BioSAXS Instrumentation and Sample Environment at BM29

Petra Pernot

15th September 2014 HERCULES Specialised Course

Non-atomic resolution scattering in biology and soft matter ESRF, Grenoble

SAXS synchrotron based in the World = at all X-ray sources

ALS Berkley SIBYLS

SLAC Stanford BL4-2

APS Chicago bioCAT

BL4-2

SIBYLS

Diamond I22

Soleil SWING

PETRA III P12

BNL NSLS II

Campinas LNLS SAXS-2

SPRING 8 BL45XU, BL40B2

Shanghai SSRF

Australian synchrotron SAXS/WAXS

SLS cSAXS

Beijing BSRF

ESRF: ID02, ID13, BM26, BM29

P12

MAX-IV

BL40B2

- optics and experimental set-up - automated data collection and analysis - future developments

X-ray source

Small Angle X- ray Scattering

1D scattering curve

model independent parameters

shape, MM, interaction,…

ab-initio model

low resolution structure

BioSAXS Instrumentation and Sample Environment at BM29

Outline

2D detector

macromolecules in solution

-

Part of ESRF Upgrade: bioSAXS went from ID14-3 to BM29

Improvements state-of-art dedicated optics: - tunability: 7-15keV; - flux: double multilayer monochromator; - focusing at detector plane: torrodial mirror

BM29 opened to users: June 2012

ID30 MASSIF

ID29 MAD

Structural Biology village

CRYO BENCH

SAMPLE PREP LAB

shutter

slits

slits

MB absorbers

mirror

monochromator

primary slits WB absorbers

Optics hutch

Monochromator: double multilayer - 2 identical coatings Ru/B4C with 3 nm period

Focusing: 1.1m long Rh coated cylindrical-torioid mirror reflecting vertically upwards at a glancing angle of 4 mrad

XBPMs

white beam 4 4 mm2 monochromatic beam E = 11 keV

beam just after torroid

needs to be slit down

Scatterless/free slits, pinholes Goal = to minimize parasitic scattering: sandwich/hybrid metal - single crystal blades

metal

a

Snapshots close to beamstop parasitic slit scattering

pinhole for cleaning “small beams”

= 100 mm diameter hole

S1 S2

PINHOLE

high q domain

high q domain: window very small, contrast only at the interface

low q domain: window very large, structure factor: interactions in the system

low q domain

What does “q-range” mean? scattered intensity

= structure of the sample

d0=2/q0

LARGE SMALL distances

observation “window”

scattered signal observed if the contrast inside

intermediary q domain

intermediary q domain: window of order of “elementary bricks” of the system, form factor: size, shape, … of 1 particle

2/q0

Experimental hutch

E = 7 keV E = 15 keV

qmin 0.019 nm-1 0.041 nm-1

qmax 2.5 nm-1 5.3 nm-1

qmin and qmax defined by the experimental setup: - = (0.82 – 1.77)Å, E = (7 – 15) keV; - detector size: distance from direct beam spot to detector edge ~ 20 cm; - sample-to-detector distance 2.87 m; - beam stop diameter ~ 3 mm.

q-range available

robot

flight tube slit box

detector

sample in capillary

First results = 2D data - detector set offset to increase the maximal angular (q)-range

- intermodulus gaps, hot pixels and beamstop shadow masked out.

- each pixel data scaled for intensity measured in diode incorporated in beamstop (transmitted intensity) and data collection time;

DIODE

X-rays

2D to 1D angle converted to q = 4sinQ/ calibrated beamline parameters needed

Pt LIII edge = 11.564 keV

I TRANSMITED

E [keV]

radial averaging = all pixels with equal angle are averaged);

1D

2

log I

q sample-to-detector distance

ring radius

= NPIXELS

172 mm

q = 4 sinQ/

- sample-to detector distance powder diffraction of Silver Behenate AgC22H43O2

d001 = 58.38 Å

d002 = 29.17 Å

d003 = 19.44 Å

d004 = 14.58 Å

- energy (energy scan with a metal foil – ex. Pt)

- beam position [X0, Y0]: direct beam spot recorded (strongly attenuated, beamstop out)

Data error handling and buffer subtraction

q [nm-1] I stddev 1.068008e-03 5.801401e-04 9.280474e-05 1.373153e-03 6.069934e-04 8.641766e-05 1.678298e-03 6.839557e-04 8.325125e-05 1.983443e-03 7.532046e-04 8.176428e-05 ….. …… ……

data file

Gauss

Poisson statistics

log I

q

data

Gauss error

Poisson error

ring

ring

i

GN

II

2

ring

ring

i

PN

I

2

log I

q

standard measurement protocol BUFFER - SAMPLE - BUFFER

SUBTRACTED curve (sample – buffer)

- intensity scaled by sample concentration

protein signals < 0.5% background level

Data quality check for particle interactions, radiation damage

repulsive particle interactions

ideal solution of particles

raw data

q

log I

attractive particle interactions

log I

q

consecutive frames

1 s each

q

usually looks as

aggregation

visible on-line

subtracted data

Sample requirements about 100 mL of stock solution needed which shows

• monodispersity (purity > 95%, no aggregation, 1 oligomeric state) - check by MALS, DLS, SEC, Analytical Ultra Centrifugation - stable day after? after freezing? - measure concentration (absorbance A280; A260 to detect nucleic acids) • no interaction between particles - reached by working at infinite dilution… - in practice: scattering pattern independent of concentration or extrapolation to c=0 - radiation damage: add free radical scavenger

Buffer should match the sample (dialyze) - salt < 1M, glycerol < 10% - if detergent and micelles created (membrane proteins): each concentration

needs its own buffer, or go for HPLC run

GEL FILTRATION

* 3 conc. 0.2-10mg/ml * 30-50ml per conc. * matching buffer

Lyzosyme 40mg/mL with NaCl concentrations

courtesy of Françoise Bonneté

droplets denerated by microfluidic chip f 300mm

no salt

200 mM

500 mM

750 mM

I

q [nm-1]

Particle mass determination

- from QR ratio (independent on particle concentration and folding)

2

0

PART

APARTPART

c

NIM

Orthaber et al (2000) J. Appl. Cryst. 33, 218-225.

partial specific volume of the macromolecule [cm3/g] protein = 0.735 , DNA/RNA = 0.54, lipids = 1.02,…

I

q

H2O in capillary

empty capillary

H2O only

I(0) at 20C = 20.33 normalization coeff. set that I(0) for a protein in kDa units

Rambo & Tainer (2013) Nature 496, 477-481.

k

c

RPART

e

QM

1

dqqqI

IV PART

C

0

g

C

RR

VQ

2

SAS invariant

integral converges for folded and unfolded particles

paramaters k and c empirically determined and specific to the class of macromolecular particle (protein-only, RNA, complex,…)

e…Euler’s number

- through intensity calibration of water (absolute)

PART

STANSTAN

STAN

PART

PARTc

Mc

I

IM

0

0 BSA measurement

- through measurement of protein standard (relative)

EDNA

SAMPLE

CHANGER/HPLC

Beamline GUI

Automated data collection and analysis

BsxCuBE

DATABASE

ISPyB: sample tracking and data flow

EXPERIMENTAL

parameters

Samples stored in: 96 well plates (up to 3), PCR tubes Thermo-regulation - storage: 4-40ºC, - exposure cell: 4-60ºC Sample loading and cleaning: 30’ In user operation from September 2010

Sample Changer for bio-SAXS

Sample Changer for bio-SAXS

ROBOT

GUI Fill – Recuperate - Clean –Mix - Transfer

Flow

Sample Volume • minimum 15 μL per exposure (in the tube, i.e. about 5 mL in the capillary) • 30 μL recommended (be able to use flow mode) • minimum 3 concentrations required per construct (1-20 mg/mL) • 1 mL of buffer for dilution/background subtractions Users recommended to bring total volume of 100 μL of stock (ideally > 10 mg/mL) solution per construct Exposure Time • standard time 10 x 1 second (= 10 s) • easily modifiable in case if needed (signal-to-noise or radiation damage issues) Beamtime required: ~ 20 sample (with the buffer) data collections per 1 hour

Data collection on BM29 a) STATIC mode = concentration series using sample changer

HPLC on-line

temperature control

(1 ÷ 8C)

thermally insulated tubing

Biophysical characterization

UV-vis spectrometer, RALS and Refractive index modules

final purification step on-line

HPLC on-line SAXS - automatic generation of 1D files - merged buffer - subtractions frames with data - merged file for each peak (using I(0) and Rg)

4 way valve = fast switching between SC/HPLC modes

Sample • minimum 50 μL per injection • 100 μL recommended • concentration ~ 5 mg/mL + 0.5 L buffer per injection and equilibration Users recommended to bring own columns and total volume of 100 μL of stock solution per run + 1 L of buffer Exposure Time • Standard time 1 second per frame (max.5s) x column elution S200 column ~ 1h, Increase column ~ 10 minutes Beamtime required: column equilibration and elution Switch between STATIC and HPLC modes quick and easy, column can be equilibrated when recording data with SC

Data collection on BM29 b) HPLC mode = SEC on-line

# of frames in 1000s

Comparison Sample Changer / HPLC use

# of samples

Most user groups combine sample changer and HPLC experiments

Sample Changer

HPLC

Data collection GUI: BsxCuBE

- data collection parameters: directory, prefix, run, n of frames, exposure; - radiation damage check option, collect using SC/HPLC switch; - transmission/filters setting and energy/wavelength setting

robot tab

operation collect user log

- energy/wavelength setting: ML angle, Pilatus threshold; - transmission/filters setting - check beam option

Data collection GUI: BsxCuBE

2D tab

- beamline ‘easy’ operation: slit, beamstop, capillary alignment, vacuum/air setting and opening/closing shutters manualy

Operation beamline

Data collection GUI: BsxCuBE

- beamline parameters: mask, pixel size, beam center,…;

-1D tab displays curves: radial integration, normalization, typically 10 frames of 1s

- ring current, vacuum state in experimental hutch

Collect using robot script

Automated use of Sample Changer with samples and buffers loaded in various micro-plates or tubes in sequence buffer-sample-buffer-... - sample positioning, volume to load, flow during exposure, transmission, kinetics measurements (waiting time), sample temperature, etc.

Bio-SAXS ISPyB: data and sample tracking

Model visualisation

Bio-SAXS ISPyB: HPLC mode

Strength and downside of SAXS measurement done in solution with little preparation (relative)

• data orientationally averaged (impossible to distinguish enantiomorphs),

scattering curve has only a small number of independent points estimated by number of Shannon channels:

usually 10-15

1st data point (lowest-resolution value describes the overall scatterer size)

, however (between two adjacent point measured in scattering curve)

SAXS data are over-sampled

effective information content is higher than predicted from

and accurate shapes can be derived from scattering curve WHEN additional constraints are imposed to the reconstruction.

MAXMINMAX

S

DqqN

)(

MAX

MIND

q

MAXD

q

SN

• Small angle X-ray scattering is now a powerful tool for determining molecular envelopes that can be combined with high-resolution structures

• The combination of high-resolution techniques with low or medium resolution approaches leads to quasi-atomic models.

• The method is complementary to EM: can be used for smaller macromolecules

EM: search volume

Crystallography: atomic models

NMR: binding site mapping and orientations

Biochemistry and FRET: interface mapping

Bioinformatics: secondary structure prediction

SANS

Complementary techniques

SAX/NS complementarity • Neutron small angle scattering provides additional information for

macromolecular complexes that are made of several types of molecules such as proteins, nucleic acids or lipids. Contrast variation experiments obtained by exchanging the solvent for deuterated or partially deuterated solvent enhances the signal from one component

• SAXS and SANS are therefore tools of major importance in tackling systems biology: joint access possible – ESRF and ILL SAS experiments during one trip to Grenoble

SAXS SANS

volume small < 50 ml larger ~ 300 ml

concentration > 0.1 mg/ml > 1 mg/ml

measuring time short ~ s longer ~ mh

radiation damage yes no

contrast variation no yes

sensitive to salts, denaturants

yes no

SANS camera D22 at the ILL

Future plans Enlarge the types of samples amenable to SAXS to those that can be purified only in limited yields: nanolitre range or to those that are soluble only at c < 1 mg/ml.

APPLICATIONS: - use of micro-screening plates for rapid, large-scale macromolecular screening: studies of protein-protein interactions or complex formation, protein crystallisation process; 96

384

1534 wells

a) b)

Slow nucleation: droplets trapped in wells arrays - measurement in each well to observe nucleation event

100mm 100mm

Fast nucleation: distance in “serpentine” ~ time (nucleation and growth with time scale down to 10ms

- droplet microfluidic chips for mapping protein phase diagrams: a) droplets generated and stored off-line b) droplets generated, mixed and analysed on-line

LTP of Sébastien Teychené and Françoise Bonneté

liquid flow X-rays

Redesign Sample Exposure Unit

Page 35

requirements

- multicolor light source

(photo-activated/sensitive

proteins)

- on-line DLS, UV/Vis and

fluorescent detector at 90

- Wide Angle Scattering

capillary pod

SEU

light

REDESIGN SEU SAXS (WAXS), UV/Vis spectrometer, fluorescence detector, DLS, Raman,…

under various illumination, temperature,… •

Page 36

photo-activated/sensitive proteins

LED source + optical fiber

(bottom or/and on-axis camera)

dark

light

light camera /

Raman probe

UV lamp

tunable LED

fluorescence

detector

UV-Vis

probe

X-ray

SAXS

WAXS

adapted from S. Haas, T.S. Plivelic & C. Dicko,

J. Phys. Chem. B 2014, 118, 226-2273.

capillary

holder

Page 37 l BM29 news l 1st July 2014 l Petra Pernot

fast valve

supports (x2)

light port n1

SEU

light port n 2

light port n 3

colour

tuneable

lighting

New SEU preliminary drawing: Frank Felisaz + Florent Cipriani EMBL

New

Modified items

bellows

bellows

X-rays

WAXS:

- SEU-to-valve

distance shortened;

- bellows offsets down

New SEU preliminary drawing - Frank Felisaz + Florent Cipriani

Page 38

Light ports for DLS, absorption spectrometry, fluorescence, Raman,...

100

DLS port n1

DLS port n2

UV-VIS lamp

UV-VIS probe

fluorescence

detector

X-rays

probed volume

thanks to whom

BioSAXS beamline exists in its current shape

• Technicians: Mario Lentini, John Surr, Franck Felisaz, Julien Huet, Hugo Caserotto, Fabien Dobias;

• Software developers: Staffan Ohlsson, Jerome Kiefer, Alejandro De Maria Antolinos, Alexandre Gobbo, Matias Guijarro, Antonia Beteva, Vicente Rey-Bakaikoa, Olof Svensson;

• Instrument support division: Dean Gibson, Philippe Retout;

• Engineers/scientists: Pascal Theveneau, Werner Schmid, Ray Barrett, Muriel Mattenet, Christian Morawe, François Torrecillas, Adam Round, Florent Cipriani, Louiza Zerrad, Martha Brennish. EMBL

ACKNOWLEDGMENTS