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Fixed-target protein serial microcrystallography with an x-ray free electron laser.

Hunter MS, Segelke B, Messerschmidt M, Williams GJ, Zatsepin NA, Barty A, Benner WH, Carlson DB, Coleman M, Graf A, Hau-Riege SP, Pardini T, Seibert MM, Evans J, Boutet S, Frank M - Sci Rep (2014)

Bottom Line: We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature.We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX.This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

View Article: PubMed Central - PubMed

Affiliation: Physics Division, Lawrence Livermore National Laboratory, 7000 East Avenue, Mail Stop L-211, Livermore, CA 94550, USA.

ABSTRACT
We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

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Experimental overview.Protein crystals embedded in Paratone-N were placed on fixed-targets and measured in vacuo at the Coherent X-ray Imaging (CXI) beam line of the LCLS.
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f1: Experimental overview.Protein crystals embedded in Paratone-N were placed on fixed-targets and measured in vacuo at the Coherent X-ray Imaging (CXI) beam line of the LCLS.

Mentions: Fixed-target supports allowed for high data-collection rates using the setup at the Coherent X-ray Imaging (CXI) beam line17 of the Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory. The fixed-target supports were silicon wafers produced by Silson Ltd (Silson Ltd, JBJ Business Park, Northampton Road, Blisworth, Northampton, NN7 3DW, England) and an individual wafer is shown in Fig. S1. The wafers were comprised of 200 μm thick silicon crystals with a 50 nm Si3N4 layer deposited on the (100) face. Long, rectangular windows measuring 200 μm × 8400 μm were etched into the silicon (leaving a 50 nm Si3N4 membrane) to allow for scanning of the sample through the LCLS x-ray beam through the use of the fixed-target stage motors. An overview of the experimental setup is shown in Fig. 1.


Fixed-target protein serial microcrystallography with an x-ray free electron laser.

Hunter MS, Segelke B, Messerschmidt M, Williams GJ, Zatsepin NA, Barty A, Benner WH, Carlson DB, Coleman M, Graf A, Hau-Riege SP, Pardini T, Seibert MM, Evans J, Boutet S, Frank M - Sci Rep (2014)

Experimental overview.Protein crystals embedded in Paratone-N were placed on fixed-targets and measured in vacuo at the Coherent X-ray Imaging (CXI) beam line of the LCLS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4129423&req=5

f1: Experimental overview.Protein crystals embedded in Paratone-N were placed on fixed-targets and measured in vacuo at the Coherent X-ray Imaging (CXI) beam line of the LCLS.
Mentions: Fixed-target supports allowed for high data-collection rates using the setup at the Coherent X-ray Imaging (CXI) beam line17 of the Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory. The fixed-target supports were silicon wafers produced by Silson Ltd (Silson Ltd, JBJ Business Park, Northampton Road, Blisworth, Northampton, NN7 3DW, England) and an individual wafer is shown in Fig. S1. The wafers were comprised of 200 μm thick silicon crystals with a 50 nm Si3N4 layer deposited on the (100) face. Long, rectangular windows measuring 200 μm × 8400 μm were etched into the silicon (leaving a 50 nm Si3N4 membrane) to allow for scanning of the sample through the LCLS x-ray beam through the use of the fixed-target stage motors. An overview of the experimental setup is shown in Fig. 1.

Bottom Line: We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature.We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX.This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

View Article: PubMed Central - PubMed

Affiliation: Physics Division, Lawrence Livermore National Laboratory, 7000 East Avenue, Mail Stop L-211, Livermore, CA 94550, USA.

ABSTRACT
We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

Show MeSH
Related in: MedlinePlus