Phasing methods for protein crystallography

Hauptman, H.

doi:10.1016/s0959-440x(97)80077-2

Cited by 30 publications

(15 citation statements)

References 47 publications

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“…Different crystal forms can be examined to rule out crystal packing artifacts. Data quality can be improved by collecting data on synchrotron sources 67 and solving structures by multiwavelength anomalous dispersion (MAD) phasing 68 or ab-initio phasing 69 which will allow for a accurate determination of the structure and of the surrounding solvent without calculation of phases from a model. Furthermore, structures at atomic resolution 70 will accurately determine the positions of protein atoms as well as the solvent with mean coordinate errors as low as 0.02 Å (for the entire structure).…”

Section: Discussionmentioning

confidence: 99%

X‐ray crystallographic studies of the denaturation of ribonuclease S

Ratnaparkhi

Varadarajan

1999

Proteins

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In an attempt to view the onset of urea denaturation in ribonuclease we have collected X-ray diffraction data on ribonuclease S crystals soaked in 0, 1.5, 2, 3, and 5 molar urea. At concentrations above 2 M urea, crystals were stabilized by glutaraldehyde crosslinking. We have also collected data on ribonuclease S crystals at low pH in an attempt to study the onset of pH denaturation. The resolution of the datasets range from 1.9 to 3.0 Å . Analysis of the structures reveals an increase in disorder with increasing urea concentration. In the 5 M urea structure, this increase in disorder is apparent all over the structure but is larger in loop and helical regions than in the ␤ strands. The low pH structure shows a very similar pattern of increased disorder. In addition there is a major change in the position of the main chain (G 1 Å ) in the 65-72 turn region. This region has previously been shown to be involved in one of the initial steps of unfolding in the reduction of ribonuclease A. Crystallographic analyses in the presence of denaturant, when combined with controlled crosslinking, can thus provide detailed structural information that is related to the initial steps of unfolding in solution. Proteins 1999;36:282-294.

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Section: Discussionmentioning

confidence: 99%

X‐ray crystallographic studies of the denaturation of ribonuclease S

Ratnaparkhi

Varadarajan

1999

Proteins

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“…The first structures to be solved by this approach were small atomic assemblies containing few atoms. Since then, a number of smallmolecule and macromolecular structures have been determined by direct methods (6,7,(9)(10)(11), including the structure of cytochrome C 3 , with 2,208 atoms (12).…”

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confidence: 99%

Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED

Sawaya

Rodríguez

Cascio

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

106

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Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined. We show with four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods.

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“…estimating structure factor phases, is particularly acute in the case of macromolecular crystals where scattering units may include several thousand atoms, so that direct methods are generally not applicable [1]. Molecular replacement [2] is a very effective phasing method, albeit applicable only when a model of the whole molecule (or at least a significant fraction) is available.…”

Section: Principle Of Experimental Phasing Methodsmentioning

confidence: 99%