Biomedical subjects

Robin Leslie Owen

Publications and source records attributed to Robin Leslie Owen.

4 recordsLinked to original sources

PubMed2006

Experimental determination of the radiation dose limit for cryocooled protein crystals.

Radiation damage to cryocooled protein crystals during x-ray structure determination has become an inherent part of macromolecular diffraction data collection at third-generation synchrotrons. Generally, radiation damage is an undesirable component of the experiment and can result in erroneous structural detail in the final model. The characterization of radiation damage thus has become an important area for structural biologists. The calculated dose limit of 2 x 10(7) Gy for the diffracting power of cryocooled protein crystals to drop by half has been experimentally evaluated at a third-generation synchrotron source. Successive data sets were collected from four holoferritin and three apoferritin crystals. The absorbed dose for each crystal was calculated by using the program raddose after measurement of the incident photon flux and determination of the elemental crystal composition by micro-particle-induced x-ray emission. Degradation in diffraction quality and specific structural changes induced by synchrotron radiation then could be compared directly with absorbed dose for different dose/dose rate regimes: a 10% lifetime decrease for a 10-fold dose rate increase was observed. Remarkable agreement both between different crystals of the same type and between apoferritin and holoferritin was observed for the dose required to reduce the diffracted intensity by half (D(1/2)). From these measurements, a dose limit of D(1/2) = 4.3 (+/-0.3) x10(7) Gy was obtained. However, by considering other data quality indicators, an intensity reduction to I(ln2) = ln2 x I(0), corresponding to an absorbed dose of 3.0 x 10(7) Gy, is recommended as an appropriate dose limit for typical macromolecular crystallography experiments.

Apoproteins↗

PubMed2005

Cryocooling and radiation damage in macromolecular crystallography.

Advances in cryocrystallographic techniques for macromolecular crystallography have been intimately intertwined with efforts to reduce the deleterious effects of X-ray damage inflicted during the collection of diffraction data. A brief overview of cryomethods and their rationale is given. This is followed by a summary of our current limited understanding of radiation damage in cryocooled crystals, investigations aimed at minimizing its effects and finally some developments which actually utilize it both for phasing and to extend structural knowledge.

Cryoprotective Agents↗

PubMed2005

Parameters affecting the X-ray dose absorbed by macromolecular crystals.

The lifetime of a macromolecular crystal in an X-ray beam is assumed to be limited by the absorbed dose. This dose, expressed in Gray (Gy = J kg(-1)), is a function of a number of parameters: the absorption coefficients of the constituent atoms of the crystal, the number of molecules per asymmetric unit, the beam energy, flux, size and profile, the crystal size, and the total irradiation time. The effects of these variables on the predicted absorbed dose, calculated using the program RADDOSE, are discussed and are illustrated with reference to the irradiation of a selenomethionine protein crystal of unknown structure. The results of RADDOSE can and will in the future be used to inform the data collection procedure as it sets a theoretical upper limit on the total exposure time at a certain X-ray source. However, as illustrated with an example for which the experimental data are compared with prediction, the actual lifetime of a crystal could become shorter in those cases where specific damage breaks down crucial crystal contacts.

Archaeal Proteins↗

PubMed2005

A new method for predetermining the diffraction quality of protein crystals: using SOAP as a selection tool.

A microscope for quantitative analysis of the birefringence properties of samples is introduced. The microscope is used to measure variations in the slow optical axis position (SOAP) across hen egg-white lysozyme, glucose isomerase and fibronectin crystals. By comparing these variations with indicators of diffraction quality, it is shown that the optical properties of a protein crystal provide a non-invasive method of determining crystal diffraction quality before any X-ray data collection is attempted.

Protein Conformation↗