Update from P12 Clement Blanchet
P12 BioSAXS beamline on PETRAIII Energy 4-20 keV (6-20keV) Flux 10 13 ph/s (>5.10 14 with MLM) Beam Size 200 x 25 μm 2 (FWHM)
P12 BioSAXS beamline on PETRAIII Scatterless slits Photon counting detector Active beamstop W Slits Scatterless slits Li et al. J. Appl. Cryst. (2008). 41 , 1134-1139 Blanchet et al. J. Synchrotron rad. (2015). 22 , 461-464 Energy 4-20 keV (6-20keV) Flux 10 13 ph/s (>5.10 14 with MLM) Beam Size 200 x 25 μm 2 (FWHM)
Sample handling Sample changer Large capacity, more than 250 samples Full cycle time ≈ 1min Sample volume: 10 – 30 μl Flow measurement Rapid and efficient cell cleaning Round et al. Acta Crystallographica Section D: Biological Crystallography , (2015) 71 (1), 67-75.
SEC-SAXS Sample handling Online purification Online spectroscopic Sample changer characterization: UV/Vis absorption MALS Refractive index 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 . Round et al. Acta Crystallographica Section D: Biological Crystallography , (2015) 71 (1), 67-75.
Automation Automated data collection… Franke et al. NIM A (2012) 689: 52-59.
Automation … and data analysis Automated data collection… Franke et al. NIM A (2012) 689: 52-59.
Users and publications Beamline statistics Papers acknowledging beamlines 400 100 90 350 80 300 70 250 60 200 50 40 150 30 100 20 50 10 0 0 3 5 7 9 1 3 5 7 9 0 2 4 6 8 0 2 4 6 8 0 0 0 0 1 1 1 1 1 0 0 0 0 0 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 P12 papers X33 papers HZG papers user-visits projects groups 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).
Highlights of recent SAXS user publications mRNA – DEAE-dextran Condensin HEAT-repeat ABC Transporter MsbA Tetrameric ATP citrate lyase polyplex delivery systems subunit Ycg1 in stealth nanodisk Verschueren et al Manalastas et al Josts et al Sievert et al Nature (2019) Structure (2018) J.Biol.Chem. (2019) Biomaterials (2019) Ammonium sensor Quaternary structures of Ectodomain of the insulin Self assembled PET- histidine kinase insulin Glargine and receptor-related receptor DDT nanoparticles Glulisine Shtykova et al J.Biol.Chem. (2019) Nagel et al Luo et al Pflüger et al Biophys Chem. (2019) Nat. Commun (2018) Nat. Commun. (2018)
Microfluidics Advanced BioSAXS Afuer: Time-resolved SAXS+WAXS 7 ms 200 ms SAXS with Pilatus 6M on P12 500 ms 1350 ms log(I), relatjve High temperature up to 26 nm -1 and aggresive chemical s, nm -1 Silica coated AuNP ASAXS High Flux option > 5.10 14 photons/s Scanning SAXS 100us exposure time Schmidt et al. Adv. Sci. 2019, 1900287.
P12 operation in 2020 Restart beginning of March PETRAIII schedule 2020
P12 operation in 2020 Restart beginning of March Operation stopped mid- march PETRAIII schedule 2020
P12 operation in 2020 Restart beginning of March Operation stopped mid- march Restart end of april for covid-related project Mail-in operation since mid-May. PETRAIII schedule 2020
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 office 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 office and instrumentation team Working with mask and social + beamline upgrade 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
Mirror upgrade P12 beam at the sample position with piezo mirror 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 replacement Adaptive bimorph mirrors replaced with fixed radius mirrors (ZEISS). 0.5 Increase of active optical area to: EMBL VFM - line3 (iteration3) VFM EMBL VFM - line3 (iteration4) 0.4 310 mm (VFM) / 460 mm (HFM). 0.3 Slope error [urad] 0.2 0.1 0 Smallest residual slope error achieved (after 4 / 3 -0.1 -0.2 IBF-metrology iterations): -0.3 96 nrad rms (VFM) / 215 nrad (HFM); better / -0.4 -150 -100 -50 0 50 100 150 Length [mm] equal specs. Installed in July during the summer break with quick commissioning, commissioning and beam characterization in September. HFM
Mirror replacement Adaptive bimorph mirrors Adaptive bimorph mirrors replaced with fixed radius mirrors (ZEISS). Smaller beam : 200 x 30 µm 2 (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. fixed radius mirrors
New sample exposure units for the sample changer SEU 2A DLS option 1.8 mm Dedicated to solution Additional fiber scattering optics port (in 1 mm Larger temperature situ illumination, range : 5°C to 60°C absorption) (80°C) 0.5 mm New pump for smaller 0.3 mm capillaries On axis camera On axis camera Smaller option (for capillaries With ESRF and EMBL Grenoble sandwiched cells) (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 Back camera lightening SEU 2B Compatible with in-air Multipurpose: microfluidics, and in scanning SAXS, in-situ vacuum illumination (light-TR SAXS), sample etc. environment Fiber optic Delivery: end 2020- beginning ports 2021 Modifiable XY piezo front and stage backplates
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 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 From PETRA IV CDR
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). Microfluidics : smaller Dead time depends on the beam size. 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. ASAXS : possible access to softer X-rays to reach Ca (4keV) or even Sulfur (2.8 keV) edge
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 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). Chemical mitigation of radiation damage Use of additives to scavenge the free radicals or slow down/reduce protein aggregation
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 Optjmizatjon The sample flow through the cell while it is of the cell size exposed and spend less time in the X-ray and geometry beam. (larger sample volume required) Small capillaries, although sub-optjmal with respect to Static X-ray scatuering intensitjes, allow one to collect betuer data with a given sample volume. Flowing
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