Journal Article

PUBDB-2018-01851

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

Gicquel, Y.CFEL*DESY* ; Schubert, R.XFEL.EU*EMBL* ; Kapis, S. ; Bourenkov, G.EMBL* ; Schneider, T.EMBL* ; Perbandt, M.Extern*EMBL* ; Betzel, C.Extern* ; Chapman, H. N.CFEL*DESY* ; Heymann, M. (Corresponding author)CFEL*DESY*

2018
[S.l.]

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Please use a persistent id in citations: doi:10.3791/57133  doi:10.3204/PUBDB-2018-01851

Abstract: This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth.After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 μm grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbancesof fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Note: PIF-2015-46

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Contributing Institute(s):

1. FS-CFEL-1 (Group Leader: Henry Chapman) (CFEL-I)
2. beauftragt von UNI (UNI/CUI)
3. EMBL (EMBL)

Research Program(s):

1. 6215 - Soft Matter, Health and Life Sciences (POF3-621) (POF3-621)
2. 6G3 - PETRA III (POF3-622) (POF3-622)
3. DFG project 194651731 - EXC 1074: Hamburger Zentrum für ultraschnelle Beobachtung (CUI): Struktur, Dynamik und Kontrolle von Materie auf atomarer Skala (194651731) (194651731)
4. 05K13GU7 - Test eines "Serial Femtosecond Crystallography (SFX)"-Messtandes am Europäischen Elektronenlaser XFEL (BMBF-05K13GU7) (BMBF-05K13GU7)

Experiment(s):

1. Experiments at CFEL
2. PETRA Beamline P14 (PETRA III)

Appears in the scientific report 2018

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Database coverage:
; ; ; BIOSIS Previews ; IF < 5 ; JCR ; NCBI Molecular Biology Database ; SCOPUS ; Science Citation Index Expanded ; Thomson Reuters Master Journal List ; Web of Science Core Collection

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