A restrained-parameter thermal-factor refinement procedure

The highly ordered crystal structure of crambin has been solved at 1.5 Å resolution directly from the diffraction data of a native crystal at a wavelength remote from the sulphur absorption edge. The molecule has three disulphide bridges among its 46 amino acid residues, of which 46% are in helices and 17% are in a β-sheet. Crambin is shown to be an amphipathic protein, inasmuch as its six charged groups are segregated from hydrophobic surface elements. Phasing methods used here will also apply elsewhere.Bijvoet's observation 1 that anomalous scattering might aid in solving the phase problem has had many implications 2 , including suggestions that departures from Friedel's law of diffraction symmetry might suffice to determine directly the atomic structures of crystals containing heavy atoms [3][4][5] . Anomalous-scattering methods have also been widely used in protein crystallography [6][7][8][9][10][11] , particularly as an adjunct to isomorphous-replacement phasing. We have now combined elements from these two traditions to solve the structure of crambin by exploiting the anomalous scattering of sulphur atoms at a single wavelength (1.54 Å of CuKα) far removed from the absorption edge of sulphur (5.02 Å).Crambin is a small protein found in the embryonic tissue (cotyledons and hypocotyledons) of seeds from Crambe abyssinica, a relative of mustard and rape commonly known as Abyssinian cabbage. It is hydrophobic in that organic solvents are required to solubilize it, Van Etten et al. 12 characterized crambin after noticing that crystals formed during evaporation of an aqueous acetone extract of defatted seed meal. The remarkable crystalline order observed by Teeter for this protein (strong diffraction from spacings <0.88 Å) 13 correlates well with the unprecedented structural stability seen in solution by Llinás et al. 14 using NMR spectroscopy. The function of crambin is not known. However, the recently completed chemical sequence 15 reveals an unmistakable homology with the plant toxins purothionin 16 and viscotoxin 17 . Crambin itself is not toxic when fed to rats 18 .We set out to solve the crystal structure of crambin in view of its exceptional potential for providing detailed structural information and in the hope of shedding light on the function of this hydrophobic protein. When our efforts to prepare heavy-atom derivatives failed, we explored alternatives to isomorphous replacement for phase determination. Our experience HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript in using anomalous scattering to locate the iron atoms in myohaemerythrin 19 led us to contemplate an attempt based on the six sulphur atoms in crambin. An estimate of the magnitude of Bijvoet differences to be expected from crambin gave a value about half that found with myohaemerythrin and showed that sulphur scattering would account for 98% of the expected anomalous signal. The expected contribution of the sulphur partial structure to the protein diffraction pattern proved to be 29% (〈|F ...

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“…deviation from bond ideality of 0.04 Å. Individual isotropic thermal parameters were varied but with tight restraints 33 .…”

Section: Model Refinement and Revisionmentioning

confidence: 99%

Structure of the hydrophobic protein crambin determined directly from the anomalous scattering of sulphur

Hendrickson

Teeter

1981

Nature

654

512

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“…The structure was refined using the XPLOR program [9] and completed by a few cycles of PROLSQ refinements [10]. The iohexol molecules were modelled using a tri-iodobenzoic acid moiety, without attempting to build the unobserved side chains.…”

Section: Methodsmentioning

confidence: 99%

Angiography contrast agents interact with serine proteinases The molecular structure of the model system elastase/iohexol

Prangé

Gaudey²,

Ohanessian³

et al. 1995

FEBS Letters

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Pancreatic elastase was co-crystallised with iohexol, a tri-iodo benzenic contrast agent used in angiography analyses. The X-ray analysis of the complex reveals the presence of three molecules of iohexol associated with the proteinase with low occupancy factors. Two iohexol molecules are located in and near the active site of the enzyme and provide a model for explaining the inhibition of the hemostatic system, one of the major and inconvenient side effect associated with these chemicals.

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“…We describe here the use of the EH parameters in the form of a computer-readable library for the PROTIN/PROLSQ package (Konnert & Hendrickson, 1980;CCP4, 1994), one of those most widely used to refine protein structures. 360.1°).…”

Section: Introductionmentioning

confidence: 99%

Dictionary of Protein Stereochemistry

Lamzin¹,

Dauter²,

Wilson³

1995

J Appl Cryst

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A computer-readable dictionary for protein structure refinement is presented. The dictionary is based on the set of interatomic bonds and bond angles previously derived from small-molecule structures by Engh & Huber [Acta Cryst. (1991), A47, 392-400]. The internal inconsistency of the set is discussed. Some preliminary results derived from several protein structures refined at atomic resolution are compared with this set.

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A restrained-parameter thermal-factor refinement procedure

Cited by 362 publications

References 6 publications

Structure of the hydrophobic protein crambin determined directly from the anomalous scattering of sulphur

Structure of the hydrophobic protein crambin determined directly from the anomalous scattering of sulphur

Angiography contrast agents interact with serine proteinases The molecular structure of the model system elastase/iohexol

Dictionary of Protein Stereochemistry

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