Serial Crystallography using x-ray Free Electron Lasers · Serial Crystallography using x-ray Free Electron Lasers ... delivery Data analysis The Cathepsin B experiment Serial Crystallography

Serial

Crystallography

using x-rayFree Electron Lasers

Milan, July 11th 2014

Francesco Stellato

I.N.F.N. – Sezione di Roma ‘Tor Vergata’

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

Summary

Structural Biology and X-rays

1E+001E+031E+061E+091E+121E+151E+181E+211E+241E+271E+301E+331E+36

1880 1910 1940 1970 2000 2030

Year

So

urc

e P

ea

k B

rilli

an

ce

Röntgen

Bragg & Bragg

reflections

von Laue

crystal diffraction

Hodgkin

penicillin,

B12

Perutz & Kendrew

myoglobin

Franklin,

Crick,

Watson

DNA

MacKinnon

Potassium

channel

Kornberg

RNA

polymerase

Jacobsen

Holography

Kirz &

Schmahl

Microscopy

Free Electron Lasers (FELs)

Radiation is generated by an undulator

Electrons are bunched up by interaction with x-rays

courtesy: Thomas Tschentscher (XFEL)

LINAC Coherent Light Source

FLASH

FLASH

Hamburg,

Germany

λ > 4.2 nm

LCLS

Stanford

USA

λ > 0.12 nm

FELs around the world

Soft x-rays

FERMITrieste Italy

SACLA

Rikken

Japan

Under construction

Hard x-rays

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

Diffraction pattern

FEL puse

Particle injection

One pulse,

one measure

R. Neutze et al, Nature 406 (2000)

A detectable signal

must be recorded

before the sample is

destroyed

Coherent x-ray ImagingDiffract and destroy proof-of-principle

First pulse

Second pulse

1 micron

SEM picture of a FIB-bed pattern

etched on a Si3N4membrane

Chapman et al. Nature Physics (2006)

Reconstructed image at

32 nm resolution

1 micron

FLASH

Diffraction patternDiffraction pattern

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

- Measurements of many (103-104)

single crystal diffraction patterns

- Indexing

- Intensities determination & merging

- Standard (and non-standard)

phasing methods0

10000

20000

30000

40000

50000

60000

70000

1972 1976 1980 1984 1988 1992 1996 2000 2004 2008

Electron

NMR

X-ray

Standard crystallography is the election

technique for structural biology

FEL Serial Crystallography

Experimental setup•FEL generated x-ray

beam

•Focusing optics

•Sample

•Sample injection system

•Detector

FEL Serial CrystallographyPilot experiment

Chapman et al. Nature 470, (2011)

Photosystem I

Sun-catcher Membrane protein

36 proteins 381 cofactors

AMO beamline

@ LCLS

Single shot at LCLS

E = 1.8 keV

80 fs pulse

2 mJ pulse energy

Upper

front

CCD

Lower

front

CCD

Resolution at corner = 8.6Å

beam center

FEL Serial CrystallographyPilot experiment

Chapman et al. Nature 470, (2011)

LCLS data allowed solving PSI structure

at 7 Å resolution

(wavelength and geometry limit)

The electron density is compatible with

the known structure one

Virtual powder data show that there is

no damage up to 70 fs pulses

Molecular replacement method is used

starting from the known structure

First FEL based pdb structure

Pdb ID: 3PCQ - www.pdb.org

Needle-

shaped

Cathepsin B

Nanocrystals

A Proteinase K

Nanocrystal

SEM images

Standard techniques can be

optimized to grow many micro-

and/or nano-crystals:

- Hanging droplet (robots)

-Batch methods

- In vivo crystallization

Nano/micro-crystals Preparation

Several techniques are used to detect

nanocrystals:

-Optical and electron Microscopy

-X-ray diffraction (XRD) (mainly powder)

-SONICC

Nano/micro crystals Screening

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

Nano/micrco crystals Characterization

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

Liquid line

Gas lineLiquid jet

100 m/s

0.5-5 μm diameter

1-10 μl/min

10% hitrate

Gas line

Ga

s b

ottle

Sa

mp

le re

serv

oir

Gas Dynamic Virtual Nozzle

De Ponte D et al. J. Phys. D 2008

water-jet-2.mov

water-jet-2.mov

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 size-

select particlesCone-Jet Mode

Electrospray/Electrospinning & Drop-on-demand

dod 50 um.mov

dod 50 um.mov

- Sample deposited on thin

Si3N4 membranes

Ideal for 2D crystallographyFrank M. et al., IUCrJ 2014

- Kapton ™ micro-cells

Good to keep samples

hydratedZarrine-Asfar A. et al., Acta D 2012

10 μm

Fix Targets

Diffraction pattern acquisition

Hit-finding

Background subtraction

Peak finding

Data Analysis Flow-chart

Only ‘hits’ are processed

Sparse patterns: average of many frames

Peaks are identified in the bkg subtracted patterns

Indexing

Intensities

Intensities merging

Structure factors

Data Analysis Flow-chart

White T. et al. J. Appl. Cryst 2012

White T. et al. Acta D 2013

Standard programs (DirAx, MOSFLM, …) called by

dedicated softwares (CrystFEL, Cctbx)

The (partial) intensity is evaluated as a locally

background subtracted sum of pixels close to the

detected (or predicted) peak position

Ring-scheme

Background

Empty region

Bragg peak

Serial Crystallography – Cathepsin B

Cathepsin B

Cysteine protease expressed by

T.brucei, organism that causes

Human African Trypanosomiasis

Luci di Sincrotrone

CNR – Roma, 22 Aprile 2014

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.

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

Luci di Sincrotrone



SEM picture of a purified

Cathepsin B crystal

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


Single crystal diffraction pattern


Measurements at the

CXI beamline - LCLS

9.4 keV

40 fs pulse-length

1011 photons/pulse

293,000 hits

175,000 indexed patterns

A virtual powder pattern

obtained as the sum of

thousand single crystals

patterns

Projection of the measured intensities on

two planes in the reciprocal space


3D structure of the fully

glycosylated protein

Redecke et al., Science

2013


Motivations

- Room temperature measurements

- Time-resolved experiments

- Outrun damage (at least partially)

Serial Crystallography at Synchrotrons

Warkentin et al. Acta Cryst. D D67 (2011)


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


Beamline P11 @ PETRA III – DESY Hamburg

Photon energy: 10 keV

Beam size: <10x10 µm2

Flux: 1012 photons/s

Detector: PILATUS 6M – 172x172 µm2 pixels


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


> 1,000,000 recorded patterns

Hit-finding

150,000 ‘hits’

Indexing

40,000 indexed patterns

2.1 Å

Bragg spots visibleup to 2 Åresolution


Lysozyme structure solved at2.1Å resolution by molecular

replacement merging

intensities from

40,000 single crystal diffraction

patterns

Pdb ID: 4O34Stellato F. et al., IUCrJ 2014


Unit cell parameters

are in excellent

agreement with

known valuesa = (79.50.3) Åb = (79.40.3) Åc = (38.40.2) Å

10,000 single crystal

diffraction patterns would

probably be enough

Stellato F. et al., IUCrJ 2014


Cathepsin B – reloaded

500 patterns from Cathepsin

B microcrystals at cryogenic

temperature

Structure solved at 3Å

resolution

Gati et al., IUCrJ 2014

Applications & Future Perspectives

- Time-resolved measurements

- Sample delivery optimized for different media

- 2D crystallography

- Spectroscopies

Luci di Sincrotrone


Serial Crystallography

Time-resolved Pump-Probe Experiments

Aquila et al. Optics Express 470 (2011) Kupitz et al. Nature (2014)

Changes observed in the

putative S3 state in the

Photosystem II complex

Serial Crystallography

ApplicationsGPCR in Lipidic Cubic Phase

Liu et al. Science (2013) Johanssonn et al. Nature Methods (2012)

Photosyntetic reaction centers in Sponge phase

Scattering & Spectroscpies

X-ray emission (XES)

X-ray absorption (XAS)

Small angle scattering (SAXS)

Wide angle scattering (WAXS)

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

Luci di Sincrotrone


Acknowledgements

The Biophysics Group in Tor Vergata

biophys.roma2.infn.it

Silvia Morante

Giancarlo Rossi

Velia Minicozzi

Francesco Stellato

Marco Pascucci

Claudia Narcisi

Emiliano De Santis

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]

Documents

Serial Crystallography using x-ray Free Electron Lasers · Serial Crystallography using x-ray Free Electron Lasers ... delivery Data analysis The Cathepsin B experiment Serial Crystallography