La Trobe Institute for Molecular Science, Department of Mathematical and Physical Sciences, School of Computing Engineering and Mathematical Science, La Trobe University, Bundoora, VIC 3086, Australia.
Australian Synchrotron, Australian Nuclear Science and Technology Organisation, 800 Blackburn Road, Clayton, VIC 3168, Australia.
J Synchrotron Radiat. 2022 May 1;29(Pt 3):602-614. doi: 10.1107/S1600577522001862. Epub 2022 Mar 21.
Serial crystallography of membrane proteins often employs high-viscosity injectors (HVIs) to deliver micrometre-sized crystals to the X-ray beam. Typically, the carrier medium is a lipidic cubic phase (LCP) media, which can also be used to nucleate and grow the crystals. However, despite the fact that the LCP is widely used with HVIs, the potential impact of the injection process on the LCP structure has not been reported and hence is not yet well understood. The self-assembled structure of the LCP can be affected by pressure, dehydration and temperature changes, all of which occur during continuous flow injection. These changes to the LCP structure may in turn impact the results of X-ray diffraction measurements from membrane protein crystals. To investigate the influence of HVIs on the structure of the LCP we conducted a study of the phase changes in monoolein/water and monoolein/buffer mixtures during continuous flow injection, at both atmospheric pressure and under vacuum. The reservoir pressure in the HVI was tracked to determine if there is any correlation with the phase behaviour of the LCP. The results indicated that, even though the reservoir pressure underwent (at times) significant variation, this did not appear to correlate with observed phase changes in the sample stream or correspond to shifts in the LCP lattice parameter. During vacuum injection, there was a three-way coexistence of the gyroid cubic phase, diamond cubic phase and lamellar phase. During injection at atmospheric pressure, the coexistence of a cubic phase and lamellar phase in the monoolein/water mixtures was also observed. The degree to which the lamellar phase is formed was found to be strongly dependent on the co-flowing gas conditions used to stabilize the LCP stream. A combination of laboratory-based optical polarization microscopy and simulation studies was used to investigate these observations.
膜蛋白的连续结晶通常采用高粘度注射器（HVI）将微米大小的晶体输送到 X 射线束中。通常，载体介质是类脂立方相（LCP）介质，它也可用于成核和生长晶体。然而，尽管 LCP 广泛用于 HVI，但注射过程对 LCP 结构的潜在影响尚未报道，因此尚未得到很好的理解。LCP 的自组装结构可能受到压力、脱水和温度变化的影响，所有这些变化都发生在连续流动注射过程中。LCP 结构的这些变化可能会反过来影响膜蛋白晶体的 X 射线衍射测量结果。为了研究 HVI 对 LCP 结构的影响，我们在大气压和真空下连续流动注射过程中研究了单油酸甘油酯/水和单油酸甘油酯/缓冲液混合物的相变。跟踪 HVI 中的储层压力，以确定其与 LCP 相行为是否存在任何相关性。结果表明，尽管储层压力（有时）会发生显著变化，但这似乎与样品流中的观察到的相变无关，也与 LCP 晶格参数的变化无关。在真空注射期间，存在向列立方相、金刚石立方相和层状相的三相共存。在大气压下注射时，也观察到单油酸甘油酯/水混合物中立方相和层状相的共存。发现层状相的形成程度强烈依赖于用于稳定 LCP 流的共流气体条件。使用基于实验室的光学偏振显微镜和模拟研究来研究这些观察结果。
