Experimental determination of the radiation dose limit for cryocooled protein crystals

Abstract

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 ؋ 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 microparticle-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 dose͞dose 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) ؋10 7 Gy was obtained. However, by considering other data quality indicators, an intensity reduction to I ln2 ‫؍‬ ln2 ؋ I0, corresponding to an absorbed dose of 3.0 ؋ 10 7 Gy, is recommended as an appropriate dose limit for typical macromolecular crystallography experiments.experimental dose limit ͉ macromolecular cryocrystallography ͉ radiation damage

Determination of X-ray flux using silicon pin diodes

“…This calculation makes several assumptions, not least concerning the beam size and shape, but does provide a rough (correct within a factor of two for most samples not containing heavy atoms) indication of the time that a macromolecular crystal exposed to such a beam will last before absorbing the experimental dose limit of 30 MGy (reduction of initial diffraction intensity, I 0 to 0.7I 0 ), after which diffraction data will have questionable value (Owen et al, 2006). For accurate dose determination, RADDOSE should be used with appropriate input values for the beam size, shape and profile, and crystal parameters (Paithankar et al, 2009;Murray et al, 2004).…”

“…is largely independent of dose rate (Owen et al, 2006;Leiros et al, 2006;Sliz et al, 2003)]. The upper dose limit that can be tolerated by a macromolecular crystal held at 100 K before half of its diffraction intensity is lost has been predicted from electron microscopy observations [20 MGy (Henderson, 1990)] and measured for MX [43 MGy (Owen et al, 2006)]. However, data collection in MX is typically formulated in units of time, so optimal planning of experiments, as well as measuring and comparing damage rates, requires that the relationship between dose and time be established for both the source and the sample in use.…”

“…This is because at cryotemperatures of around 100 K the damage appears to depend on the accumulated energy absorbed by the sample, regardless of the time taken to deposit it [i.e. is largely independent of dose rate (Owen et al, 2006;Leiros et al, 2006;Sliz et al, 2003)]. The upper dose limit that can be tolerated by a macromolecular crystal held at 100 K before half of its diffraction intensity is lost has been predicted from electron microscopy observations [20 MGy (Henderson, 1990)] and measured for MX [43 MGy (Owen et al, 2006)].…”

Diffraction data analysis in the presence of radiation damage

“…Macromolecular crystals inherently scatter weakly, so reaching the highest possible scattering intensity is always desirable in order to minimize random effects Popov & Bourenkov, 2003;Bourenkov & Popov, 2006). The decay of half the total diffraction intensity defines the upper limit for X-ray dose (Henderson, 1995;Owen et al, 2006;Kmetko et al, 2006). However, chemical and physical changes induced by X-ray photons during data collection may result in the deterioration of merging statistics, sometimes long before reaching half the intensity decay (Borek et al, 2007;Zwart et al, 2004), prompting experimenters to limit the exposure.…”

NSLS-II biomedical beamlines for micro-crystallography, FMX, and for highly automated crystallography, AMX: New opportunities for advanced data collection

“…FMX will surpass the currently brightest MX beamlines by up to two orders of magnitude in achievable dose rate ( Figure 1). The time to deliver the dose corresponding to the Garman dose limit [4] can then be as low as 10 ms at FMX. This performance is a direct consequence of the NSLS-II's world-leading emittanceimplicitly this means that comparable performance improvements are expected from MX beamlines planned at upgraded storage rings for example at the ESRF and at the APS.…”