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Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography
Published on: September 20, 2016
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Polyacrylamide injection matrix for serial femtosecond crystallography
Jaehyun Park1, Sehan Park2, Jangwoo Kim2
1Pohang Accelerator Laboratory, Pohang, Gyeongbuk, Republic of Korea. jaehyun.park@postech.ac.kr.
Scientific Reports
|February 23, 2019
Summary
A new polyacrylamide (PAM) injection matrix significantly reduces sample consumption for serial femtosecond crystallography (SFX) experiments. This advancement enables high-resolution macromolecular structure determination using X-ray free-electron lasers with less material.
Area of Science:
- Structural Biology
- Biophysics
- Crystallography
Background:
- Serial femtosecond crystallography (SFX) offers room-temperature macromolecular dynamics studies with reduced radiation damage.
- High sample consumption is a major limitation in SFX, hindering broader application.
- Existing viscous media can react with specific crystal samples, limiting their utility.
Purpose of the Study:
- To develop and characterize a novel polyacrylamide (PAM) based injection matrix for SFX.
- To assess PAM's efficacy in reducing sample consumption and enabling high-resolution structure determination.
- To compare PAM's performance with existing viscous media like monoolein.
Main Methods:
- Preparation of a polyacrylamide (PAM) solution as a carrier matrix.
- Delivery of protein microcrystals (lysozyme, thermolysin) in PAM using an injector system.
- Data collection using an X-ray free-electron laser (XFEL) and subsequent structure determination.
Main Results:
- Successfully obtained numerous indexed images for lysozyme and thermolysin delivered in PAM.
- Determined high-resolution crystal structures (1.7 Å for lysozyme, 1.8 Å for thermolysin).
- Observed lower background scattering from PAM compared to monoolein.
Conclusions:
- Polyacrylamide (PAM) is a viable and effective sample delivery medium for SFX.
- PAM facilitates reduced sample consumption and high-resolution structure determination.
- This new matrix expands the applicability of SFX techniques.
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