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.

[1]  M. Rossmann,et al.  Crystallographic studies on lactate dehydrogenase at –75°C , 1970 .

[2]  W A Hendrickson,et al.  Radiation damage in protein crystallography. , 1976, Journal of molecular biology.

[3]  D. Rice,et al.  Ferritin: design and formation of an iron-storage molecule. , 1984, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[4]  C. Nave,et al.  Radiation damage in protein crystals at low temperature. , 1994, Acta crystallographica. Section D, Biological crystallography.

[5]  D. Rodgers.  [13] Practical cryocrystallography. , 1997, Methods in enzymology.

[6]  P. Andrew Karplus,et al.  Improved R-factors for diffraction data analysis in macromolecular crystallography , 1997, Nature Structural Biology.

[7]  B. Gallois,et al.  Comparison of the structures of the cubic and tetragonal forms of horse-spleen apoferritin. , 1997, Acta crystallographica. Section D, Biological crystallography.

[8]  P. Harrison,et al.  Mineralization in ferritin: an efficient means of iron storage. , 1999, Journal of structural biology.

[9]  A G Leslie,et al.  Biological Crystallography Integration of Macromolecular Diffraction Data , 2022 .

[10]  J L Sussman,et al.  Specific chemical and structural damage to proteins produced by synchrotron radiation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[11]  R. Ravelli,et al.  The 'fingerprint' that X-rays can leave on structures. , 2000, Structure.

[12]  W. Burmeister,et al.  Structural changes in a cryo-cooled protein crystal owing to radiation damage. , 2000, Acta crystallographica. Section D, Biological crystallography.

[13]  J. Sussman,et al.  Solvent behaviour in flash-cooled protein crystals at cryogenic temperatures. , 2001, Acta crystallographica. Section D, Biological crystallography.

[14]  S. McSweeney,et al.  Atomic resolution structures of trypsin provide insight into structural radiation damage. , 2001, Acta crystallographica. Section D, Biological crystallography.

[15]  B. J. Hsieh,et al.  X-ray beam/biomaterial thermal interactions in third-generation synchrotron sources. , 2001, Acta crystallographica. Section D, Biological crystallography.

[16]  M. Caffrey,et al.  Unit-cell volume change as a metric of radiation damage in crystals of macromolecules. , 2002, Journal of synchrotron radiation.

[17]  E. Garman,et al.  Investigation of possible free-radical scavengers and metrics for radiation damage in protein cryocrystallography. , 2002, Journal of synchrotron radiation.

[18]  Sean McSweeney,et al.  Zero-dose extrapolation as part of macromolecular synchrotron data reduction. , 2003, Acta crystallographica. Section D, Biological crystallography.

[19]  Sean McSweeney,et al.  Specific radiation damage can be used to solve macromolecular crystal structures. , 2003, Structure.

[20]  Fei Long,et al.  REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. , 2004, Acta crystallographica. Section D, Biological crystallography.

[21]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[22]  James W. Murray,et al.  X-ray absorption by macromolecular crystals: the effects of wavelength and crystal composition on absorbed dose , 2004 .

[23]  Parameters affecting the X-ray dose absorbed by macromolecular crystals. , 2005, Journal of synchrotron radiation.

[24]  Olof Svensson,et al.  Automation of macromolecular crystallography beamlines. , 2005, Progress in biophysics and molecular biology.

[25]  O. Carugo,et al.  When X-rays modify the protein structure: radiation damage at work. , 2005, Trends in biochemical sciences.

[26]  E. Garman,et al.  Elemental analysis of proteins by microPIXE. , 2005, Progress in biophysics and molecular biology.

[27]  Elspeth F Garman,et al.  Cryocooling and radiation damage in macromolecular crystallography. , 2006, Acta crystallographica. Section D, Biological crystallography.