, but in the best case shows no overfitting and a resolution of ~6 Å. (382 words)

The PDB_REDO server for macromolecular structure model optimization

The PDB_REDO pipeline aims to improve macromolecular structures by optimizing the crystallographic refinement parameters and performing partial model building. Here, algorithms are presented that allowed a web-server implementation of PDB_REDO, and the first user results are discussed.