Radiation-damage- Radiation-damage- induced phasing induced phasing with anomalous with anomalous scattering scattering Peter Zwart Peter Zwart Physical biosciences division Physical biosciences division Lawrence Berkeley National Lawrence Berkeley National Laboratories Laboratories Not long ago: BNL/ANL/SAIC; Dauter group Not long ago: BNL/ANL/SAIC; Dauter group
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Radiation-damage- induced phasing with anomalous scattering Peter Zwart Physical biosciences division Lawrence Berkeley National Laboratories Not long.
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Radiation-damage-Radiation-damage-induced phasing induced phasing with anomalous with anomalous scatteringscattering
Lawrence Berkeley National LaboratoriesLawrence Berkeley National Laboratories
Not long ago: BNL/ANL/SAIC; Dauter groupNot long ago: BNL/ANL/SAIC; Dauter group
IntroductionIntroduction Radiation damage has been seen as a Radiation damage has been seen as a
curse in macromolecular crystallographycurse in macromolecular crystallography Diffraction power is lost over the course of Diffraction power is lost over the course of
data collectiondata collection Cell dimensions changeCell dimensions change Introduction of non-isomorphism over the Introduction of non-isomorphism over the
course of data collectioncourse of data collection Can we turn a problem into an Can we turn a problem into an
opportunity?opportunity? What are the signs of radiation damage?What are the signs of radiation damage? How does it affect the structure?How does it affect the structure? How can we use radiation damage?How can we use radiation damage?
IntroductionIntroduction
X-rays cause ionization events in unit X-rays cause ionization events in unit cellcell Protein is ionized and electronic Protein is ionized and electronic
rearrangements take placerearrangements take place Primary damagePrimary damage
Solvent is ionized and reacts with Solvent is ionized and reacts with proteinprotein
Secondary damageSecondary damage Secondary damage is limited by Secondary damage is limited by
cryo-coolingcryo-cooling
IntroductionIntroduction
Common rearrangements / reactions Common rearrangements / reactions due to radiation damage includedue to radiation damage include Disulfide breakageDisulfide breakage Dehalogenation of halogenated aromatic Dehalogenation of halogenated aromatic
compounds (brominated uracil)compounds (brominated uracil) Decarboxylation of side chainsDecarboxylation of side chains Associated main and side chain Associated main and side chain
P4(1)2(1)2: Easy to P4(1)2(1)2: Easy to get a complete data get a complete data set fairly quicklyset fairly quickly
Collect the same Collect the same angular range angular range several times to several times to investigate radiation investigate radiation damagedamage
Data collected at Data collected at NSLS X9B NSLS X9B
ResolutionResolution 1.45 Å1.45 Å
WavelengthWavelength 0.979 Å0.979 Å
SpacegroupSpacegroup P4(1)2(1)2P4(1)2(1)2
Osc. RangeOsc. Range 90° ; 1.0 ° 90° ; 1.0 °
Redundancy / Redundancy / compcomp
7 / 99%7 / 99%
# Data sets# Data sets 2020
I/sigma (HR)I/sigma (HR) 7.4 -> 2.37.4 -> 2.3
RRmergemerge 4.3 % -> 5.8 %4.3 % -> 5.8 %
Signs of radiation damageSigns of radiation damage
Structural changes imply changes Structural changes imply changes in intensitiesin intensities A simple model: Protein + heavy atomA simple model: Protein + heavy atom Assume only heavy atom is affected Assume only heavy atom is affected
by RDby RD Assume RD diminishes occupancyAssume RD diminishes occupancy
Make Argand diagram and see Make Argand diagram and see what happenswhat happens
FFHeavyHeavy becomes becomes smaller due smaller due to damage to damage resulting in resulting in FFTotTot to to decreasedecrease
FFHeavyHeavy becomes becomes smaller due smaller due to damage to damage resulting in resulting in FFTotTot to to increaseincrease
FFHeavyHeavy becomes becomes smaller due to smaller due to damage damage resulting in Fresulting in F++ and Fand F-- to to increase, while increase, while anomalous anomalous difference difference becomes becomes smallersmaller
Signs of radiation damageSigns of radiation damage Say we have two main processes over Say we have two main processes over
the course of our data collectionthe course of our data collection Major change (disulphide breakage + Major change (disulphide breakage +
carboxyl diss.+ etc)carboxyl diss.+ etc) Involves lots of electronsInvolves lots of electrons
Minor change (carboxyl dissociation+etc) Minor change (carboxyl dissociation+etc) Imagine the following scenario:Imagine the following scenario:
State 0 State 1 State 2
Major change Minor change
Signs of radiation damageSigns of radiation damage
Structure at image X End of data collection
Zero dose
I(h)
Time
Time averaged I(h)
Large difference
Small difference
Signs of radiation damageSigns of radiation damage Roughly spoken:Roughly spoken:
22 (I(t) - <I(t)>) (I(t) - <I(t)>)22
RR |I(t) - <I(t)>| |I(t) - <I(t)>|
This implies that the presence of This implies that the presence of radiation damage can be detected from radiation damage can be detected from the R values / Chi-squares vs frame the R values / Chi-squares vs frame number (in favorable cases)number (in favorable cases)
Signs of radiation damageSigns of radiation damage
‘‘Late’ radiation Late’ radiation damagedamage State 1 @ frame State 1 @ frame
100100 ‘‘Early’ radiation Early’ radiation
damagedamage State 1 @ frame State 1 @ frame
4040 ‘‘Very Early’ radiation Very Early’ radiation
damagedamage State 1 @ frame State 1 @ frame
2020
0
0 20 40 60 80 100
Frame number (%)
R-v
alu
e
Late
Early
Very Early
Numbers for figure obtained via simulation techniquesNumbers for figure obtained via simulation techniquesState 0: 207 residues, 17 SulphursState 0: 207 residues, 17 Sulphurs
State 1: Protein: 0.1 State 1: Protein: 0.1 ÅÅ rmsd; Sulphurs: 0.8 rmsd; Sulphurs: 0.8 ÅÅ rmsd rmsd
State 2: Protein: 0.1+0.05 State 2: Protein: 0.1+0.05 ÅÅ rmsd; Sulphurs: 0.8+0.1 rmsd; Sulphurs: 0.8+0.1 ÅÅ rmsd rmsd
Signs of radiation damageSigns of radiation damage
Scaling the 20 individual data sets indicates radiation damage
Signs of radiation damageSigns of radiation damage
Signs of radiation damageSigns of radiation damage
Increase in Wilson B value indicates loss of diffraction power due to radiation damage
10
11
12
13
14
15
16
17
0 5 10 15 20Data set
BW
ilson
Structural changesStructural changes
Visualise structural changes by Visualise structural changes by isomorphous difference mapsisomorphous difference maps Refine model against first data setRefine model against first data set Use phases and isomorphous differences to Use phases and isomorphous differences to
Disulfide breakage involves large Disulfide breakage involves large rearrangement of electrons: X-ray rearrangement of electrons: X-ray induced derivative induced derivative
|F|
|Fmod|
Substructure solutionPhasingDensity modificationModel building
Map resulting Map resulting from (1,15) iso from (1,15) iso differences after differences after shelxd, sharp and shelxd, sharp and DMDM
(1,5) iso was (1,5) iso was enough as wellenough as well
Radiation-damage induced Radiation-damage induced phasing with anomalous phasing with anomalous scattering (RIPAS)scattering (RIPAS)
What would be the effect of What would be the effect of anomalous dispersion in anomalous dispersion in substructure solution and phasing?substructure solution and phasing?
RIP: SIR like; RIPAS: SIRAS likeRIP: SIR like; RIPAS: SIRAS like Both anomalous difference in ‘native’ Both anomalous difference in ‘native’
and ‘derivative’ thoughand ‘derivative’ though Most damaged data is treated as Most damaged data is treated as
‘native’‘native’
Radiation-damage induced Radiation-damage induced phasing with anomalous phasing with anomalous scattering (RIPAS)scattering (RIPAS)
Iodinate tyrosine by treating protein Iodinate tyrosine by treating protein with N-Iodo-succinamidewith N-Iodo-succinamide Either prior to or after crystallisationEither prior to or after crystallisation
Iodinated tyrosines are sensitive to Iodinated tyrosines are sensitive to radiation damageradiation damage Se-Met not extremely sensitive to RDSe-Met not extremely sensitive to RD
Collect 4 data sets on two derivativesCollect 4 data sets on two derivatives Thaumatin Thaumatin
Iodinated prior to crystallization: IC (co-Iodinated prior to crystallization: IC (co-crystal)crystal)
Iodinated after crystallization: CS (soak)Iodinated after crystallization: CS (soak)
Radiation-damage induced Radiation-damage induced phasing with anomalous phasing with anomalous scattering (RIPAS)scattering (RIPAS)
Radiation-damage induced Radiation-damage induced phasing with anomalous phasing with anomalous scattering (RIPAS)scattering (RIPAS)
Radiation Radiation damage was damage was apparent apparent within a within a single data single data setset
Radiation-damage induced Radiation-damage induced phasing with anomalous phasing with anomalous scattering (RIPAS)scattering (RIPAS)
Anom diffs Anom diffs indicate indicate presence of presence of iodine (green)iodine (green)
(1,2) iso diffs (1,2) iso diffs indicate loss of indicate loss of iodines (red)iodines (red)
Blue: 2Fo-FC Blue: 2Fo-FC
Radiation-damage induced Radiation-damage induced phasing with anomalous phasing with anomalous scattering (RIPAS)scattering (RIPAS)
Iso and Ano diffs can be used Iso and Ano diffs can be used simultaneously in substructure simultaneously in substructure solution in Xprep (analogous to solution in Xprep (analogous to MAD)MAD) Substructure solution success rate Substructure solution success rate
increases compared to SAD/RIPincreases compared to SAD/RIP Iso and Ano diffs can be used Iso and Ano diffs can be used
simultaneously in SHARP for phasingsimultaneously in SHARP for phasing RIP/SAD phase ambiguity is brokenRIP/SAD phase ambiguity is broken
Radiation-damage induced Radiation-damage induced phasing with anomalous phasing with anomalous scattering (RIPAS)scattering (RIPAS)
CS data (2.5 Å) directly after SHARP. No CS data (2.5 Å) directly after SHARP. No DM/solomonDM/solomon
SAD RIP RIPASSAD RIP RIPAS
Radiation-damage induced Radiation-damage induced phasing with anomalous phasing with anomalous scattering (RIPAS)scattering (RIPAS)
IC data (2.0 Å) directly after SHARP. No IC data (2.0 Å) directly after SHARP. No DM/solomonDM/solomon
SAD RIP RIPASSAD RIP RIPAS
Other Possible derivatives Other Possible derivatives for RIP(AS)for RIP(AS)
International Tables for Crystallography International Tables for Crystallography Volume F., Page 752Volume F., Page 752 A Mercury derivative for LysozymeA Mercury derivative for Lysozyme
Other Possible derivatives for Other Possible derivatives for RIP(AS)RIP(AS)
The Mercury-S bond is sensitive to The Mercury-S bond is sensitive to radiation damage as wellradiation damage as well Ramagopal et al, last year ACA Ramagopal et al, last year ACA
Anomalous signal so Anomalous signal so strong and Osmium strong and Osmium so large, that so large, that combining with RIP combining with RIP signal does not signal does not improve phasingimprove phasing
ConclusionsConclusions The presence of radiation damage can be The presence of radiation damage can be
spotted in several waysspotted in several ways Disulfides break and push / release Disulfides break and push / release
surrounding main and side chainssurrounding main and side chains Radiation damage induced isomorphous Radiation damage induced isomorphous
differences can be the sole source of differences can be the sole source of information in substructure solution and information in substructure solution and phasing and AS enhances itphasing and AS enhances it
Iodinated tyrosines are susceptible to Iodinated tyrosines are susceptible to radiation damage radiation damage
Other RIPAS type derivatives are availableOther RIPAS type derivatives are available
AcknowledgementsAcknowledgements
Banumathi SankaranBanumathi Sankaran
Mirka DauterMirka Dauter
Zbigniew DauterZbigniew Dauter
Announcing the secondAnnouncing the second
INTERNATIONAL SYMPOSIUM ONINTERNATIONAL SYMPOSIUM ON
RECENT TRENDS IN RECENT TRENDS IN
MACROMOLECULAR STRUCTURE AND FUNCTIONMACROMOLECULAR STRUCTURE AND FUNCTION