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SAXS and SANS facilities and experimental practice Clement Blanchet EMBL Hamburg Small Angle Scattering experiment Detector Sample X-ray or neutron 2 2 Beam s Buffer The beam hits the sample, X-rays/neutrons interact with the

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SAXS and SANS facilities and experimental practice

Clement Blanchet – EMBL Hamburg

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Small Angle Scattering experiment

2θ 2θ

s

X-ray or neutron Beam

Sample Buffer Detector

The beam hits the sample, X-rays/neutrons interact with the sample and are scattered, providing structural information on the sample. Same formalism but different scattered particles  Different instrument.

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Outline

  • X-rays / neutrons
  • SAS instruments
  • Sample environment
  • Sample requirements and collection strategy
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X-rays and neutrons

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X-rays

Roengten, 1895

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Electromagnetic wave

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How are X-ray produced?

  • Brehmstrahlung

– When a charge is accelerated charge, electromagnetic radiation is produced

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X-ray sources - Synchrotron

  • Synchrotrons
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X-ray sources - synchrotron

  • Synchrotron radiation – Insertion devices
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Insertion devices

Dipole bending magnet (APS) Undulator (PetraIII)

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Synchrotrons around the world

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X-ray sources - FEL

  • Free electron laser

– Electrons are accelerated and send into a long undulator (several 100s meters) – Self amplified spontaneous emission: electrons group themselves into small bunches. – Production of very short and intense X-ray pulses

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X-ray sources - FEL

  • Free electron laser
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Lab sources

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Lab sources

  • Principle : electrons, produced by heating a

cathode are accelerated in an electric field and projected on a metallic anode. – Brehmstrahlung – Fluorescence

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X-ray sources

  • Lab source (rotating anode, liquid jet)
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Neutron

λ=h/mv

James Chadwick

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Neutron production

  • Nuclear reaction
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Neutron production

  • Spallation source

– Accelerated protons hit a heavy metal target.

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Neutrons Facilities

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SAXS and SANS Instruments

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Optics

Polychromatic divergent beam from the source Monochromatic focused (parallel) beam for SAS

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Monochromatic X-ray

  • Bragg diffraction on a crystal

nλ = 2dsinθ

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Monochromator

  • Before
  • Polychromatic
  • After
  • One wavelength + harmonics
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Focusing/low divergence

  • Small beam at the detector position
  • Small beam at the sample position

2θ 2θ

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Focusing X-ray

  • Compound refractive lenses
  • X-ray mirrors
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Focusing X-ray

  • Focussing mirror
  • Reflectivity

Energy [eV]

10000

Transmission

0,0 0,2 0,4 0,6 0,8 1,0

0.15 Degree 0.25 Degree 1 Degree

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Focussing mirror – harmonics filter

Monochromatic, focused x-ray beam

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Monochromatic neutrons

  • De Broglie equation: λ=h/mv

The wavelength of a neutron is related to its velocity.

  • Velocity selector

∆λ/λ=5-10%

  • For pulsed source, TOF
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Collimation neutrons

  • The collimator is used to obtain a parallel

beam

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Get rid of parasitic scattering: slits

Beam defining slits Guard or anti-scatter slits

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Hybrid slits

  • Idea: use a crystal for the tip of the blade:

 no scattering but diffraction

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Hybrid slits

  • On the P12 beamline

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Sample environment

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Flight tube

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Beamstop

  • Prevent the direct beam from hitting the

detector

– Big enough to stop the direct beam – Small enough to collect the small angle

  • Measure transmitted beam
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Active Beamstop

  • SAXS images needs to be accurately

scaled to allow for proper buffer subtraction and extraction of the solute SAXS pattern

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Detectors

Gas detector

X-rays ionize a gas (argon) producing of ions and free electrons. A voltage applied to the gas chamber, ions and electrons are moved in opposite direction and produce a detectable current.

X-ray film

X-rays interacts with silver halide crystals, electrons are produced by photoelectric effect. They gather around crystal defects, dragging the silver atoms that can be revealed during the development phase.

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Photostimulated luminescence

X-rays interacts electrons of a photostimulable phosphore plate. After exposure, the electrons are trapped in a high energy state. They can be untrapped by laser illumination and go back to their initial state. While doing so, they produce light that can be detected

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Hybrid pixel detectors

  • Each pixel is readout individually:

– No readout noise – Fast readout – high frame rate – High dynamic range

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Pilatus Eiger

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  • Count rate capability 107 photons/s/pixel
  • High spatial resolution with 75 µm pixel size
  • Kilohertz frame rate with dead-time-free readout
  • Two energy discriminating thresholds
  • Gateable detection for pump & probe experiments
  • Large active area for wide angular coverage
  • Single-pixel point-spread function
  • No readout noise or dark current
  • Compact housing
  • Room temperature operation of all components

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Up to 2000 Hz for smaller detectors

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Neutron detection

  • He3 detector:

n + 3He → 3H + 1H + 0.764 MeV

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Sample environment

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Samples

Metal alloys Nanomagnetic materials Sufactants Polymers Tissues Bio-macromolecules in solution

SAS applicable to many type of samples.

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10/15/2019 SAXS and SANS facilities Clement Blanchet 48 Rapid mixing device Heating stages Magnetic field system Sample changers

Sample environment

Example ID02 (ESRF) multipurpose beamline

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Sample environment

  • Bio-macromolecules in solution are weakly

scattering sample.

  • For biological macromolecules in solution:

– fragile – Preferably in vacuum – Thermostated

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Sample cell

  • Cell material: low absorption and scattering

– Mica, quartz, polycarbonate

  • Sample thickness (t): compromise between

scattering and absorption

– Scattering α t – absorption α exp(-ut)

  • For neutron, cell are rather thin (<1mm to avoid

multiple scattering)

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Solution SAXS

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15 years ago: Manual sample loading

  • Buffer and sample should be

measured in the same cell

  • Difficult to implement in vacuum
  • 10-15 minutes per measurement
  • High sample consumption
  • Non-optimized cleaning procedure
  • Tedious, energy and attention

consuming

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SAXS sample changer @EMBL Hamburg

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SAXS sample changer

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Sample changer performances

  • Large storage capacity
  • Full cycle time (loading, exposure,

flushing, cleaning, drying) ≈ 1 min

  • Volume 5-20 microliter
  • Very efficient cleaning
  • Flow measurement

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Online size exclusion column

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SEC + SAXS

Defined buffer region

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SEC + SANS

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Experimental practice

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Buffer subtraction

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Buffer subtraction

  • Biological sample scatters very weakly,

SAXS curves collected on the buffer should be carefully subtracted

– Exactly matching buffer (dialysis, elution buffer) – Sample and buffer measured in the same cell

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Monodispersity

  • SAS is very sensible to aggregation, the

sample should be monodisperse

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Monodispersity

  • Check the monodispersity of your sample

before coming to the beamline.

(native gel, dynamic light scattering, ultracentrifugation,…)

  • Use online chromatography
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Inter-particle interactions

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Inter-particle interactions

  • Change solution (pH, salt concentration) to

limit interactions

  • Measure different concentrations and

extrapolate

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Measure also water and/or standard protein…

  • … to estimate the molecular mass of your

sample using the forward scattering

– For data on an absolute scale (water measurement)

  • M=I(0)*NA/(C*ν∗∆ρ)

– Using a protein standard

  • M=MBSA*I(0)/IBSA(0)
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X-rays - Radiation damage!!!

Water 2011, 3(1), 235-253

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X-rays - Radiation damage!!!

  • Monitor radiation damage: collect several

frames and compare them.

Radiation damage! Only use undamaged frames No radiation damage, All the frames can be used.

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Radiation damage

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Different RD on different proteins

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Mitigate radiation damage.

Flow measurement Beam attenuation Use of additives Rnase – Static measurement

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Contrast in neutron

  • Neutrons interact with the nucleus of

atoms

  • Each atoms has its own scattering length:

H D C N O P S

  • .3742

0.6671 0.6651 0.940 0.5804 0.517 0.2847

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Labeling

  • Protein and nucleic acids have different

scattering length densities and the different components can readily be studied by SANS

  • To study protein-protein complex, one of the

components needs to be deuterated (hydrogen exchanged with deuterium) to changed its scattering length density

  • A deuterated protein is obtained by producing

the protein in a deuterated medium.

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Neutron collection strategy

  • Solvent matching

– Find the solvent composition that match the contrast of the component you want to hide – Measure the sample in this solvent

  • Contrast variation

– Measure the sample in solvent with different D/H ratio, different scattering length. – Using Stuhrmann analysis you can access the curves of the different components.

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Summary: SAS sample

  • Protein concentration: 0.1-10 mg/ml
  • Volume: 5-50 microliter (SAXS), 200-300

microliter (SANS)

  • Time:
  • lab source: 5-60 min
  • Synchrotron: seconds
  • Neutrons: 30 minutes - hours
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Summary: SAS sample

  • Pure and monodisperse sample
  • Exactly matching buffer
  • Measure concentration series
  • For SAXS:

– Be aware of radiation damage

  • For SANS:

– Carefully design your experiment, think of your collection strategy.

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Conclusion

  • To optimally use your beamtime: carefully plan your

experiment, prepare your sample and characterize them before coming to the beamline

  • SAXS is now widely used. Dedicated instruments with

low background and high level of automation, high quality lab instruments.

  • SANS more difficult: consume more time and sample,

requires good planning of your experiments and collection strategy, but can provides unique information.

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Structural and biophysical methods for biological macromolecules in solution

EMBO Global Exchange Lecture Course 14 – 20 October 2019 | Santiago, Chile