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Macromolecular ab initio phasing enforcing secondary and tertiary structure.

Millán C, Sammito M, Usón I - IUCrJ (2015)

Bottom Line: Beyond α-helices, other fragments can be exploited in an analogous way: libraries of helices with modelled side chains, β-strands, predictable fragments such as DNA-binding folds or fragments selected from distant homologues up to libraries of small local folds that are used to enforce nonspecific tertiary structure; thus restoring the ab initio nature of the method.Using these methods, a number of unknown macromolecules with a few thousand atoms and resolutions around 2 Å have been solved.In the 2014 release, use of the program has been simplified.

View Article: PubMed Central - HTML - PubMed

Affiliation: Structural Biology, Molecular Biology Institute of Barcelona , Baldiri Reixac 15, Barcelona, 08028, Spain.

ABSTRACT
Ab initio phasing of macromolecular structures, from the native intensities alone with no experimental phase information or previous particular structural knowledge, has been the object of a long quest, limited by two main barriers: structure size and resolution of the data. Current approaches to extend the scope of ab initio phasing include use of the Patterson function, density modification and data extrapolation. The authors' approach relies on the combination of locating model fragments such as polyalanine α-helices with the program PHASER and density modification with the program SHELXE. Given the difficulties in discriminating correct small substructures, many putative groups of fragments have to be tested in parallel; thus calculations are performed in a grid or supercomputer. The method has been named after the Italian painter Arcimboldo, who used to compose portraits out of fruit and vegetables. With ARCIMBOLDO, most collections of fragments remain a 'still-life', but some are correct enough for density modification and main-chain tracing to reveal the protein's true portrait. Beyond α-helices, other fragments can be exploited in an analogous way: libraries of helices with modelled side chains, β-strands, predictable fragments such as DNA-binding folds or fragments selected from distant homologues up to libraries of small local folds that are used to enforce nonspecific tertiary structure; thus restoring the ab initio nature of the method. Using these methods, a number of unknown macromolecules with a few thousand atoms and resolutions around 2 Å have been solved. In the 2014 release, use of the program has been simplified. The software mediates the use of massive computing to automate the grid access required in difficult cases but may also run on a single multicore workstation (http://chango.ibmb.csic.es/ARCIMBOLDO_LITE) to solve straightforward cases.

No MeSH data available.


Related in: MedlinePlus

Dual-space recycling Shake-and-Bake algorithm for ab initio phasing at atomic resolution.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4285884&req=5

fig1: Dual-space recycling Shake-and-Bake algorithm for ab initio phasing at atomic resolution.

Mentions: One hundred years have passed since Max von Laue was awarded the Nobel Prize in Physics for his discovery of the diffraction of X-rays by crystals (Friedrich et al., 1912 ▶; von Laue, 1912 ▶). Since that discovery, crystallography has become an essential tool of investigation throughout the sciences, as it provides information on molecular structure down to the atomic level with a degree of detail and accuracy that is unsurpassed by any other structural technique. X-ray diffraction was first used by the Braggs to determine the three-dimensional structure of crystals (Bragg & Bragg, 1913 ▶). In a diffraction experiment only the intensities of the diffracted X-ray beams are recorded, whereas their phases are not. Nevertheless, phases are required to compute an electron-density map from which an atomic model can be derived. Providing the missing phases has been a quest since the beginning of crystallography and phasing still constitutes a bottleneck in many crystallographic studies. In the field of macromolecular crystallography, initial phases are usually derived either experimentally from a substructure of reference atoms, intrinsic to the structure or incorporated, and data collected at one or more particular wavelengths (Hendrickson, 1991 ▶), or from the placement in the asymmetric unit of a model related to the target structure (Rossmann, 1972 ▶). In chemical crystallography, for structures composed of fewer than 200 independent atoms, direct methods (Hauptman & Karle, 1953 ▶; Karle & Hauptman, 1956 ▶) are generally able to provide an initial model exclusively from the experimental intensities measured on a native crystal. Unlike in macromolecular crystallography, no previous stereochemical knowledge or additional experimental data from modified crystals or selected wavelengths are needed. Direct methods are therefore termed ab initio methods. They solve the phase problem exploiting probabilistic relations and the possibility of evaluating many starting phase sets through reliable figures of merit. The extension of direct methods to larger structures of around 1000 independent atoms was accomplished by the introduction of the Shake-and-Bake algorithm (Miller et al., 1993 ▶) implemented in the programs SnB (Miller et al., 1994 ▶) and SHELXD (Usón & Sheldrick, 1999 ▶). Fig. 1 ▶ shows a scheme of the Shake-and-Bake algorithm (Sheldrick et al., 2011 ▶). Starting from an initial hypothesis, usually a set of randomly generated atoms, phases are calculated and modified according to direct methods relationships. The modified phases are used to calculate an electron-density map and a new set of atoms is selected from the maxima in this map. In favourable cases, iteration of this process leads to a structure solution, which can be identified by a reliable figure of merit called the correlation coefficient (CC) (Fujinaga & Read, 1987 ▶). It should be noted that all steps in the procedure described enforce atomicity as a constraint: the initial phase set is calculated from a (random) atomic model, the tangent formula and minimal function are derived from atomicity and the calculated maps are interpreted by picking atoms from which to calculate a new set of phases. It is therefore not surprising that such methods were limited by the requirement of atomic resolution data. Table 1 ▶ summarizes the previously unknown structures with more than 300 independent atoms which were solved ab initio using SHELXD. Remarkably, the table features a large number of nonstandard macromolecules, such as antibiotics or large disulfide-rich peptides for which classic protein methods did not provide an adequate alternative as neither suitable models nor easy ways of derivatization were an option. For example, the structure of the antibiotic vancomycin had long been awaited, as its crystallization had been described many years before a solution was independently achieved with SHELXD (Schäfer et al., 1996 ▶) and SnB (Loll et al., 1997 ▶).


Macromolecular ab initio phasing enforcing secondary and tertiary structure.

Millán C, Sammito M, Usón I - IUCrJ (2015)

Dual-space recycling Shake-and-Bake algorithm for ab initio phasing at atomic resolution.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4285884&req=5

fig1: Dual-space recycling Shake-and-Bake algorithm for ab initio phasing at atomic resolution.
Mentions: One hundred years have passed since Max von Laue was awarded the Nobel Prize in Physics for his discovery of the diffraction of X-rays by crystals (Friedrich et al., 1912 ▶; von Laue, 1912 ▶). Since that discovery, crystallography has become an essential tool of investigation throughout the sciences, as it provides information on molecular structure down to the atomic level with a degree of detail and accuracy that is unsurpassed by any other structural technique. X-ray diffraction was first used by the Braggs to determine the three-dimensional structure of crystals (Bragg & Bragg, 1913 ▶). In a diffraction experiment only the intensities of the diffracted X-ray beams are recorded, whereas their phases are not. Nevertheless, phases are required to compute an electron-density map from which an atomic model can be derived. Providing the missing phases has been a quest since the beginning of crystallography and phasing still constitutes a bottleneck in many crystallographic studies. In the field of macromolecular crystallography, initial phases are usually derived either experimentally from a substructure of reference atoms, intrinsic to the structure or incorporated, and data collected at one or more particular wavelengths (Hendrickson, 1991 ▶), or from the placement in the asymmetric unit of a model related to the target structure (Rossmann, 1972 ▶). In chemical crystallography, for structures composed of fewer than 200 independent atoms, direct methods (Hauptman & Karle, 1953 ▶; Karle & Hauptman, 1956 ▶) are generally able to provide an initial model exclusively from the experimental intensities measured on a native crystal. Unlike in macromolecular crystallography, no previous stereochemical knowledge or additional experimental data from modified crystals or selected wavelengths are needed. Direct methods are therefore termed ab initio methods. They solve the phase problem exploiting probabilistic relations and the possibility of evaluating many starting phase sets through reliable figures of merit. The extension of direct methods to larger structures of around 1000 independent atoms was accomplished by the introduction of the Shake-and-Bake algorithm (Miller et al., 1993 ▶) implemented in the programs SnB (Miller et al., 1994 ▶) and SHELXD (Usón & Sheldrick, 1999 ▶). Fig. 1 ▶ shows a scheme of the Shake-and-Bake algorithm (Sheldrick et al., 2011 ▶). Starting from an initial hypothesis, usually a set of randomly generated atoms, phases are calculated and modified according to direct methods relationships. The modified phases are used to calculate an electron-density map and a new set of atoms is selected from the maxima in this map. In favourable cases, iteration of this process leads to a structure solution, which can be identified by a reliable figure of merit called the correlation coefficient (CC) (Fujinaga & Read, 1987 ▶). It should be noted that all steps in the procedure described enforce atomicity as a constraint: the initial phase set is calculated from a (random) atomic model, the tangent formula and minimal function are derived from atomicity and the calculated maps are interpreted by picking atoms from which to calculate a new set of phases. It is therefore not surprising that such methods were limited by the requirement of atomic resolution data. Table 1 ▶ summarizes the previously unknown structures with more than 300 independent atoms which were solved ab initio using SHELXD. Remarkably, the table features a large number of nonstandard macromolecules, such as antibiotics or large disulfide-rich peptides for which classic protein methods did not provide an adequate alternative as neither suitable models nor easy ways of derivatization were an option. For example, the structure of the antibiotic vancomycin had long been awaited, as its crystallization had been described many years before a solution was independently achieved with SHELXD (Schäfer et al., 1996 ▶) and SnB (Loll et al., 1997 ▶).

Bottom Line: Beyond α-helices, other fragments can be exploited in an analogous way: libraries of helices with modelled side chains, β-strands, predictable fragments such as DNA-binding folds or fragments selected from distant homologues up to libraries of small local folds that are used to enforce nonspecific tertiary structure; thus restoring the ab initio nature of the method.Using these methods, a number of unknown macromolecules with a few thousand atoms and resolutions around 2 Å have been solved.In the 2014 release, use of the program has been simplified.

View Article: PubMed Central - HTML - PubMed

Affiliation: Structural Biology, Molecular Biology Institute of Barcelona , Baldiri Reixac 15, Barcelona, 08028, Spain.

ABSTRACT
Ab initio phasing of macromolecular structures, from the native intensities alone with no experimental phase information or previous particular structural knowledge, has been the object of a long quest, limited by two main barriers: structure size and resolution of the data. Current approaches to extend the scope of ab initio phasing include use of the Patterson function, density modification and data extrapolation. The authors' approach relies on the combination of locating model fragments such as polyalanine α-helices with the program PHASER and density modification with the program SHELXE. Given the difficulties in discriminating correct small substructures, many putative groups of fragments have to be tested in parallel; thus calculations are performed in a grid or supercomputer. The method has been named after the Italian painter Arcimboldo, who used to compose portraits out of fruit and vegetables. With ARCIMBOLDO, most collections of fragments remain a 'still-life', but some are correct enough for density modification and main-chain tracing to reveal the protein's true portrait. Beyond α-helices, other fragments can be exploited in an analogous way: libraries of helices with modelled side chains, β-strands, predictable fragments such as DNA-binding folds or fragments selected from distant homologues up to libraries of small local folds that are used to enforce nonspecific tertiary structure; thus restoring the ab initio nature of the method. Using these methods, a number of unknown macromolecules with a few thousand atoms and resolutions around 2 Å have been solved. In the 2014 release, use of the program has been simplified. The software mediates the use of massive computing to automate the grid access required in difficult cases but may also run on a single multicore workstation (http://chango.ibmb.csic.es/ARCIMBOLDO_LITE) to solve straightforward cases.

No MeSH data available.


Related in: MedlinePlus