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%