radiation-damage- induced phasing with anomalous scattering peter zwart physical biosciences...

Radiation-damage-Radiation-damage-induced phasing induced phasing with anomalous with anomalous scatteringscattering

Peter ZwartPeter Zwart

Physical biosciences divisionPhysical biosciences division

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

movement movement

Frying ThaumatinFrying Thaumatin Thaumatin: 207 Thaumatin: 207

Residues, 8 disulfide Residues, 8 disulfide bridgesbridges

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


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)


‘‘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


Scaling the 20 individual data sets indicates radiation damage


Increasing cell parameters indicates 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

compute mapcompute map

{|F{|F11|-|F|-|FX>1X>1|, |, 1 1 } synthesis} synthesis Positive peaks: disappearing electrons (red)Positive peaks: disappearing electrons (red) Negative peaks: appearing electrons (green)Negative peaks: appearing electrons (green)


Disulfide Disulfide breakage breakage over the over the course of the course of the experimentexperiment

(Elastase (Elastase data)data)


Disulfide Disulfide breakage in breakage in thaumatin thaumatin results in results in main and main and side chain side chain movementmovement

Radiation-damage induced Radiation-damage induced phasing (RIP)phasing (RIP)

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

Radiation-damage induced Radiation-damage induced phasing (RIP)phasing (RIP)

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’


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)


CSCS ICIC

ResolutionResolution 2.5 Å2.5 Å 2.0 Å2.0 Å

WavelengthWavelength 1.54 Å1.54 Å 1.54 Å1.54 Å

SpacegroupSpacegroup P4(1)2(1)2P4(1)2(1)2 P4(1)2(1)2P4(1)2(1)2

Osc. RangeOsc. Range 60° ; 1.0° 60° ; 1.0° 90° ; 0.5 °90° ; 0.5 °

Ano red. / Ano red. / comp. comp.

2.4 / 97%2.4 / 97% 3.8 / 97%3.8 / 97%

# Data sets# Data sets 44 44

I/sigma (HR)I/sigma (HR) 2.4 -> 1.92.4 -> 1.9 4.0 -> 1.44.0 -> 1.4

RRmergemerge 5.6% -> 5.6% -> 5.2%5.2%

5.9 % -> 7.2 5.9 % -> 7.2 %%


Radiation Radiation damage was damage was apparent apparent within a within a single data single data setset


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


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


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


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

meeting.meeting. p-Iodo-Phenylanaline; p-Iodo-Phenylanaline;

First talk of the dayFirst talk of the day Selenated ribose moiety / Selenated ribose moiety /

Brominated Uracil (DNA/RNA)?Brominated Uracil (DNA/RNA)?

Other Possible derivatives Other Possible derivatives for RIP(AS)for RIP(AS)

Osmium ChlorideOsmium Chloride Over the course of 5 Over the course of 5

datasets, Osmium datasets, Osmium Chloride clusters Chloride clusters disappeardisappear

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

Cl

Cl

Cl

Cl

Cl

Cl Os

Red: Isomorphous difference map (2 sigma)Blue: Anomalous difference map (15 sigma)

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

Jan 18-20, 2006Jan 18-20, 2006

Chennai, IndiaChennai, India

For information, contact:Prof. D. Velmurugan:

[email protected]@rediffmail.com

radiation-damage- induced phasing with anomalous scattering peter zwart physical biosciences...

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