The kinetic dose limit in room-temperature time-resolved macromolecular crystallography.

0.95 MB·English

by  Schmidt, M.

research papers
Journalof The kinetic dose limit in room-temperature
Synchrotron
time-resolved macromolecular crystallography
Radiation
ISSN0909-0495
M. Schmidt,a* V. Sˇrajer,b N. Purwara and S. Tripathia
Received22September2011
Accepted23December2011 aDepartmentofPhysics,UniversityofWisconsin-Milwaukee,Milwaukee,WI53211,USA,and
bCenterforAdvancedRadiationSources,TheUniversityofChicago,Chicago,IL60637,USA.
E-mail:[email protected]
Protein X-ray structures are determined with ionizing radiation that damages
theproteinathighX-raydoses.Asaresult,diffractionpatternsdeterioratewith
the increased absorbed dose. Several strategies such as sample freezing or
scavengingofX-ray-generatedfreeradicalsarecurrentlyemployedtominimize
this damage. However, little is known about how the absorbed X-ray dose
affects time-resolved Laue data collected at physiological temperatures where
theproteinisfullyfunctionalinthecrystal,andhowthekineticanalysisofsuch
data depends on the absorbed dose. Here, direct evidence for the impact of
radiationdamageonthefunctionofaproteinispresentedusingtime-resolved
macromolecular crystallography.Theeffectofradiationdamage on thekinetic
analysis oftime-resolved X-ray data is also explored.
Keywords:radiationdamage;X-raydose;roomtemperature;time-resolvedcrystallography;
Lauecrystallography.
1. Introduction by time-resolved crystallography. The photocycle of the
photoactiveyellowprotein(PYP;Meyer,1985)providessuch
Time-resolvedmacromolecularcrystallography(Moffat,1989)
areaction.PYPabsorbsabluephotonandentersaphotocycle
isauniquemethodthatisabletodetermineatomicstructure
with several intermediates spanning timescales from pico-
andchemicalkineticsatthesametime(Schmidt,2008).X-rays
seconds to milliseconds (Fig. 1). The photocycle is very well
canpotentially affecttheprotein activesites (Dubnovitskyet
al.,2005; Adamet al.,2004;Purwar etal.,2011; Schlichtinget
al., 2000). Techniques like cryo-cooling (Kuzay et al., 2001;
Nicholsonetal.,2001)andfree-radicalscavengers(Murray&
Garman,2002)areusedinmacromolecularcrystallographyto
reduceradiationdamage.However,littledirectinformationis
available onwhetherandhowtheprotein kineticsisaffected
by radiation damage. Synchrotron beamlines, which are
specialized in time-resolved crystallography (Graber et al.,
2011), provide very specific experimental capabilities for the
collectionoftime-resolvedX-raydata:thebeamsizeismuch
smaller than the crystal size typically used for time-resolved
datacollection,reciprocalspaceiscoveredatrandomtoavoid
orientational preferences, the crystal is translated along its
axis after the collection of a diffraction pattern to expose a
fresh crystal volume to the X-rays, and the X-ray beam
impinges the crystal as close as possible to the surface where
the extent of reaction initiation by a laser pulse is at a
maximum. In addition, extremely intense and ultra-short
pulses of polychromatic narrow-bandwidth (pink) X-ray
radiation are employed. All of these experimental details Figure 1
characteristicoftime-resolvedstudieshaveneverbeentaken ThePYPphotocycle.Afterabsorptionofabluephotonthephotocycle
proceeds through a number of intermediates that are occupied on
into account in the calculations of the absorbed dose. To
differenttime-scales.Reddashedarrow:partofthephotocyclecovered
addresstheimpactofradiationdamageontheproteinkinetics
by our short time-series consisting of eight time delays from 256ms to
areactionneedstobeselectedthatcanbereadilyinvestigated 32msplusthedarkdata.
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doi:10.1107/S090904951105549X J.SynchrotronRad.(2012).19,264–273
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investigated by both time-resolved spectroscopy (Hoff et al., time points (delay times of 256ms, 512ms, 1ms, 2ms, 4ms,
1994,1999;Rubinstennetal.,1998;Ujjetal.,1998;Hendrikset 8ms,16msand32ms)plusoneLauepatternwherethelaser
al., 1999; Brudler et al., 2001; Xie et al., 2001; Borucki et al., was not fired (dark data). After a time-series was collected
2002;Shimizuetal.,2006;vanWilderenetal.,2006)andtime- fromonecrystalsetting,thecrystalorientationwaschangedto
resolvedcrystallography(Schmidtetal.,2004;Andersonetal., cover more of the reciprocal space. Twenty different crystal
2004; Ihee et al., 2005; Genick et al., 1997). The late inter- orientations (N ) were used for a dataset. For each new
set
mediatesarepB1andpB2(Iheeetal.,2005),whicharealmost crystalorientationthecrystalwasalsotranslatedalongitslong
identicalinstructureexceptattheN-terminalend(Harigaiet axis to expose a fresh volume. For the 20 orientations the
al., 2003; Ihee et al., 2005; Ramachandran et al., 2011). The crystalwastranslatedatotalof440mm.Hence,crystalsettings
majorityofPYPmoleculesinthecrystalrevertdirectlytothe wereseparatedby440mm/N =22mm.Inordertocompare
set
darkstate(pG)fromthepB1state.Therelaxationtimefrom theproteinkineticsandassesstheeffectofradiationdamage,
pB1topGcanbeobservedexquisitelywellwithtime-resolved this experiment was repeated 12 times.
crystallographic methods. The idea here is to collect a short As a control, a crystal of size 150mm (cid:2) 150mm (cid:2) 900mm
time-seriesconsistingofaverylimitednumberoftimepoints was illuminated by laser pulses only with the same pulse
on the slower part of the PYP photocycle (see red dashed energyasabove.Theprotocolusedwasessentiallythesameas
arrowinFig.1)sothattheabsorbeddosecanberegardedas above,whichincludes20crystalorientations,thesamecrystal
roughly constant through the short time-series. This time- translations (440mm) and four laser pulses per setting with
series is subsequently recollected multiple times until the 5mJmm(cid:3)2perpulseintoaspotofabout200mm,exceptthat
diffraction patterns fade away. The kinetic analysis on each we did not expose the crystals to X-rays after the laser illu-
short time-series will be based on the singular value decom- mination. A total of 128 virtual time-resolved datasets were
position (SVD) of time-resolved X-ray data (Schmidt et al., collectedwhichcorrespondsto16virtualtime-series,withthe
2003) and kinetic refinement will be attempted by ‘posterior crystalexposedonlytolaserpulses.Theprogressofthelaser
analysis’ (Schmidt et al., 2004; Schmidt, Ihee et al., 2005; damage was monitored by several Laue datasets that were
Schmidt,2008). collectedexposingthecrystalstoX-raysinthedark.Afteran
initialdarkdatasetwascollected,wecollectedfourmoredark
datasetsinintervalsoffourvirtualtime-seriesequivalentto32
virtual datasets or 128 laser pulses per crystal setting. At the
2. Material and methods
end of the experiment the crystal was exposed to a total
2.1. Time-resolved experiments
numberof10240laserpulses,with512laserpulsespercrystal
Crystals of PYP were grown as described elsewhere setting and five dark Laue datasets collected at equal time
(Borgstahletal.,1995).Apencil-shapedcrystalofdimensions intervals.
170mm (cid:2) 170mm (cid:2) 700mm was equilibrated in stabilization
buffer at pH7 and mounted in a capillary, with the long
2.2. Average absorbed dose calculation
dimension along the length of the capillary. This crystal was
usedinatime-resolvedcrystallographicexperimentusingthe TocalculatetheabsorbeddoseinJkg(cid:3)1=Gyweusedthe
Laue method at BioCARS beamline 14-ID-B (Graber et al., program Raddose (Murray et al., 2004). If a crystal of PYP
2011) at the Advanced Photon Source, Argonne National (hexagonal P6 , a= 66.9A˚, b= 66.9A˚, c= 40.8A˚, six mole-
3
Laboratory, USA. The crystal was kept at 288K using an culesintheunitcell)isirradiatedbya90mm(h)(cid:2)60mm(v)
Oxford Instruments CryoJet. A reaction in the crystal was (full width at half-maximum, FWHM) single X-ray pulse
initiated using a 4ns (FWHM) laser pulse from an Opolette containing 3.2 (cid:2)1010photons withan average wavelengthof
HEIIlasersynchronizedtothe100psX-raypulses.Thepulse 1.05A˚ the absorbed dose is 0.244 (cid:2) 104Gy for the X-ray-
energydensityofthelaseratthecrystalwas5mJmm(cid:3)2,with illuminatedvoxelequaltothebeamsizetimesthethicknessof
the laser beam focused into a 205mm spot perpendicular to the crystal. The total irradiated crystal volume in the experi-
the X-ray beam [see Fig. 2 and also Fig. 3 in Schmidt et al. mentisshowninFig.2(a).Itisgivenbytheverticalbeamsize,
(2010) for the crystal–laser X-ray geometry]. Single 100ps thethicknessofthecrystalandthelengthL.Lisgivenbythe
X-ray pulses were extracted by an ultrafast X-ray chopper total translation of the beam plus half the beam size at the
(Graber et al., 2011) in the hybrid mode of operation of the startandendpositionseach.Inourcase,Lis530mm.Hence,
synchrotron storage ring. One single pulse contained 3.2 (cid:2) each single X-ray pulse adds Lh =L (cid:2) 0.244 (cid:2) 104Gy =
beam
1010 polychromatic photons with an average wavelength of 0.0414(cid:2)104Gytothetotalirradiatedvolume,whereLh is
beam
1.05A˚, which corresponds to an average photon energy of the horizontal beam size of 90mm. Each short time-series
11.8keV.Thebandwidthwas10%.TheX-raybeamsizeatthe consisting of N = 8 time delays, the dark data and the edge
t
sample was 90mm (horizontal) (cid:2) 60mm (vertical). The scat- scan (see below) was collected with N = 4 pulses per crystal
p
teredradiationfromN =4X-raypulseswasaccumulatedona settingforatotalofN =20crystalsettingsspanningthetotal
p set
Mar165CCDdetectortoconstituteadiffractionpattern.The translation of the crystal. An average of N N (N + 2) (cid:2)
P set t
laserwasfiredatatimedelay(cid:2)tbeforeeachX-raypulse.The 0.0414(cid:2)104Gy=3.3(cid:2)105Gywouldbeabsorbedpershort
delay time was varied from 256ms to 32ms on a logarithmic time-series by the total irradiated crystal volume. Since the
timescale.Consequently,ashorttime-seriesconsistedofN =8 experiment was repeated 12 times, complete datasets for 12
t
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Table1
Absorbeddose(in105Gy)anddatastatisticsfor12consecutiveshorttime-series,eachconsistingofthesameeighttimedelaysandadarkdataset.
hy0i:averageverticaltranslationofthecrystal.TheaverageIandI/(cid:5) intheresolutionshell1.9–1.8A˚ weredeterminedfromthe32mstimepointofeachshort
I
time-series.Nisthenumberofreflectionsinthisresolutionshell.Foreachshorttime-series,characteristictimesforthedecayphasearegivenfromthefittothe
RSV((cid:6) )andfromposterioranalysis(1/k).RAO(amplitudetooffsetratio)observedinthefirstRSVoftheSVDanalysisarealsoshownforeachtime-series.
RSV 2
NormalizeddatafromthistableareshowninFig.4.
Shorttime-series
1 2 3 4 5 6 7 8 9 10 11 12
Averagedose,uncorrected 3.3 6.6 9.9 13.2 16.5 19.8 23.1 26.4 29.7 33.0 36.3 39.6
(i)Dosecorrected:crystalsettings(V =77%) 2.54 5.08 7.62 10.16 12.71 15.25 17.79 20.32 22.87 25.41 27.95 30.49
C
hy0i(mm) 0 0 0 0 2 5 8 11 12 13 14 16
(ii)Dosecorrected(D ):+edgescan 2.54 5.08 7.62 10.16 12.29 13.98 15.41 16.59 18.30 19.90 21.42 22.36
corr
N 3852 3860 3861 3861 3861 3859 3853 3864 3889 3908 3888 3894
reflections
hIi 330 297 271 237 211 185 160 151 123 110 88 84
32ms
hIi 337 310 289 258 233 206 181 172 142 128 104 100
free
hI/(cid:5)i 11.07 10.22 9.35 8.23 7.33 6.50 5.71 5.31 4.42 3.98 3.33 3.26
I32ms
hI/(cid:5)i 11.31 10.67 9.97 9.00 8.10 7.28 6.47 6.07 5.12 4.66 3.94 3.89
Ifree
(cid:6) (ms) 22 20 26 30 24 21 29 35 27 25 18 24
RSV
1/k ((cid:6) )(ms) 23 23 27 30 48 65 95 89 168 136 113 nd
2 post
RAO 6.04 7.27 5.72 6.12 3.91 3.04 2.49 2.45 2.07 1.8 1.57 1.53
shorttime-seriesconsistingofeighttimepointsplusthedark (orange) which depends on the angular difference (cid:2)(cid:2) of the
data were collected. This amounts to 108 complete Laue beam directions,and whichcan be calculated as
datasets collected from the same crystal. The total absorbed (cid:2) (cid:3)
(cid:3) ð1(cid:3)2v=tÞ
doseistherefore12(cid:2)3.3(cid:2)105Gy=39.6(cid:2)105Gyorabout F ¼ðt=2Þ2 (cid:4)(cid:3) sin(cid:3) ; ð1Þ
C 180(cid:5) cosð(cid:2)(cid:2)=2Þ
4MGy. Since one X-ray pulse lasts only 100ps, the instanta-
neous(pulse)doserateR is0.244(cid:2)104Gy/(100(cid:2)10(cid:3)12s) with(cid:3)=90(cid:5) (cid:3)(cid:2)(cid:2)/2(cid:3)arcsin(1(cid:3)2v/t).Equation(1)andthe
inst
= 2.4 (cid:2) 1013Gy s(cid:3)1. The average dose rate R is 0.244 (cid:2) equation fortherelativecommonvolume(in %)arederived
ave
104Gy/4s=610Gys(cid:3)1becausethewaitingtimebetweenthe in the supplementary material.1
single pulses was 4s, and the time delays are negligible in Fig.2(c)showsanactualsequenceofcrystalsettingsusedin
comparison. ourexperiments,eachofwhichisseparatedbyanangle(cid:2),the
above-mentioned translation of 22mm. The X-ray beam is
shown for the central setting at (cid:3)29(cid:5). Since the horizontal
2.3. Corrections to the dose absorbed beam diameter is 90mm, the beam covers three full settings
Corrections to the average dose calculated above are plus the two flanking one only partially. So, five settings are
necessary as explained in this section. As they are due to affected.Allfiveneighboringsettingssharecommonvolumes
additionalfreshcrystalvolumebeingexposedtoX-rays,they dependingontheirangularsettings.Thecorrectionfactorfor
effectivelyreducetheaverageabsorbeddosecalculatedabove thedoseabsorbedbyanentiresmalltime-seriesistheaverage
(Table 1). overallsetsoffive-memberedneighboringcommonvolumes.
2.3.1.Crystalrotations.Ourgoalwastofindouthowmany 2.3.2. Edge scan. A convenient feature of the data collec-
datasets can be safely collected on a PYP crystal exploiting tion protocol is the edge scan. Here, the crystal is translated
the capabilities of the BioCARS 14-ID beamline and data along the vertical direction (across the X-ray beam) and one
collection protocol. A very useful feature is the random single X-ray pulse is used to produce a weak diffraction
coverage of reciprocal space. The idea is that subsequent pattern.OncetheX-raybeamcrossestheedgeofthecrystal,
settings of the crystal are maximally spaced across reciprocal thediffractionpatternsfadeaway.Theanalysisofsuchaseries
space until a complete dataset is collected. If the experiment of diffraction patterns collected across the crystal edge is
terminates prematurely, the data have no preferred orienta- basedonthetotallyscatteredintensityineachpattern.Itcan
tion and, although incomplete, might already provide mean- determine the position of the edge precisely. The crystal is
ingfuldifferenceelectrondensitymaps.Aconsequenceofthis then positionedin the X-ray beam such that only thesurface
feature is that subsequent crystal orientations are largely layer of the crystal is probed by the X-rays (Schotte et al.,
different. As we rotate the crystal and the crystal is thick 2003). Fig. 2(a) shows the geometry. This is necessary, since
comparedwiththeverticalX-raybeamsize,somefreshcrystal protein crystals are exquisitely optically thick and the laser
volume is introduced. We used a relatively simple model to light penetration into the crystal is therefore shallow. As a
correct for this. In Fig. 2(b) a circle is shown representing a result, the laser pulses initiate the reaction primarily close to
crosssectionthroughthecrystal.Weimpingethiscrystalwith the illuminated crystal surface. The edge scan helps to maxi-
anX-raybeam[redinFig.2(b)]whoseverticalsizevissmaller mizetheoverlapoftheX-raybeamwiththelaser-illuminated
thantheradiust/2ofthecrystal.Whenthecrystalisrotatedby
1SupplementarydataforthispaperareavailablefromtheIUCrelectronic
an angle (cid:2)(cid:2), another volume is illuminated by the yellow
archives(Reference:RX5003).Servicesforaccessingthesedataaredescribed
beam.TheredandtheyellowbeamsshareacommonareaFC atthebackofthejournal.
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owing to edge scan and that owing to crystal rotation can be
applied independently, and that their effects add. For the
correction, weassumedarectangular X-ray beamshapewith
the same area and the same vertical size as the ellipsoidal
realistic X-ray beam (Fig. 2a). A displacement of 10mm
exposes17%offreshvolumeand83%isexposedtothedose
fromtheprevioussweep(s).Usinghy0iwecancorrectforthe
dose,subsequentlyforeachsweep.Thedosenecessaryforthe
edge scan itself is roughly equal to that of one regular
diffraction pattern (about four single X-ray shots were used
within the crystal volume) and this was already taken into
account in the averageabsorbeddose calculation.
We also include the edge scan in our control experiment
whereweexposedthecrystaltothelaserpulsesonly.Theedge
scanwasperformedforthefivedarkdatasetscollectedatthe
mentioned regular time intervals. The crystal positions from
those edge scans are stored and applied for the virtual time-
series with laser exposures only. In this way we assessed
whether subsequent laser damage results in displacements of
thecrystal deeper into the X-ray beam.
2.4. Data reduction and data analysis
Laue data were indexed and integrated using the program
Precognition and scaled using Epinorm (both RenzReserach,
http://renzresearch.com/). Difference structure factor ampli-
tudeswerecalculatedasreported(Renetal.,2001;Iheeetal.,
2005).ProteinDataBank(Bermanetal.,2002)entry2phywas
usedtoprovidephasestocalculatetime-dependentdifference
Figure2
maps.Twelveshorttime-serieswereobtained,eachsubjectto
(a)GeometryofthecrystalsettingandX-rayandlaserillumination.The
X-raybeam(redellipse)probesthevolumenearthesurfaceofthecrystal ahigherabsorbeddose(seeTable1).Eachofthesetime-series
thatisilluminatedbythelaserlight.Thelaserbeam(blue)issubstantially wasanalyzedbySVD(Schmidtetal.,2003;Zhao&Schmidt,
larger than the X-ray beam to facilitate alignment. Arrow: positive y0- 2009). For this analysis only the difference electron density
displacement of the crystal. The region shaded orange shows the new
in the chromophore region was included. Grid points that
beam position after y0-displacement. The crystal translation along the
longaxisis440mm.ThelengthLisusedforthedosecalculation.Dashed includethechromophoreandtheaminoacidresiduesthatline
box: approximation of the beam with a rectangular box. Dotted box: thechromophorepocketsuchasTyr-42,Glu-46,Met-100and
displacement of the box to the new beam position. (b) Model used to
Arg-52 (in total 188 atoms) were masked out. The mask was
determinethecommonareaF usedtocalculatethecommonvolumeV .
C C modified by allowing only grid points that are above 2.5(cid:5) or
ArectangularX-raybeam(red)whoseverticalsizeissmallerthanhalf
thecrystaldiameterfallsonacrystalwithacircularcrosssection.Asthe below(cid:3)2.5(cid:5)inatleastoneofthedifferencemapsofthesmall
crystal is re-oriented by (cid:2)(cid:2) it is irradiated from another direction time-series. The masked difference maps were arranged into
(yellow). The orange area is the common area F that determines the
C data matrix A, which was decomposed by SVD into the left
common volume V . F is the first term and 2F the second term in
equation (1), respecCtiveSly (see also the supplemenTtary material). (c) A (U) and right (V) singular vectors and the corresponding
sequenceofangularsettings(indegrees).Theangularsettingsarealso singularvalues(S)accordingtoA=USVT.Thekineticsofthe
separated by translations of 22mm. Five settings are fully or partially reactionisobservedintherightsingularvectors(RSVs).The
exposed given the horizontal size of the X-ray beam. The orange bars
firstRSVforeachshorttime-serieswasfittedbyasumoftwo
denote the relative sizes of the common volumes V ,for each angular
C
setting,valuesofwhicharegivenasapercentageatthebottom. exponentials, one exponential for a rising phase (if present)
andanotherforadecayingphase.Thecharacteristictime(cid:6)
RSV
forthe decaying phase is reported.
volume.Theedgescanwasperformedonce,atthebeginning
of each crystal setting, hence 20 times per short time-series.
2.5. Posterior analysis
Thepositionx,yofthegoniometerisrecordedaftereachedge
scan. These coordinates were used together with the angular Posterior analysis refines the rate coefficients for a given
setting (cid:2) of the goniometer to calculate the relative vertical mechanism by comparing calculated difference maps that
displacementsy0ofthecrystalacrosstheX-raybeamforeach depend on the rate coefficients of the mechanism with the
crystal setting. The individual y0 were averaged (hy0i, see observed difference maps. In the case of this study the inter-
Table 1). The crystal displacement about hy0i exposes a new mediate that is dominant throughout the time-series is pB1.
crystalvolumetothebeam.Thedosecalculationneedstobe There isalsosome smallpRremnant atthebeginningofour
corrected. For simplicity, we assumed that the correction time-series (256ms), but the majority of the electron density
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hence the different crystal orientations (settings) exposed
18% of fresh volume on the average in addition to the
translation. The dose was further adjusted based on our
recorded vertical displacements of the crystal (see Fig. 2 and
Figure3 Table 1). For the first five time-series the displacements are
SimplekineticmechanismwithasourceSandtworatecoefficientsk1and negligible,thenthecrystalstartedtomoveup.Attime-series
k2forkineticrefinementwithposterioranalysis.Dashedbox:mechanism 12 (the last sweep), hy0i was 16mm relative tothe position of
usedifnorisingphaseisdetectedintheRSV.
time-series 1; hence, from time-series 5 to 12 a total of 25%
of new volume is eventually exposed (see Table 1 for the
features must be interpretable by only pB1. Since the pB1
corrected dose).
structure is known (Ihee et al., 2005; Tripathi et al., 2012), we
Inourcontrolexperiment(laserpulsesonly)weobserveda
cancalculatethetime-independentpB1(cid:3)pGdifferencemap
linear decay of the hIi as well as of the hI/(cid:5) i values as a
(cid:2)(cid:7)calc. If a raising phase was observed in the first RSV (see I
PB functionofthelaserpulses.InFig.4(b)wehavetranslatedthe
below), we calculated the time-dependent fractional concen-
number of laser pulses into virtual X-ray dose, which is the
tration c (k,t) based on amechanism involving a rate coef-
frac doseabsorbedbythesamecrystalifitwereexposedtoX-rays.
ficient k for thepB1 state formation from thesourcestate S
1 This way we can plot hI/(cid:5)i (or hIi) observed in our control
andaratecoefficientk forasubsequentpB1statedecay(see I
2 experimentinthesameframeastheaverageintensitiesofthe
Fig. 3). The time-dependent difference maps are then calcu-
truetime-series[seesolidtrianglesinFig.4(b)].Wecorrected
lated as
the virtual dose by a common volume calculation similar to
(cid:2)(cid:7)calcðkÞ¼c ðk;tÞ(cid:2)(cid:7)calc: ð2Þ thatfortherealdata,onlyhereweusedasmallercrystalwith
t frac PB
t = 150mm. The common volume is slightly larger (80%
By fitting these maps to the observed difference maps
compared with 77% in the real time-series), and the corre-
[equation (3)], the concentration profile of pB1 is reflected
spondingvirtualdoseisalsoslightlylarger.Therewasnoneed
properlyeven without knowingthe structure ofthesourceS,
to further correct for crystal displacements into the X-ray
PT PM (cid:4)(cid:2)(cid:7)otbs(cid:3)sc(cid:2)(cid:7)ctalcðkÞ(cid:5)2 !min: ð3Þ buenatimlt.hIenfifancatl,dthaerkcrXy-srtaaylpeoxspiotisounrer.eTmhaeinheIdivsaulrupersis(isnoglliydsbtalabclke
t¼1m¼1 triangles)werefitbyastraightlinewithslopeS =(cid:3)0.0113(cid:2)
L
Here the fit is executed at M grid points in a mask similar to 10(cid:3)5Gy(cid:3)1 which is normalized to unity at zero virtual dose.
theoneusedtoperformtheSVD,andT=8representingtime Thislinereacheshalfofitsinitialvalueatthevirtualvaluehalf
pointsfrom256msto32ms.Thescalefactorscrepresentsthe value dose D1=2 of about 4.5MGy. In the dose range of our
L
peak fractional concentration of molecules in the pB1 state real data [red solid squares in Fig. 4(b)] the intensity decay
andis,aswellask andk ,afitparameter.Iftherisingphase owing to laser damage is small but meaningful as it reaches
1 2
wasnotobservedinthefirstRSV,weusedamechanismwhere 20% at (cid:6)2.0MGy. Assuming that laser damage and X-ray
pB1 only decays with k (dashed box in Fig. 3). In all cases damage are independent events, we can correct the intensity
2
the magnitude of k is compared depending on the absorbed of our true time-series for the laser damage and obtain
2
X-ray dose. Posterior analysis was performed using the intensitiesthatarefreeoflaserdamage:hIi =hIi((cid:3)S D
free L corr
programGetMech(Schmidt et al.,2004; Schmidt,2008). +1),whereD isthedoseofourtime-seriescorrectedbythe
corr
common volume and the edge scan and S is given above.
L
SinceS is negative,the hIi values are slightly higher than
3. Results L free
those determined from the raw intensities of our time-series
ThemeanintensityhIiisthegenerallyusedmetrictoaddress (see Table 1).
radiationdamage(Owenetal.,2006;Southworth-Daviesetal., In Fig. 4(a) (red squares) the hIi and hI/(cid:5) i values from
I
2007). Our data were processed up to 1.6A˚. However, since datasets collected at the 32ms time-delay from 12 repeated
dataqualityispoorinthelastresolutionshell,weuseddatato time-series are shown as a function of the uncorrected dose
1.8A˚ tocalculatethedifferencemaps.Inordertoaddressthe (solidandopensquares,respectively).Thedataarefittedbya
radiationdamagewecalculatedhIiaswellasthemeanofthe single exponential [solid lines in Fig. 4(a)]. The half-value
ratio of the intensity over its experimental uncertainty hI/(cid:5) i uncorrected dose D is obtained when this exponential
I 1/2,nc
in aresolutionshell from 1.9 to1.8A˚.Weused unscaledraw decaystohalfofitsinitialvalue,whichisafter18.4(cid:2)105Gy
reflection intensities derived from integrating the Laue spots whenthehIivaluesand19.7(cid:2)105GywhenthehI/(cid:5)ivalues
I
of the diffraction pattern.In this way we avoided any scaling are used (see Table 2). In Fig. 4(b) the dose has been
andmergingandacleanestimateofhIiaswellashI/(cid:5) iofthe corrected. The laser-damage-free hIi (solid red squares)
I free
Laue data was obtained. The uncorrected dose increased in and the hI/(cid:5) i from Table 1 (open red squares) were
I free
stepsof3.3(cid:2)105Gypertime-seriesuptoabout4MGyinour normalizedtounityatzerodoseandthevaluesareplottedas
final time-series. afunctionofthecorrecteddose.Inthedoseintervalassessed
Theabsorbeddosewasadjustedbasedonanestimateofthe byourexperimentsthedecayfollowsmoreastraightline[see
extent of fresh crystal volume exposed owing to subsequent thefitofanexponentialwhichisshownbythethindashedline
crystalsettings.77%ofthetotalvolumeisshared(V =77%), in Fig. 4(b)]. Also here, the half value dose D can be
C 1/2
268
M.Schmidtetal. (cid:4) Kineticdoselimitinmacromolecularcrystallography J.SynchrotronRad.(2012).19,264–273
Table 2
D andDK derivedfromuncorrectedandcorrecteddata.
1/2 1=2
The dose was corrected by the common volume and the extent of vertical
crystal translation. X-ray intensities and I/(cid:5) were corrected in addition to
I
accountforthelaserdamage.
D DK
1/2 1=2
hIi,uncorrected 18.4(cid:2)105Gy
hI/(cid:5)i,uncorrected 19.7(cid:2)105Gy
I
hIi ,corrected 16.8(cid:2)105Gy
free
hI/(cid:5)i ,corrected 16.8(cid:2)105Gy
Ifree
RAO,doseuncorrected† 16.0(cid:2)105Gy
RAO,dosecorrected 9.6(cid:2)105Gy
† NotshowninFig.4.
obtained when the data decay to half their initial magnitude.
D is the same, 16.7 (cid:2) 105Gy, regardless of whether hIi or
1/2
hI/(cid:5) ivaluesare used(Table 2).
I
However, it is unknown whether the D value is also a
1/2
meaningful limit for the PYP photocycle kinetics. To address
this wesubjected the light–darkdifference maps tothe SVD.
Fig.5showstheRSVsforthefirsttime-series,withanaverage
absorbed dose of 2.5 (cid:2) 105Gy, compared with those of the
tenthtime-serieswith20(cid:2)105Gy.Sinceourtime-seriesspan
theslowpartofthePYPphotocycleandonlypB1isdominant,
onlyonesignificantRSV(RSV1)ispresentineachsmalltime-
series. The difference between RSV1(1) and RSV1(10) is
immediately evident. For both time courses the first singular
vectors were fit by trial functions, which were independently
determinedbasedontheappearanceofthefirstRSV.Fortime
course 1 two exponentials were employed, one with a rising
phase and another displaying a decay with a relaxation time
(cid:6)(1) (Fig.5a).Forcomparison,asingleexponentialisalso
RSV
shown in Fig.5(a)(longdashed lines) with thesame(cid:6)(1) .
RSV
Intimecourse10arisingphasecanbarelybeobservedinthe
firstRSV1(10)andafitofanexponentialtothisrisingphase
was not possible. Consequently, only one exponential with
the characteristic time (cid:6)(10) was used. Interestingly, both
RSV
(cid:6)(1) and(cid:6)(10) areverysimilar,althoughtheamplitudes
RSV RSV
andoffsetofRSV1(1)andRSV1(10)differ grossly[compare
Figs. 5(a) and 5(b)]. Typically, with increasing radiation
Figure4 damage thereis an increasingly higher level ofoverall differ-
(a)RawmeanintensitieshIi(solidsquares)andhI/(cid:5)i(opensquares)asa enceelectrondensityinthemaps,whichisaccumulatedinthe
I
function of the uncorrected dose. Black and dashed lines: fits by firstleftsingularvector.Thiscanalsobeobservedintheoffset
exponentialfunctions.Verticaldashedanddashed-dotted linesindicate
of the corresponding first RSVand is also a reported effect
theD obtainedwiththeuncorrecteddose.(b)Normalizedquantities
M plo1/t2tedasafunctionofadjusteddose.AverageintensityhIi (red in spectroscopy for data with reduced signal-to-noise ratio
n free
solidsquares)andhI/(cid:5)i (redopensquares)areplottedasafunctionof (Henry & Hofrichter, 1992). This offset is large in RSV1(10)
I free
adjusted dose.Redsolidline:fitbyastraightline.Thindotted line:fit andsmallinRSV1(1)(Fig.5).Consequently,theratio,RAO,of
by an exponential function. The dashed vertical line indicates the
the amplitude of the first RSV to the offset is large for
correspondingD .Blacksolidandblackopentriangles:variationofthe
mean intensity h1I/2i and hI/(cid:5)i , respectively, observed in the control RSV1(1)andmuchsmallerforRSV1(10).ForRSV1(1),RAO
L I L
experimentasafunctionofvirtualdose.Theblacksolidanddashedlines is 6 and, for RSV1(10), RAO is only 1.5. In Fig. 6(a) the first
arefitsbyastraightline.(c)NormalizedRAOvalues(blackspheres)asa RSVs for all the time courses are shown in a three-dimen-
functionofadjusteddose.Blackdashedcurve:fitbyasingleexponential.
sionalplot.ThecorrespondingRAOareshowninFig.4(c)and
Thehorizontal/verticaldashedlinesindicateRAOandthecorresponding
kinetic dose limit DK . (d) Red squares: rela1=x2ation times (cid:6) from listed in Table 1. The decaying RAO can be fit by a single
1=2 RSV1
the SVD analysis as a function of adjusted dose. The red dashed line exponential[blackdashedcurveinFig.4(c)].Fromthefit,RAO
1=2
is a guide to the eye. Green triangles: inverse of the rate coefficient isdetermined.Onlythefirstfourtime-serieshaveRAOvalues
(relaxationtime(cid:6) )obtainedfromposterioranalysis.Thegreendashed
post larger than RAO. We call the corresponding dose (9.7 (cid:2)
line is a guide to the eye. Black spheres: difference between (cid:6)post and 1=2
(cid:6) .ThedashedverticallineindicatesthekineticdoselimitDK . 105Gy) the kinetichalf-valuedose (DK ;see also Table 2).
RSV1 1=2 1=2
269
J.SynchrotronRad.(2012).19,264–273 M.Schmidtetal. (cid:4) Kineticdoselimitinmacromolecularcrystallography
research papers
Figure5
Rightsingularvectors(RSV)resultingfromtheSVDanalysisoftheshort
time-series.Allsingularvectorsareshown.(a)Firstshorttime-serieswith
the lowest absorbed dose. Solid spheres: first singular vector; solid
squares:secondsingularvector;solidtriangles:thirdsingularvector;blue Figure 6
crosses:fourthsingularvector.RSV5to8areshownasthinlines.Solid Three-dimensionalplotoffirstrightsingularvectorsforall12shorttime-
black line: fit of two exponentials with a source and decaying phase. seriesshownasafunctionofdose.DK isreachedafter36datasetsorfour
1=2
Verticaldashedline:relaxationtimeofthedecayingphasefromafitof shorttime-series.TheoffsetsintheRVSareindicatedbythedottedlines.
thesumoftwoexponentials;longdashedline:fitofonlyoneexponential Green dotted line: small offsets; orange dotted line: offset increases
withthesamerelaxationtime;verticaldashed-dottedline:amplitudeof slightly;reddottedline:offsetincreasesstrongly.Theorangeregimeends
RSV1;horizontaldashed-dottedline:offsetofRSV1.Insert:reddashed after72datasets.Theredlineindicatesthatposterioranalysisofthedata
curve: concentration profile of pB1; black dashed line: relaxation time beyond thisdosewillnot bepossible.D isalso indicated.(b)Three-
1/2
fromtheinverseoftheratecoefficientk.(b)Tenthshorttime-serieswith dimensionalplotofallfittedtimecoursesfromtheposterioranalysisas
2
ahighabsorbeddose.Solidspheres:firstsingularvector;solidsquares: a function of dose. The green, orange and red regimes as well as the
secondsingularvector;solidtriangles:thirdsingularvector;bluecrosses: approximaterelaxationtimesthatcanbeexpectedintheseregimesare
fourthsingularvector.RSV5to8areshownasthinlines.Verticaldashed marked.DK ,D aswellasOwen’slimitarealsoshown.
1=2 1/2
line: relaxation time of the decaying phase from fit of only one
exponential;verticaldashed-dottedline:amplitudeofRSV1;horizontal
dashed-dottedline:offsetofRSV1.Insert:reddashedcurve:concentra- 4. Discussions
tionprofileofpB1;blackdashedline:relaxationtimefromtheinverseof
theratecoefficientk. We inspected the difference map calculated from the dark
2
data at time-series 1 and 12 (D1 (cid:3) D12 difference maps) for
RAO is a good indicator of the outcome of the post-SVD site-specific radiation damage of the dark PYP (Fig. S2,
refinementofthemechanism.UptoDK ,therelaxationtimes supplementary material). However, we were unable to iden-
1=2
(cid:6) found in the RSV(1) [Fig.4(d), red squares] agree with tifydamageonanyaminoacidresiduesofthePYP(including
SVD
relaxation times (cid:6) which are the inverse of the rate coef- Asp,Gluorthesulfur-bearingaminoacids).Theonlyeffectis
post
ficients k from the posterior analysis [green triangles in thatthedifferencemapsbecomenoisier.Thenoiselevelinthe
2
Fig.4(d)].AfterDK ,(cid:6) and(cid:6) diverge.Hence,although D1 (cid:3) D2 difference map is 0.012e(cid:3) A˚(cid:3)3 which in the D1 (cid:3)
1=2 RSV post
thetimescaleofthereactionisfoundtobe25msbyfittingthe D12 difference map is higher by a factor of three (0.032e(cid:3)
RSV1,posterioranalysiswillshiftthistimescaletomorethan A˚(cid:3)3). We also do not observe any specific damage when the
150msiftheabsorbeddoseistoohigh[Table1andFig.6(b)]. difference map D5 (cid:3) D1 from our control experiment (laser
On the other hand, at low to moderate doses up to 9.7 (cid:2) only)isinspected(seealsoFig.S2,supplementarymaterial).It
105Gy (DK ), time-scales from the SVD and posterior appears that in this case all damage is non-specific.Probably,
1=2
analysis agree very well [Fig.4(d),blackspheres]. unlike at cryogenic temperatures where the molecules are
270
M.Schmidtetal. (cid:4) Kineticdoselimitinmacromolecularcrystallography J.SynchrotronRad.(2012).19,264–273
research papers
stabilized, at room temperature the PYP molecules quickly optimize the overlap between the X-ray beam and the laser-
lose their structural integrity when damaged. Even if there is illuminated volume of the crystal.
specific radiation damage, its signature disappears as if the Afterthedoseadjustmentstheinitialexponentialdecrease
entiremoleculeisremovedfromthecrystalresultingincrystal oftheintensities(orI/(cid:5) values)appearstobelinear[compare
I
imperfection and increasingly larger B-factors (Rajendran et Figs. 4(a) and 4(b)]. Our linear decay may indicate that the
al.,2011).Thisisfurthercorroboratedbytheobservationthat mechanismfordamageatroomtemperatureandclosetothe
the PYP Laue reflection patterns become more streaky with surface is different from proposed first-order models (see
higher X-ray doses. Streaks in the Laue pattern result from Southworth-Daviesetal.,2007).Tofullyaccountforthis,more
enlarged crystal mosaicity. A possible reason for this is that experiments are necessary.
damaged PYP molecules create defects in the crystals which The SVD analysis of time-resolved data on PYP is amaz-
inturngeneratelong-rangedisorderandincreasedmosaicity. ingly robust against radiation damage. The relaxation times
Interestingly, others observe specific damage also at room extracted from the SVD show almost no dose dependence
temperature (Kmetko et al., 2011) even at moderately high even after 2MGy at room temperature (Fig. 4d). This is
doses around 1 (cid:2) 105Gy which is one order of magnitude because the SVD deals properly with the increasingly larger
lower than our D . Damage is most prominent on disulfide difference electron density background (offset) on which a
1/2
bonds, of which PYP has none, and there are, as in our subsequently smaller signal is sitting (see Fig. 5). Since the
difference maps,noise features scattered throughout. SVDisabletoaccuratelydealwithverysmalloccupanciesof
When monochromatic data are processed, the number of theorderof3–5%(Schmidtetal.,2003),extractionofaccurate
reflections found by the software typically decreases with relaxation times is still feasible at very high doses, far higher
decreasing scattering power (Owen et al., 2006; Rajendran et than the dose limit D . However, to extract and refine a
1/2
al.,2011).ThisdiscouragestheuseofhI/(cid:5) ivaluesandfavors proper kinetic mechanism, posterior analysis is necessary. Its
I
the use of mean intensities to study dose effects. In Laue outcome is strongly dose-dependent, since at high doses the
crystallography, however, the data reduction software deter- calculated noise-freedifference maps have tobe fitted to the
mines the number of reflections based on the form of the very noisy observed difference maps. At higher doses the
wavelength normalization curve (Ren & Moffat, 1995). The posterior analysis attempts to fit the noise and produces rate
numberofpredictedreflectionsstaysapproximatelythesame coefficientsthatareatleastafactoroffivelowerthanthoseat
even if the dose is increased and the scattering power of the thelowestdoses(Table1).Socarehastobetakentolimitthe
crystal decreases (see Table 1). As a consequence, the D dose.InFig.6(b)resultsfromtheposterioranalysisareshown
1/2
determinedwiththehIiorthehI/(cid:5) iareessentiallythesame. as a function of dose. We can distinguish three regimes: the
I
WithLauedatathehI/(cid:5) icanbeusedinsteadof,orinaddition green regime goes up to the DK , 9.7 (cid:2) 105Gy, where the
I 1=2
to,the mean intensities. relaxationratesofbothSVDandposterior analysis agree.In
SincethecrystalwasnotrotatedduringX-rayexposure,the theorangedose-regime,up to17(cid:2)105Gy,which is equal to
dose calculated by Raddose for each exposure is accurate the D , some kinetic information can be extracted. Relaxa-
1/2
(Garman&Weik,2011).However,theaverageabsorbeddose, tiontimesbetweenSVDandposterioranalysisdifferbyupto
based on the simple initial calculation that took into account afactorofthree.Above thatregime (in the redregime),itis
crystal translation during collection of each short time-series questionable whether a complex kinetic analysis is feasible.
(see x2), was adjusted twice to account for effects caused by ForPYPthedoseshouldbekeptlowerthanDK tostayinthe
1=2
theadditionalintroductionoffreshcrystalvolumeduringthe green regime ofFig.6.
data collection. The first contribution comes from the fresh Room-temperature absorbed dose effects were recently
crystal volume that is exposed each time the crystal orienta- reported (Southworth-Davies et al., 2007; Rajendran et al.,
tion, the angular setting, is changed. Maximizing this volume 2011;Kmetkoetal.,2011;Barkeretal.,2009).Resultsstrongly
willdecreasethedoseperdatasetandwillallowmoredatasets depend on the dose-rate employed to collect the data. We
to be collected. Taking crystal symmetry and space-group were using an average dose rate of 600Gy s(cid:3)1 for PYP (see
considerations into account, subsequent crystal orientations above) and our D is 16.7 (cid:2) 105Gy which is remarkably
1/2
need to be as far apart as possible to make use of the entire similar to the D for lysozyme reported by Southworth-
1/2
availablecrystalvolume.Thesecondcontributiontothedose Davies et al. (2007) (16.3 (cid:2) 105Gy at 10Gy s(cid:3)1) but much
adjustment comes from the vertical translation of the crystal higherthantheD valuesreportedbyRajendranetal.(2011)
1/2
relativetotheX-raybeamwhenthedoseincreases.Theedge on insulin (2.2 (cid:2)105Gy at their lowest dose rate of 1430Gy
scan,whichisusedtopositionthecrystalintheX-raybeam,is s(cid:3)1).Thelatterstudyreportsanegativeeffectonthedoserate,
basedonthetotalscatteredintensitywhichinturnisaffected hence D decreases with increasing dose-rate. Single-pulse
1/2
by the dose. The result is that the X-ray beam moves deeper Laue experiments use the highest peak dose-rates available
intothecrystal,awayfromthecrystalsurface.Thismightpose (2.3 (cid:2) 1013Gy s(cid:3)1) at synchrotrons. Obviously, it is not the
a problem for time-resolved experiments, since the X-ray high peak dose-rate that shows a detrimental effect, but it is
beamincreasinglyprobesdeeperregionsofthecrystalthatare theaveragedose-rate,whichwasmoderateinourexperiments
notoptimallyilluminatedbythelaserbeam.However,below ((cid:6)600Gy s(cid:3)1). It was suggested that high dose-rates dispro-
DK ,thecrystaldisplacementsremainnegligible,smallerthan portionately heat up the crystal, which leads to damage.
1=2
1mm on average, and the edge scan can be safely used to Furthermore, hydrogen is presumably produced, which accu-
271
J.SynchrotronRad.(2012).19,264–273 M.Schmidtetal. (cid:4) Kineticdoselimitinmacromolecularcrystallography
research papers
mulates in the crystal at higher dose-rates and which may be Our results also show that by carefully setting up a time-
oneofthecauses,ifnotthemaincause,forthenegativedose- resolved experiment the number of kinetically meaningful
rate dependence (Meents et al.,2010; Rajendran et al., 2011). datasets available from a single crystal can be maximized.
Although our instantaneous dose-rates were extreme, the 4s Although a thicker crystal absorbs more energy for a given
waitingtimebetweentheX-raypulsesisenoughforthecrystal incidentintensityowingtothelongerpathoftheX-raybeam
to cool down (Moffat et al., 1992) and for any potential through the thicker crystal, the dose remains the same since
hydrogen to diffuse out of the crystal. This maintains crystal the absorbing volume is also proportionally larger. For
integrity at higher doses. intensities scattered into Bragg reflections the situation is
OurD isamongthelargestreportedonaproteincrystal different. The intensity scales linearly with mass because the
1/2
at room temperature so far. Still, it is more than an order of latticefactorisproportionaltothenumberofunitcells.Hence
magnitude smaller than the Henderson limit (200 (cid:2) 105Gy), acrystalthatistwiceasthickscatterswithtwicetheintensity.
which, however, is valid only at cryogenic temperatures To match the average intensity scattered by a thin crystal, a
(Henderson, 1990). Owen et al. (2006) determined a roughly thick crystal requires lower incident X-ray beam intensity.
two times higher cryogenic D value of 430 (cid:2) 105Gy. It is Consequently,lowerdoseisabsorbedperdiffractionpattern.
1/2
thought that secondary damage effects such as diffusion of To reach the dose limit D which is the same for a thin and
1/2
radicals are strongly inhibited at these low temperatures. We a thick crystal, twice as many diffraction pattern can be
achieved here about 1/25 of Owen’s limit although we were collected from a crystal that is twice as thick as a corre-
operating at room temperature, where free radicals and spondingthincrystal.SincethekineticdoseDK issubjectto
1=2
solvatedelectronsmaydiffusefreely.Itmaywellbethatwith the same reasoning, we can expect to collect even more
theexperimentalconditionspresentedwereachedadoselimit kineticallymeaningfultime-resolveddiffractionpatternsfrom
for room-temperature X-ray data collection on crystals that a thick PYP crystal up to a reasonable crystal thickness, and
arenottreatedwithradicalscavengers.Ashasbeenshownby theuseofthincrystalsisdiscouraged.Thethicknessislimited
others(Barkeretal.,2009),addingradicalscavengerssuchas bythelaserbeamdiameteralongtheaxisoftheX-raybeam.
ascorbate may even increase this limit. A properly set up BioCARS 14-ID-B currently features ellipsoidal laser beam
single-pulsedLaueexperimentthenbecomesatooltocollect profileswiththelargeaxisupto600mm(Graberetal.,2011)
diffraction data on a dose-sensitive specimen that also obsti- along the X-ray beam direction, so a crystal thickness in the
nately resists freezing. Experiences with cytochrome-c nitrite range300–400mmwouldbeoptimal.Withthis,morethan400/
reductase crystals, which deteriorate quickly in a monochro- 170 (cid:2) 36 = 85 kinetically meaningful time points can be
matic X-ray beam at ambient temperatures, seem to corro- collected from one PYP crystal. The dose would still only be
borate this observation. With short polychromatic X-ray around DK . On the other hand, if the number of kinetically
1=2
pulses it was possible to collect a complete high-resolution meaningful datasets (time points) drops below 27, a compre-
dataset at 273K (Youngblut et al., 2012). However, to firmly hensivekineticallymeaningfultime-seriescannotbecollected
establish this observation, systematic experiments are neces- from a single crystal. This would happen when the crystal
sary. One could presume that the dose limit given by short- thickness falls below about 120mm. In this case, however,
pulsed Laue crystallography may only be surpassed by longer crystals can be selected where larger lengths L0 are
diffract-and-destroy experiments at a free-electron laser possible,andthenumberofusefuldatasetsincreasesbyL0/L.
(Chapman et al., 2011; Spence & Hawkes, 2008), where Theseresultspavethewaytofive-dimensionalcrystallography
radiation damage occurs after the scattering event and (Schmidtetal.,2010),whereinadditiontospaceandtimean
diffraction patterns are essentially damage-free (Chapman et additional parameter such as the temperature or the pH
al.,2006). (Tripathietal.,2012)isvaried.Here,successdependscritically
Nevertheless, for PYP we determined a kinetic dose limit, ontheabilitytocollectanentirekineticallymeaningfultime-
DK . Roughly 36 complete Laue datasets can be collected to series from only one crystal.
1=2
1.6A˚ fromonemoderatelysizedPYPcrystal,thesizeofwhich ForPYPthevaluesofDK andD differ,withDK ofthe
1=2 1/2 1=2
is very well suited to time-resolved experiments, without order of 40% smaller (Table 2). This relationship might also
disturbingthekineticanalysisbypost-SVDanalysis(seeFigs.4 holdforproteinsotherthanPYP,whichwereinvestigatedbya
and6).Theruleofthumbistocollectthreedatasetsperorder long and comprehensive time-series of time-resolved crystal-
ofmagnitudeintimeforasuccessfulSVDanalysis(Schmidtet lographicdatasuchasmyoglobin(Srajeretal.,2001;Schmidt,
al.,2003).The36datasetswithintheDK limit,therefore,will Nienhausetal.,2005)orclamhemoglobin(Knappetal.,2006).
1=2
allow the coverage of 12 orders of magnitude in time. This SinceD canberelativelyeasilydeterminedbeforehand,the
1/2
requirement can be easily fulfilled when a time-series from number of kinetically useful datasets can then be estimated
1ns to 0.5s is collected on one crystal. This requires 27 time from the size of the crystal mounted for the time-resolved
points (datasets) equidistantly spread on a logarithmic time- crystallographicexperiment.
scale plus the dark. There is even some room to increase the
numberoftimepointsfurtherintothepicosecondregime.The MS is supported by NSF CAREER grant 0952643. Use
experiments shown here demonstrate that entire time-series of the BioCARS Sector 14 was supported by the National
from picoseconds to seconds can be collected from a single InstitutesofHealth,NationalCenterforResearchResources,
crystal (Schmidt et al.,2010). under grant number RR007707. The time-resolved set-up at
272
M.Schmidtetal. (cid:4) Kineticdoselimitinmacromolecularcrystallography J.SynchrotronRad.(2012).19,264–273
research papers
Sector 14 was funded in part through a collaboration with Moffat,K.,Chen,Y.,Ng,K.M.,Mcree,D.&Getzoff,E.D.(1992).
PhilipAnfinrud(NIH/NIDDK).UseoftheAdvancedPhoton Philos.Trans.R.Soc.A,340,175–189.
Murray,J.&Garman,E.(2002).J.SynchrotronRad.9,347–354.
Source,anOfficeofScienceUserFacilityoperatedfortheUS
Murray,J.W.,Garman,E.F.&Ravelli,R.B.G.(2004).J.Appl.Cryst.
Department ofEnergy(DOE) Office of Science by Argonne
37,513–522.
National Laboratory, was supported by the US DOE under Nicholson,J.,Nave,C.,Fayz,K.,Fell,B.&Garman,E.(2001).Nucl.
ContractNo.DE-AC02-06CH11357. Instrum.MethodsPhys.Res.A,467,1380–1383.
Owen,R.L.,Rudin˜o-Pin˜era,E.&Garman,E.F.(2006).Proc.Natl
Acad.Sci.USA,103,4912–4917.
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