Update from P12 Clement Blanchet P12 BioSAXS beamline on PETRAIII - - PowerPoint PPT Presentation

Update from P12

Clement Blanchet

P12 BioSAXS beamline on PETRAIII

Energy 4-20 keV (6-20keV) Flux 1013 ph/s (>5.1014 with MLM) Beam Size 200 x 25 μm2 (FWHM)

P12 BioSAXS beamline on PETRAIII

Energy 4-20 keV (6-20keV) Flux 1013 ph/s (>5.1014 with MLM) Beam Size 200 x 25 μm2 (FWHM) Scatterless slits

Li et al. J. Appl. Cryst. (2008). 41, 1134-1139

W Slits Scatterless slits

Active beamstop

Blanchet et al. J. Synchrotron rad. (2015). 22, 461-464

Photon counting detector

Sample handling

Round et al. Acta Crystallographica Section D: Biological Crystallography, (2015) 71(1), 67-75.

Sample changer

• Large capacity, more than 250

samples

• Full cycle time ≈ 1min
• Sample volume: 10 – 30 μl
• Flow measurement
• Rapid and efficient cell cleaning

Sample handling

Round et al. Acta Crystallographica Section D: Biological Crystallography, (2015) 71(1), 67-75.

Sample changer

• Large capacity, more than 250

samples

• Full cycle time ≈ 1min
• Sample volume: 10 – 30 μl
• Flow measurement
• Rapid and efficient cell cleaning

Graewert et al. Scientific Reports 5 (2015) 10734.

SEC-SAXS

• Online purification
• Online spectroscopic

characterization:

• UV/Vis absorption
• MALS
• Refractive index

Automation

Franke et al. NIM A (2012) 689: 52-59.

Automated data collection…

Automation

Franke et al. NIM A (2012) 689: 52-59.

Automated data collection… … and data analysis

Users and publications

• Beamyear 2019: 355 user-visits, 272 unique users (including mail-in), 75 projects, 68 unique groups

(counting a BAG as one group). Proprietary measurements were conducted for 7 companies.

• Publications referencing EMBL BioSAXS beamlines: 90 papers in 2019 (including 9 HZG CRG

publications).

2 2 2 2 4 2 6 2 8 2 1 2 1 2 2 1 4 2 1 6 2 1 8 50 100 150 200 250 300 350 400

Beamline statistics

user-visits projects groups 2 3 2 5 2 7 2 9 2 1 1 2 1 3 2 1 5 2 1 7 2 1 9 10 20 30 40 50 60 70 80 90 100

Papers acknowledging beamlines

P12 papers X33 papers HZG papers

Highlights of recent SAXS user publications

Condensin HEAT-repeat subunit Ycg1

Manalastas et al J.Biol.Chem. (2019) Shtykova et al J.Biol.Chem. (2019)

Ectodomain of the insulin receptor-related receptor

Nagel et al Biophys Chem. (2019)

Quaternary structures of insulin Glargine and Glulisine Tetrameric ATP citrate lyase

Verschueren et al Nature (2019)

mRNA – DEAE-dextran polyplex delivery systems

Sievert et al Biomaterials (2019)

Self assembled PET- DDT nanoparticles

Luo et al

• Nat. Commun (2018)

ABC Transporter MsbA in stealth nanodisk

Josts et al Structure (2018)

Ammonium sensor histidine kinase

Pflüger et al

• Nat. Commun. (2018)

Advanced BioSAXS

SAXS+WAXS with Pilatus 6M on P12

up to 26 nm-1

ASAXS

Silica coated AuNP

log(I), relatjve s, nm-1

Afuer: 7 ms 200 ms 500 ms 1350 ms

100us exposure time

Schmidt et al. Adv. Sci. 2019, 1900287.

Time-resolved SAXS

High Flux option

> 5.1014 photons/s

High temperature and aggresive chemical Scanning SAXS Microfluidics

P12 operation in 2020

PETRAIII schedule 2020

 Restart beginning of

March

P12 operation in 2020

PETRAIII schedule 2020

 Restart beginning of

March

 Operation stopped mid-

march

P12 operation in 2020

PETRAIII schedule 2020

 Restart beginning of

March

 Operation stopped mid-

march

 Restart end of april for

covid-related project

 Mail-in operation since

mid-May.

Beamline operation in Corona time

Many tools for remote working already existed before shutdown, mail in operation could be quickly implemented Since mid-May : mail-in only but « quasi-normal » schedule (5 groups/week), although with restrictions on the number of samples. Thanks to the colleagues from the SAXS group, user

• ffice and instrumentation team

Working with mask and social distances.

Beamline operation in Corona time

Many tools for remote working already existed before shutdown, mail in operation could be quickly implemented Since mid-May : mail-in only but « quasi-normal » schedule (5 groups/week), although with restrictions on the number of samples. Thanks to the colleagues from the SAXS group, user

• ffice and instrumentation team

+ beamline upgrade

Working with mask and social distances.

Mirror upgrade

P12 mirror until July 2020, adaptative bimorph mirror :

 Piezo elements integrated in the mirror

substrate :

 Bending radius can be modify to adjust the focal

distance,

 But problems at the piezo junction

Alcock, S. G., Sutter, J. P., Sawhney, K. J., Hall, D. R., McAuley, K., & Sorensen, T. (2013). NIM A: 710, 87-92.

Mirror upgrade

P12 mirror until July 2020, adaptative bimorph mirror :

 Piezo elements integrated in the mirror

substrate :

 Bending radius can be modify to adjust the focal

distance,

 But problems at the piezo junction

P12 beam at the sample position with piezo mirror

Mirror replacement

• 150
• 100
• 50

50 100 150

Length [mm]

• 0.4
• 0.3
• 0.2
• 0.1

0.1 0.2 0.3 0.4 0.5

Slope error [urad]

EMBL VFM - line3 (iteration3) EMBL VFM - line3 (iteration4)

 Adaptive bimorph mirrors replaced with fixed radius mirrors (ZEISS).

 Increase of active optical area to:  310 mm (VFM) / 460 mm (HFM).  Smallest residual slope error achieved (after 4 / 3 IBF-metrology iterations):  96 nrad rms (VFM) / 215 nrad (HFM); better / equal specs.

 Installed in July during the summer break with quick commissioning, commissioning and beam characterization in September.

HFM VFM

Mirror replacement

 Adaptive bimorph mirrors replaced with fixed radius mirrors (ZEISS).

 Smaller beam : 200 x 30 µm2(FWHM)   → much higher brilliance   → Radiation damage   Currently, we are often measuring with an attenuated beam but the sample cell can be further reduced to be adjusted to the beam in the new sample exposure unit.

Adaptive bimorph mirrors fixed radius mirrors

New sample exposure units for the sample changer

SEU 2A



Dedicated to solution scattering



Larger temperature range : 5°C to 60°C (80°C)



New pump for smaller capillaries



On axis camera

On axis camera

• ption (for

sandwiched cells) DLS option Additional fiber

• ptics port (in

situ illumination, absorption) Smaller capillaries

1.8 mm 1 mm 0.5 mm 0.3 mm

With ESRF and EMBL Grenoble (Cipriani team)

Installation SEU 2A

SEU 2A



Installed on the beamline last week.

A special thanks to Raphael Cohen-Aberdam, from EMBL Grenoble, who went through multiple covid tests and spent several days in quaratine to install the SEU in Hamburg)

SEU 2B

On axis camera XY piezo stage Modifiable front and backplates Back lightening Fiber optic ports Compatible with in-air and in vacuum sample environment

SEU 2B



Multipurpose: microfluidics, scanning SAXS, in-situ illumination (light-TR SAXS), etc.



Delivery: end 2020- beginning 2021

What’s next ?

Replacement of the PETRA III storage ring with a state-of-the-art MBA-based ultra-low-emittance storage ring.

PETRAIV - timeline

From PETRA IV CDR

 Last years : preparation of the

conceptual design report (accessible on DESY website)

 This year, preparation of the scientific

case :



PETRAIV workshop « Soft matter, health and life science » (Oct 28-30)



Preparation of the scientific instrument proposal (deadline December 1st)

 Next year : beamline selection and

preparation of the technical design report

PETRAIV

Advanced BioSAXS with PETRAIV beam

Time resolved : Resolution ultimately depends on the beam flux (a a single 5 us exposure could be enough to collect proper BioSAXS data). Dead time depends on the beam size. ASAXS : possible access to softer X-rays to reach Ca (4keV) or even Sulfur (2.8 keV) edge Microfluidics: smaller beamsize : better suited for microfluidics. Scanning SAXS : with small beam with low divergence the gap between real and reciprocal space can be bridged with micron size beam.

Petra IV challenge : Radiation damage

In biomolecular solutjon, solvated electrons and free radicals created when the X-rays radiolyse water molecules, leads to protein damage and

• aggregatjon. While damages to monomeric

proteins are generally not notjceable, aggregates quickly spoil the SAXS patuern.

Mitigate radiation damage

Chemical mitigation of radiation damage

Use of additives to scavenge the free radicals or slow down/reduce protein aggregation

Radiation damage highly depends on the sample and buffer composition and it is not possible to estimate the sensitivity to radiation damage from the protein sequence. Chemical mitigation works, however, additional molecules need to be added to the sample (and might perturb it).

In flow measurement

The sample flow through the cell while it is exposed and spend less time in the X-ray

• beam. (larger sample volume required)

Static Flowing

In flow measurement

The sample flow through the cell while it is exposed and spend less time in the X-ray

• beam. (larger sample volume required)

Static Flowing

Optjmizatjon

• f the cell size

and geometry

Small capillaries, although sub-optjmal with respect to X-ray scatuering intensitjes, allow one to collect betuer data with a given sample volume.

In flow measurement

The sample flow through the cell while it is exposed and spend less time in the X-ray

• beam. (larger sample volume required)

Static Flowing

Optjmizatjon

• f the cell size

and geometry

New sample chamber with on- axis microscope to explore additjonal cell geometries.

Small capillaries, although sub-optjmal with respect to X-ray scatuering intensitjes, allow one to collect betuer data with a given sample volume.

Collecting bioSAXS with brilliant X-ray beam?

Multilayer monochromator on P12 : Beam flux > 5.1014 photons/s

Collecting bioSAXS with brilliant X-ray beam?

Multilayer monochromator on P12 : Beam flux > 5.1014 photons/s

Collecting bioSAXS with brilliant X-ray beam?

Data collected in 1.35 ms exposure time!

1.35 ms frame (after 0, 13.5, 27, 40.5 and 54 ms)

BSA 2.5 mg/ml Severe radiation damage within 5 to 10 ms, but enough photons scattered in 1 ms for proper data analysis.

100us exposure time

Collecting bioSAXS with brilliant X-ray beam?

Slow ?

Using a high flux beam, more photons are scattered before the proteins start to aggregate. → Sample can be kept at fixed position during exposure, the sample volume is reduced, well suited for microfluidics application. → SAXS data collection with 100nl sample volume on PetraIV.

With Jesse Hopkins and Richard Gillilan MacCHESS

Improve sensitivity: further background reduction

• I. Steinke et al.; Review of

Scientific Instruments 2016, 87

Windowless setup

P12 instrumental background comes mostly from the capillary scattering. Can one measure without capillary?

Protein in vacuum?

Most photons are scattered by the buffer, not the biomolecules in solution  proteins in vacuum Aerosol, electrospray.

• J. Phys. B: At. Mol. Opt. Phys. 43 (2010)

Meyer et al, WIRe: Computational Molecular Science. 3(4) 2013

Conclusion

Given the current pandemic situation, P12 is doing quite well:

 Mail-in operation could be maintained for a large part of the year, and is still

expected to be the only mode of operation for the coming months.

 Several beamline developments could be finalized this year (Sample exposure

unit, mirror)

 In the coming years, further developments on P12 toward PETRAIV : test of

smaller capillaries, new microfluidics, radiation damage test with multilayer beam with new mirror, new sample environments, etc.

Acknowledgments

SAXS Group EMBL Hamburg

Dmitri Svergun Clemente Borges Taja Cheremnykh Stefano Da Vela Daniel Franke Melissa Gräwert Tobias Gräwert Andrey Gruzinov Cy Jeffries Al Kikhney Petar Markov Haydyn Mertens Ahmed Mohammed Dima Molodenskiy Martin Schroer

Instrumentation team EMBL Hamburg

Stefan Fiedler Elias Ben Boehme Moises Bueno Thomas Gehrmann Doris Jahn Liliana Kolwicz-Chodak Jochen Meyer Marina Nikolova Vamsee Krishna Palnati Uwe Ristau

Instrumentation team EMBL Grenoble

Florent Cipriani Raphael Cohen-Aberdam Gergely Papp Marcos Lopez Marrero

User office

Lola Jablanovic Sarah Marshall-Bensch Ivanka Araujo

Virtual beamline tour