Electrically stimulated droplet injector for reduced sample consumption in serial crystallography

15 pags., 6 figs., 1 tab.With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its "off-time" between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.Financial support from the STC Program of the National Science Foundation through BioXFEL under agreement no. 1231306, NSF ABI Innovations award no. 1565180, and the National Institutes of Health award no. R01GM095583 is gratefully acknowledged. The use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is generously supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The HERA system for in-helium experiments at MFX was developed by Bruce Doak and funded by the Max Planck Institute for Medical Research. This work was also supported by The Center for Structural Dynamics in Biology, NIH grant P41GM139687.Peer reviewe

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[2] Principle: In isocratic HPLC the analyte is forced through a column of the stationary phase (usually a tube packed with small round particles with a certain surface chemistry) by pumping a liquid (mobile phase) at high pressure through the column. The sample to be analyzed is introduced in a small volume to the stream of mobile phase and is retarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. Nature of the analyte, stationary phase and mobile phase composition accounts for the retardation. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time and is considered a reasonably unique identifying characteristic of a given analyte. Pressure increases the linear velocity (speed) of the component providing it less time to diffuse within the column, which leads to improved resolution in the resulting chromatogram. Any miscible combinations of water or various organic liquids (the most common are methanol and acetonitrile) serves as the solvent system. Water may contain buffers or salts to assist in the separation of the analyte components, or compounds such as Trifluoroacetic acid which acts as an ion pairing agent In spite of being considered somewhat mature, new developments in HPLC still continue. There have been improvements in column construction, packing material design, bonded phase chemistry and formats. In addition to this, new phases have extended the pH range (high and low), providing it more versatility. [3] Instrumentation: The heart of a HPLC system is the column. The column contains the particle that contains the Stationary phase. Pump forces the mobile phase to pass through the column and injector injects the mixture to be separated into the flowing mobile phase. Molecules that are adsorbed maximum by stationary phase are eluted slowest through the column, whereas the molecules which are High Performance Liquid Chromatography (HPLC) is a highly improved form of column chromatography in which instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres(500-5000 p.s.i). The sensitivity and range of the technique depends on the choice of column and on the efficiency of the overall system. Column technology has seen great developments over the years. The transition from large porous particles and pellicular materials to small porous particles occurred in the early 1970s, when micro particulate silica gel (10 -mm dp) came into light and appropriate packing methods were developed. Monolithic columns are a promising alternative to packed columns in the future but much of the research is yet to be done. The use of CAPILLARY COLUMNS (4 mm) has increased in recent years, in part because small-diameter columns use much less solvent and provide higher sensitivity. While speaking of the future of packaging development, silica gel with chemically bonded phases will be around for a long time. The field is a promising one with great scope for research and development

Electrically stimulated droplet injector for reduced sample consumption in serial crystallography

With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its “off-time” between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time

Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme

18 pags. 7 figs., 1 tab. -- This article is part of the themed collection: Lab on a Chip HOT Articles 2023Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS). Under optimized droplet injection conditions, we demonstrate that up to 4-fold sample consumption savings can be achieved with the droplet injector. In addition, we collected a full data set with droplet injection for NQO1 protein crystals with a resolution up to 2.7 Å, leading to the first room-temperature structure of NQO1 at an XFEL. NQO1 is a flavoenzyme associated with cancer, Alzheimer's and Parkinson's disease, making it an attractive target for drug discovery. Our results reveal for the first time that residues Tyr128 and Phe232, which play key roles in the function of the protein, show an unexpected conformational heterogeneity at room temperature within the crystals. These results suggest that different substates exist in the conformational ensemble of NQO1 with functional and mechanistic implications for the enzyme's negative cooperativity through a conformational selection mechanism. Our study thus demonstrates that microfluidic droplet injection constitutes a robust sample-conserving injection method for SFX studies on protein crystals that are difficult to obtain in amounts necessary for continuous injection, including the large sample quantities required for time-resolved mix-and-inject studies.Financial support from the STC Program of the National Science Foundation through BioXFEL (under agreement # 1231306), ABI Innovations award (NSF # 1565180), IIBR award (# 1943448), MCB award (1817862), and the National Institutes of Health award # R01GM095583 is gratefully acknowledged. The use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is generously supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract # DE-AC02-76SF00515. The authors would like to acknowledge the instrument group and facility staff for their assistance in the use of the MFX instrument during proposal MFXLW7919 at LCLS. The HERA system for in-helium experiments at MFX was developed by Bruce Doak and funded by the Max Planck Institute for Medical Research. This work was also supported by The Center for Structural Dynamics in Biology, NIH grant P41GM139687. Alice Grieco and Jose M. Martin-Garcia were supported by the “Ayuda de Atracción y Retención de Talento Investigador” from the Community of Madrid, Spain (REF: 2019-T1/BMD-15552). JLPG and ALP acknowledge funding from the ERDF/Spanish Ministry of Science, Innovation, and Universities—State Research Agency (grant RTI2018-096246-BI00), Consejería de Economía, Conocimiento, Empresas, y Universidad, Junta de Andalucía (grant P18-RT-2413), and ERDF/Counseling of Economic transformation, Industry, Knowledge, and Universities (grant B-BIO-84-UGR20).Peer reviewe

Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser.

NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance

Co-flow injection for serial crystallography at X-ray free-electron lasers.

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macro-molecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX

Co-flow injection for serial crystallography at X-ray free-electron lasers

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macro­molecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX