FEL Serial Crystallography

Serial
Crystallography
using x-ray
Free Electron Lasers
Francesco Stellato
I.N.F.N. – Sezione di Roma ‘Tor Vergata’
Milan, July 11th 2014
Summary

Structural biology and X-rays

From synchrotrons to Free Electron Lasers

Diffract-and-destroy measurements

Serial Crystallography at FELs

Sample Preparation and Charcterization

Sample delivery

Data analysis


The Cathepsin B experiment
Serial Crystallography at synchrotrons

Applications & Future perspectives
Structural Biology and X-rays
Source Peak Brilliance
Perutz & Kendrew
myoglobin MacKinnon
1E+36
Potassium
channel
Franklin,
1E+33
Crick,
1E+30
Watson
Bragg & Bragg
DNA
1E+27 reflections Hodgkin
penicillin,
1E+24
B12
von Laue
1E+21 crystal diffraction
1E+18
1E+15 Röntgen
1E+12
Kornberg
1E+09
RNA
Kirz
&
1E+06
polymerase
Schmahl
Jacobsen
1E+03
Microscopy
Holography
1E+00
1880 1910 1940 1970 2000 2030
Year
Free Electron Lasers (FELs)
Radiation is generated by an undulator
Electrons are bunched up by interaction with x-rays
courtesy: Thomas Tschentscher (XFEL)
FELs around the world
Hard x-rays
Soft x-rays
FLASH
FLASH
LCLS
Hamburg,
Stanford
Germany
LINAC Coherent Light
Source
USA
λ > 4.2 nm
λ > 0.12 nm
SACLA
FERMI
Trieste
Italy
Rikken
Japan
Under construction
Synchrotrons and FELs
-Similar average brilliance,
very different peak brilliance
1012 photons in ~0.05 μm2
FEL Pulse-rate FEL: 100 Hz (so far…)
-Different pulse length:
10 -100 fs FEL
10-100 ps sinchrotrons
-Short wavelength:
Up to 10 keV FELs (first harmonic)
Up to 100 keV sinchrotrons
Diffraction before destruction
Particle injection
One pulse,
one measure
FEL puse
Diffraction pattern
A detectable signal
must be recorded
before the sample is
destroyed
R. Neutze et al, Nature 406 (2000)
Coherent x-ray Imaging
Diffract and destroy proof-of-principle
1 micron
Diffraction pattern
SEM picture of a FIB-bed pattern
etched on a Si3N4membrane
Diffraction pattern
Second pulse
Reconstructed image at
32 nm resolution
FLASH
First pulse
Chapman et al. Nature Physics (2006)
1 micron
Diffraction before destruction
The diffract-and-destroy principle can be exported
for in principle all synchrotron x-ray techniques
- Coherent Imaging
- SAXS / WAXS
- X-ray Spectroscopies
- Crystallography
200 nm
2D reconstruction of a
mimi-virus from a single
200 fs LCLS pulse
Seibert et al. Nature 470, p.78 (2011)
FEL Serial Crystallography
70000
60000
- Measurements of many (103-104)
single crystal diffraction patterns
50000
40000
30000
- Indexing
- Intensities determination & merging
20000
Electron
NMR
1972 1976
X-ray
10000
1980
1984
1988
1992
1996
2000
2004
2008
0
Standard crystallography is the election
technique for structural biology
- Standard (and non-standard)
phasing methods
FEL Serial Crystallography
Experimental setup
•FEL generated x-ray
beam
•Focusing optics
•Sample
•Sample injection system
•Detector
FEL Serial Crystallography
Pilot experiment
Photosystem I
AMO beamline
Sun-catcher Membrane protein
@ LCLS
Upper
front
CCD
beam center
36 proteins 381 cofactors
Lower
front
CCD
Resolution at corner = 8.6Å
Single shot at LCLS
E = 1.8 keV
80 fs pulse
2 mJ pulse energy
Chapman et al. Nature 470, (2011)
FEL Serial Crystallography
Pilot experiment
Virtual powder data show that there is
no damage up to 70 fs pulses
Molecular replacement method is used
starting from the known structure
LCLS data allowed solving PSI structure
at 7 Å resolution
(wavelength and geometry limit)
The electron density is compatible with
the known structure one
First FEL based pdb structure
Pdb ID: 3PCQ - www.pdb.org
Chapman et al. Nature 470, (2011)
Nano/micro-crystals Preparation
SEM images
Standard techniques can be
optimized to grow many microand/or nano-crystals:
- Hanging droplet (robots)
-Batch methods
- In vivo crystallization
Needleshaped
Cathepsin
B
Nanocrystals
A Proteinase K
Nanocrystal
Nano/micro crystals Screening
Several techniques are used to detect
nanocrystals:
-Optical and electron Microscopy
-X-ray diffraction (XRD) (mainly powder)
-SONICC
Nano/micrco crystals Characterization
Several techniques are used to
characterize nanocrystals in terms of
quality, concentration and size distribution
-Dynamic light scattering (DLS)
- Optical and electron Microscopy
-Differential mobility analysis (DMA)
-Nanoparticle tracking analysis (NTA)
-X-ray diffraction (XRD) (mainly powder)
- SONICC
Sample Delivery
A good sample delivery system
should:
-Keep the sample as close as possible
to native conditions
-Have low background
-Deliver a fresh crystal at every FEL
pulse
-Use as few crystals as possible
-Allow pump-probe measurements
-Be as stable as possible
Systems used so far at FELs:
-Gas Dynamic Virtual Nozzle
-Lipidic cubic phase nozzle
-Aerosol injector
-Electrospinning
-Fix targets
-…
Sample Delivery Systems
Hitrate (fraction of FEL pulses that hit a
sample) is determined by
-Sample concentration
-Beam diameter
-Particle beam diameter
-Particle beam stability
Examples of hitrate at LCLS
Gas Dynamic Virtual Nozzle
Gas line
Liquid jet
100 m/s
0.5-5 μm diameter
1-10 μl/min
10% hitrate
Liquid line
Gas line
Sample reservoir
Gas bottle
De Ponte D et al. J. Phys. D 2008
Electrospray/Electrospinning & Drop-on-demand
Cone-Jet Mode
A drop-on-demand
system can be used
to generate 20-40 μm
diameter droplets
An electrospray
source can generate
small droplets and an
associated
Differential Mobility
Analyzer can sizeselect particles
Fix Targets
- Sample deposited on thin
Si3N4 membranes
Ideal for 2D crystallography
Frank M. et al., IUCrJ 2014
- Kapton ™ micro-cells
Good to keep samples
hydrated
Zarrine-Asfar A. et al., Acta D 2012
10 μm
Data Analysis Flow-chart
Diffraction pattern acquisition
Hit-finding
Only ‘hits’ are processed
Background subtraction
Sparse patterns: average of many frames
Peak finding
Peaks are identified in the bkg subtracted patterns
Data Analysis Flow-chart
Indexing
Standard programs (DirAx, MOSFLM, …) called by
dedicated softwares (CrystFEL, Cctbx)
Intensities
The (partial) intensity is evaluated as a locally
background subtracted sum of pixels close to the
detected (or predicted) peak position
Ring-scheme
Intensities merging
Background
Empty region
Bragg peak
Structure factors
White T. et al. J. Appl. Cryst 2012
White T. et al. Acta D 2013
Luci di Sincrotrone
CNR – Roma, 22 Aprile 2014
Serial Crystallography – Cathepsin B
Cathepsin B
Cysteine protease expressed by
T.brucei, organism that causes
Human African Trypanosomiasis
The structure of the protein in the
non-native form is known, the
glycosylated one not
Baculovirus infection of
insect cells is commonly
used for the expression
of proteins requiring
post-translational
modifications.
Luci di Sincrotrone
CNR – Roma, 22 Aprile 2014
Serial Crystallography – Cathepsin B
Needle-shaped crystals were observed
in the cells over-expressing the protein
They were purified and concentrated
to reach about 109 #/ml
10 ml of concentrated solution were
obtained
SEM picture of a purified
Cathepsin B crystal
Serial Crystallography – Cathepsin B
Synchrotron data
60s exposure pattern have been
collected at DORIS, Hamburg
1010 photons/s in 200x200 μm2
There is a clearly visible ring at 60 Å
Faint rings at higher (20-40 Å)
resolution.
1s exposure pattern have been
collected at SLS, Switzerland
1011 photons/s in 20x20 μm2
Bragg spots are visible up to 8 Å
Why such a low resolution?
Essentially, because of damage
Serial Crystallography – Cathepsin B
Measurements at the
CXI beamline - LCLS
9.4 keV
40 fs pulse-length
1011 photons/pulse
293,000 hits
175,000 indexed patterns
Single crystal diffraction pattern
A virtual powder pattern
obtained as the sum of
thousand single crystals
patterns
Serial Crystallography – Cathepsin B
Projection of the measured intensities on
two planes in the reciprocal space
Serial Crystallography – Cathepsin B
Redecke et al., Science
2013
3D structure of the fully
glycosylated protein
Serial Crystallography at Synchrotrons
Motivations
- Room temperature measurements
- Time-resolved experiments
- Outrun damage (at least partially)
Warkentin et al. Acta Cryst. D D67 (2011)
Serial Crystallography at Synchrotrons
The serial approach can be used at synchrotrons
Peak brilliance is lower than FEL‘s one
Exposure time must be longer
Rotation during the exposure
helps integrating the Bragg peak
Serial Crystallography at Synchrotrons
Beamline P11 @ PETRA III – DESY Hamburg
Photon energy: 10 keV
Beam size: <10x10 µm2
Flux:
1012 photons/s
Detector: PILATUS 6M – 172x172 µm2 pixels
Serial Crystallography at Synchrotrons
Lysozyme microcrystals grown
in batch in high-salt and high
viscosity medium
Crystal suspension flowing in
a thin-walled SAXS capillary
at 2.5 l/min
Exposure time: 10 ms
Serial Crystallography at Synchrotrons
 > 1,000,000 recorded patterns
 Hit-finding
 150,000 ‘hits’
 Indexing
 40,000 indexed patterns
Bragg spots visible
up to  2 Å
resolution
2.1 Å
Serial Crystallography at Synchrotrons
Lysozyme structure solved at
2.1Å resolution by molecular
replacement merging
intensities from
40,000 single crystal diffraction
patterns
Pdb ID: 4O34
Stellato F. et al., IUCrJ 2014
Serial Crystallography at Synchrotrons
Unit cell parameters
are in excellent
agreement with
known values
a = (79.50.3) Å
b = (79.40.3) Å
c = (38.40.2) Å
10,000 single crystal
diffraction patterns would
probably be enough
Stellato F. et al., IUCrJ 2014
Serial Crystallography at Synchrotrons
Cathepsin B – reloaded
500 patterns from Cathepsin
B microcrystals at cryogenic
temperature
Structure solved at 3Å
resolution
Gati et al., IUCrJ 2014
Luci di Sincrotrone
CNR – Roma, 22 Aprile 2014
Applications & Future Perspectives
- Time-resolved measurements
- Sample delivery optimized for different media
- 2D crystallography
- Spectroscopies
Serial Crystallography
Time-resolved Pump-Probe Experiments
Changes observed in the
putative S3 state in the
Photosystem II complex
Aquila et al. Optics Express 470 (2011)
Kupitz et al. Nature (2014)
Serial Crystallography
Applications
GPCR in Lipidic Cubic Phase
Liu et al. Science (2013)
Photosyntetic reaction centers in Sponge phase
Johanssonn et al. Nature Methods (2012)
Scattering & Spectroscpies
X-ray emission (XES)
X-ray absorption (XAS)
Small angle scattering (SAXS)
Wide angle scattering (WAXS)
Luci di Sincrotrone
CNR – Roma, 22 Aprile 2014
Outlook
- Less beamtime: higher repetition rate FELs (XFEL)
- More sources: brighter sinchrotrons (ESRF, PETRA III)
- Less sample: improved sample injection systems
- More science: time-resolved experiments on different proteins
Higher and higher brilliance will
enable approaching the limit of
high-resolution single molecule
imaging
Acknowledgements
The Biophysics Group in Tor Vergata
Silvia Morante
Giancarlo Rossi
Velia Minicozzi
Francesco Stellato
Marco Pascucci
Claudia Narcisi
Emiliano De Santis
biophys.roma2.infn.it
CFEL-DESY
H. Chapman, J. Schulz, A. Barty, M. Liang, A. Aquila, T. White,
D. Deponte, S. Bajt, M. Barthelmess, A. Martin, C. Caleman, K.
Nass, F. Stellato, H. Fleckenstein, L. Galli, R. Kirian, K. Beyerlein
Arizona State Univeristy
J. Spence, P. Fromme, U. Weierstall, B. Doak, M. Hunter, C.
Kupitz
SLAC
M. Bogan, S. Boutet, G. Williams, D. Starodub, R. Sierra,
C. Hampton, J. Kryzwinski, C. Bostedt, M. Messerschmidt
Uppsala Univeristy
J. Hajdu, Nic Timneanu, J. Andreasson, M. Seibert, F. Maia, M.
Svenda, T. Ekeberg, J. Andreasson, A. Rocker, O. Jonsson, D.
Westphal
University of Tübingen, Hamburg and Lübeck
C. Betzel, L. Redecke, D. Rehders, K. Cupelli, R. Koopmann, M.
Duszenko, T. Stehle
Max Planck Heidelberg, LBNL, LLNL
European XFEL Massimo Altarellii
Thank you for
the attention
Contacts
Francesco Stellato
I.N.F.N. Sezione di Roma Tor Vergata
Via della Ricerca Scientifica, 1
Tel: 0039 06 7259 4284
[email protected]