From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography

Dods, Robert; Båth, Petra; Arnlund, David; Beyerlein, Kenneth R.; Nelson, Garrett; Liang, Mengling; Harimoorthy, Rajiv; Berntsen, Peter; Malmerberg, Erik; Johansson, Linda C.; Andersson, Rebecka; Bosman, Robert; Carbajo, Sergio; Claesson, Elin; Conrad, Chelsie E.; Dahl, Peter; Hammarin, Greger; Hunter, Mark S.; Li, Chufeng; Lisova, Stella; Milathianaki, Despina; Robinson, Joseph S.; Safari, Cecilia; Sharma, Amit; Williams, Garth J.; Wickstrand, Cecilia; Yefanov, Oleksandr; Davidsson, Jan; DePonte, Daniel P.; Barty, Anton; Brändén, Gisela; Neutze, Richard

doi:10.1016/j.str.2017.07.002

Cited by 21 publications

(29 citation statements)

References 49 publications

(57 reference statements)

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“…Most XFEL experiments require several thousands to many millions of homogeneously sized protein crystals. These are often prepared by batch-crystallization approaches , including seeding to achieve homogeneity and control of crystal size (Nass et al, 2016;Coquelle et al, 2017;Dods et al, 2017). Given the small size of the crystals, light microscopy may not suffice to characterize the samples, and alternative/complementary characterization techniques may be required.…”

Section: Introductionmentioning

confidence: 99%

Sample delivery for serial crystallography at free-electron lasers and synchrotrons

Grünbein

Kovács

2019

Acta Cryst Sect D Struct Biol

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The high peak brilliance and femtosecond pulse duration of X-ray free-electron lasers (XFELs) provide new scientific opportunities for experiments in physics, chemistry and biology. In structural biology, one of the major applications is serial femtosecond crystallography. The intense XFEL pulse results in the destruction of any exposed microcrystal, making serial data collection mandatory. This requires a high-throughput serial approach to sample delivery. To this end, a number of such sample-delivery techniques have been developed, some of which have been ported to synchrotron sources, where they allow convenient low-dose data collection at room temperature. Here, the current sample-delivery techniques used at XFEL and synchrotron sources are reviewed, with an emphasis on liquid injection and high-viscosity extrusion, including their application for time-resolved experiments. The challenges associated with sample delivery at megahertz repetition-rate XFELs are also outlined.

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Section: Introductionmentioning

confidence: 99%

Sample delivery for serial crystallography at free-electron lasers and synchrotrons

Grünbein

Kovács

2019

Acta Cryst Sect D Struct Biol

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“…This has been demonstrated by rapid titration of the protein into precipitant solution [70,71] or careful mapping of the crystallization phase diagram as shown with ferric uptake transporter A (FutA) and flavin prenyltransferase UbiX [72]. Conditions from vapor diffusion can also produce high density of microcrystals by controlled evaporation to push the protein into supersaturation [73], multiple rounds of seeding [74], or by direct crushing of macrocrystals using seed beads [74,75]. Sample preparation of crystals grown in LCP (which is in batch) follows a similar principle [76].…”

Section: Serial Crystallography Sample Preparationmentioning

confidence: 99%

Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

Cheng¹

2020

Crystals

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The advent of the X-ray free electron laser (XFEL) in the last decade created the discipline of serial crystallography but also the challenge of how crystal samples are delivered to X-ray. Early sample delivery methods demonstrated the proof-of-concept for serial crystallography and XFEL but were beset with challenges of high sample consumption, jet clogging and low data collection efficiency. The potential of XFEL and serial crystallography as the next frontier of structural solution by X-ray for small and weakly diffracting crystals and provision of ultra-fast time-resolved structural data spawned a huge amount of scientific interest and innovation. To utilize the full potential of XFEL and broaden its applicability to a larger variety of biological samples, researchers are challenged to develop better sample delivery methods. Thus, sample delivery is one of the key areas of research and development in the serial crystallography scientific community. Sample delivery currently falls into three main systems: jet-based methods, fixed-target chips, and drop-on-demand. Huge strides have since been made in reducing sample consumption and improving data collection efficiency, thus enabling the use of XFEL for many biological systems to provide high-resolution, radiation damage-free structural data as well as time-resolved dynamics studies. This review summarizes the current main strategies in sample delivery and their respective pros and cons, as well as some future direction.

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“…The reaction center from Blastochloris viridis (molecular weight 142 kDa) was produced and purified as described previously (Wö hri et al, 2009;Dods et al, 2017). For crystallization, thawed protein was centrifuged (16 900g, 15 min), concentrated to 0.3 mM and mixed with microcrystal seeds (Dods et al, 2017) at a protein:seed ratio of 100:5. The seeded protein was mixed with monoolein (9.9 MAG) doped with 0.5%(v/v) ubiquinone-2 in a 40:60 protein:lipid ratio to form the LCP.…”

Section: Crystallization Of a Bacterial Reaction Centermentioning

confidence: 99%

“…Since then it has served as a model system for the study of photosynthetic reactions. Previous SFX experiments have been carried out with reaction-center crystals produced in lipidic sponge phase (Johansson et al, 2013), a swollen analog of LCP, and by the detergent-based vapor-diffusion method (Dods et al, 2017). However, these crystals need to be injected using a liquidbased injector such as the gas dynamic virtual nozzle system, and are thus not suitable for experiments at synchrotrons, where a slower flow rate is required.…”

Section: Well Diffracting Microcrystals Of a Bacterial Reaction Centermentioning

confidence: 99%

Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments

Andersson¹,

Safari²,

Båth³

et al. 2019

Acta Cryst Sect D Struct Biol

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Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.

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From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography

Cited by 21 publications

References 49 publications

Sample delivery for serial crystallography at free-electron lasers and synchrotrons

Sample delivery for serial crystallography at free-electron lasers and synchrotrons

Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments

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