LCLS

Structural Studies on Single Particles and Biomolecules

Janos Hajdu, Uppsala University

Gyula Faigel, Institute for Solid State Physics and Optics, Budapest

Keith Hodgson, Stanford Synchrotron Radiation Laboratory

Chris Jacobsen, State University of New York at Stony Brook

Janos Kirz, State University of New York at Stony Brook

Gerd Materlik, HASYLAB, DESY, Hamburg

John Miao, Stanford Synchrotron Radiation Laboratory

Richard Neutze, Uppsala University

Carol V. Robinson, New Chemistry Laboratory, Oxford University

David Sayre, State University of New York at Stony Brook

Abraham Szöke, Lawrence Livermore National Laboratory

Edgar Weckert, HASYLAB, DESY, Hamburg

David van der Spoel, Uppsala University

LCLSMycoplasmasMYCOPLASMAS

The smallest creatures capable of self-replication

1 DNA (genome size: 600 - 1,300 kbp) 400 ribosomes 10,000 RNA molecules 50,000 protein molecules 400,000,000 water/solute molecules

• ~300 nm Ø (cell membrane: 8 nm)• Solvent content: 60-70%

LCLS

• Biological samples are highly radiation sensitive

• Conventional methods cannot achieve atomic resolution on non- repetitive (or non-reproducible) structures

• The limit to damage tolerance is about 200 X-ray photons/Å2 in crystals (conventional experiments)

• The conventional damage barrier can be stretched by very fast imaging

Structural Biology: The Damage Problem

(Neutze, R., Wouts, R., van der Spoel, D., Weckert, E. Hajdu, J. (2000) Nature 406, 752-757)

LCLS

Planar section, simulated imageSINGLE MACROMOLECULEPROTEIN CRYSTAL

Max. resolution does not depend on sample quality

Max. resolution is a function of crystal quality

Scattering by a Crystal and by a Single Molecule

LCLSScattering and Damage by X-rays (biological samples: C,N,O,H,S)

• (1) Photoelectric effect (~90%) followed by Auger emission, shake-up excitations, and interactions between decay channels

• (2) Elastic scattering (~7-10%)

• (3) Inelastic scattering (~3%)

The LCLS Beam Interacts with the Matter Through Scattering and Absorption:

LCLS - a “never seen regime”, for which only predictions and simulations exist

LCLSCoulomb Explosion of Lysozyme (50 fs) LCLS

Radiation damage interferes with atomic

positions and the atomic scattering

factors

LCLS

Heating conserving momentum

Bond break through Morse potential

Ionisation primary and secondary effects

Ionisation dynamics calculate changes in the elastic, inelastic and photoelectric cross-sections for each atom during exposure

Modelling Sample Dynamics

XMD interfaced with GROMACS (van der Spoel et al.)

Inventory kept on all electrons in the sample

(Neutze, R., Wouts, R., van der Spoel, D., Weckert, E. Hajdu, J. (2000) Nature 406, 752-757)

LCLS

Ionisation events Energy

Ionisation and Coulomb Explosion of a Protein Molecule (Lysozyme) in Intense X-ray Pulses

3x1012 photons/100 nm diameter spot (3.8x106 photons/Å2, 12 keV)

.

Time (fs) Time (fs)0-50 500-50 50

15002000

1000500

0 0

1x108

2x108

# e

ve

nts

kJ/m

ole

Total energyPotential

0-10 100-10 10

Ionisation events Energy

0-2 2 0-2 2

# e

ve

nts

# e

ve

nts

15002000

1000500

0

15002000

1000500

0

0

1x108

2x108

0

1x108

2x108

kJ/m

ole

kJ/m

ole

Auger emissionPrimary ionisation events

I(t) Kinetic

LCLSP

ho

ton

s/p

uls

e/10

0 n

m s

po

t

40%

30%

20%

15%

Relec

1010

1011

1012

1013

1014

1 10 100 1000

b Relectronic

Tolerable damage(single exposures)

Initial LCLSparameters

20% 30% 40%

Landscape of Damage Tolerance

Ionisation and subsequent sample explosion causes diffraction intensities to change

Agreement factor:

Time (fs)

I(t) - Io Io

R =

LCLSSample Size and Scattering

RUBISCO 562,000 Da

HRV ~3,000,000 DaLYSOZYME 19,806 Da

Structure of content unknown

LCLS

Puls e dura tion (FWHM) 10 fs 50 fs 100 fs 230 fs

Phot ons/pu lse (100 nm spot)(R = 15%)

5x1012 8x1011 3x1011 5x1010

Sing le lyso zyme molec uleMW: 19,806

26 Å 30 Å >30 Å >30 Å

3x3x3 cl uster of lyso zymesTotal MW: 535,000

<2.0 Å 3.0 Å 6.5 Å 12 Å

Sing le RUBISCO molec uleMW: 562,000

2.6 Å 4.0 Å 20 Å 30 Å

Sing le viral capsid (TBSV)MW: ~3,000,000

<2.0 Å <2.0 Å <2.0 Å 2.4 Å

Single virus particles look very promising

Calculated Limits of Resolution with Relectronic = 15 %

LCLSFirst Experiments

• Single viral particles - structure of the viral genome

• Nanoclusters

• Structural kinetics on nanometer-sized samples

• Nanocrystals

• Two dimensional crystalline arrays

• X-ray diffraction tomography of whole cells

• X-ray scattering from intact cells

LCLSSample Handling

1. Spraying Techniques

2. Sample embedded in vitreous ice

• Native proteins,• Viruses,• Nanoclusters,• Nanocrystals, • Cell organelles,• Intact cells

Sample selection and injectionNanodroplets, Cryogenic Temperatures, High Vacuum

• Intact cells, cell organelles

Goniostat, Cryogenic Temperatures, High Vacuum

LCLSPrototype High Mass Q-TOF Mass Spectrometer

COLLECTOR PROBE

REFLECTRON

TIME-OF-FLIGHT ANALYSER

LOW FREQUENCY QUADRUPOLE MASS FILTER

SAMPLE INJECTOR

EXTRACTION LENS

RF HEXAPOLE RF HEXAPOLE

PUSHERDETECTOR

SA

MP

LE

GR

ID

10000 15000 20000 25000 30000(m/z)

50%

0%

100%

FIRST MASS MEASUREMENT:MS2 virus: 2,484,700 Da

(Tito et al. (2000) J. Am. Chem. Soc. 122, 3550-3551)

LCLS

To MASS/CHARGE ANALYSIS and ELECTRON MICROSCOPY

DIAGNOSTICSand LASER PORT

X-RAY PULSE

SAMPLE INJECTION

DETECTOR 1DETECTOR 2

INTELLIGENTBACK-STOP

Interaction Chamber and Detector Arrangement

LCLS

2 Å resolution

A Planar Section across the Molecular Transform of TBSV

LCLSA Planar Section across the Molecular Transform of TBSV

8 Å resolution

LCLS

• Continuous molecular transforms (oversampling)

• Tools of classical crystallography (these should work with reproducible structures)

• Holography

The Phase Problem

LCLSHow is Nucleic Acid Organised inside a Virus?

Courtesy of D. I. Stuart, Oxford

Experiment:

Artist’s view:

Gouet et al. Cell 97, 481-490 (1999)

Nucleic acid is released at the right time in the right order

LCLS

Most biochemical processes involve diffusion of reactants

Decorated nanoclusters may help to overcome this limitation

Implications for Functional Genomics

Kinetic studies in crystals suffer from “the mixing problem”

Structural Kinetics on Nanocrystals and Nanoclusters

LCLSMycoplasmasMYCOPLASMAS

The smallest creatures capable of self-replication

1 DNA (genome size: 600 - 1,300 kbp) 400 ribosomes 10,000 RNA molecules 50,000 protein molecules 400,000,000 water/solute molecules

• ~300 nm Ø (cell membrane: 8 nm)• Solvent content: 60-70%