KR102416509B1 - Polyacrylamide injection matrix for serial crystallography - Google Patents

Abstract

The present invention relates to X-ray crystallography sample delivery, and more particularly, to the development of a technology using polyacrylamide as a delivery material to reduce sample consumption in serial crystallography research.
Since the protein samples do not interact with PAM, as a result, the protein crystal sample also does not undergo a chemical reaction with PAM. This means that when conducting SFX studies, all protein crystal samples can use PAM as a shipping material. In addition, since there is no X-ray background scattering from the material, it can increase the experimental success rate by providing an excellent signal / noise ratio (SNR) in the data for which phasing is to be found in crystallography. As a result, it is possible to apply a variety of crystal samples compared to previously reported delivery materials, and to ensure excellent data quality.

Description

Polyacrylamide injection matrix for serial crystallography

The present invention relates to X-ray crystallography sample delivery, and more particularly, to the development of a technology using polyacrylamide as a delivery material to reduce sample consumption in serial crystallography research.

X-ray free-electron lasers (XFELs) are X-rays with unprecedented levels of brilliance, excellent spatial coherence, and femtosecond pulse durations. Serial femtosecond crystallography (SFX) using XFEL offers new opportunities to visualize high-resolution structures, which not only provide biologically more reliable crystal structures at room temperature compared to conventional radiation accelerators, but also provide new opportunities to visualize high-resolution structures. It provides molecular dynamics information through time-resolved studies using ultrafast X-ray pulses.

In the initial stage of SFX research, crystal samples were delivered to X-rays using a liquid jet sample injector using a gas dynamic virtue nozzle (GDVN) (Korea Patent Publication No. 2016). -0035783). Through this delivery method, we were able to successfully identify the high-resolution crystal structure. However, there was a problem in that a large amount of crystal samples was consumed because the samples were delivered at a high flow rate.

Accordingly, sample delivery methods such as electrospinning, lipidic cubic phase (LCP) injector, acoustic injector, or fixed-target scanning have been developed to reduce this sample consumption and deliver crystal samples as X-ray pulses. was developed These methods reduce the rate at which the sample exits the injector or increase the hit rate between the crystal sample and the X-ray pulse. Among the many ways to reduce sample consumption, shipping with a high-viscosity material dramatically reduced crystal sample consumption by lowering the sample flow rate in the injector.

Previously, monoolein, grease, agarose, carbohydrate-based materials and polymer-based delivery materials have been successfully used in protein crystal structure studies. However, these delivery media are often not usable for specific protein samples due to chemical reactions or physical effects. For example, when a lipid-binding protein or a protein such as cellulase is mixed with a monoolein or celluose-based delivery material, the protein crystal suspension may be weakened or dissolved by chemical reaction with the delivery material.

When conducting SFX studies using the delivery material as described above, (i) the stability of the crystal sample in the delivery material, and (ii) the minimization of the X-ray scattering intensity from the delivery material should be considered. Taking as an example the problems of materials previously reported as shipping materials, monoolein dissolves in the crystal sample when mixed with a lipid-binding protein crystal sample and is damaged, and X-ray scattering generated from monoolein around 4.5 Å when exposed to X-rays provides The grease-based delivery material has low uniformity of the material itself, and X-ray background scattering occurs in the low-resolution area similar to monoolein. In the case of carbohydrate-based substances, when mixed with a sugar-related crystal sample, the crystal sample is dissolved through a specific or non-specific reaction, thereby inhibiting the crystal stability. As a result, when the stability of the crystal sample is inhibited by a chemical reaction in the delivery material, the diffraction intensity of the crystal sample decreases or the crystal melts. In addition, if the background of X-ray scattering generated from the material is large, the quality of the research data is deteriorated.

An object of the present invention is to provide a composition for X-ray crystallography analysis, including polyacrylamide, which can overcome the above problems, and an X-ray crystallography analysis method using the composition.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a composition for X-ray crystallography analysis, comprising polyacrylamide.

In one embodiment of the present invention, the polyacrylamide is characterized in that the sample to be analyzed is delivered to X-rays.

In another embodiment of the present invention, the polyacrylamide is characterized in that it is in the form of a fragment.

In another embodiment of the present invention, the X-ray crystallography analysis is characterized in that the continuous crystallographic analysis.

In another embodiment of the present invention, the sample to be analyzed is a protein sample, and the protein sample is characterized in that it is lysozyme or thermolysin.

In addition, the present invention provides an X-ray crystallography analysis method using polyacrylamide as a delivery material, comprising the following steps:

preparing a polyacrylamide injection matrix by mixing polyacrylamide and a sample to be analyzed (S1); and

delivering the polyacrylamide injection matrix to X-rays (S2); and

Analyzing the crystal structure of the sample to be analyzed by collecting the diffraction image after the X-ray transmission (S3).

The present invention provides an X-ray crystallography analysis method using polyacrylamide as a delivery material, and more particularly, a continuous crystallographic analysis method using polyacrylamide as a delivery material, wherein the polyacrylamide of the present invention is a protein sample and Since there is no interaction, it has the advantage of stably delivering protein samples, and has minimized the intensity of X-ray scattering from the delivery material.

1 shows the process of preparing a PAM delivery material according to the present invention.
Figure 2 relates to a diffraction image of Lysozyme obtained using the PAM delivery material according to the present invention.
Figure 3 relates to an electron density map of Lysozyme obtained using the PAM delivery material according to the present invention.
Figure 4 shows a photograph confirming the crystals of Lysozyme, thermolysin and broken thermolysin.
5 relates to a diffraction image of thermolysin obtained using the PAM delivery material according to the present invention.
6 relates to an electron density map of thermolysin obtained using the PAM delivery material according to the present invention.
7 relates to a result of comparing the X-ray background scattering of PAM and monoolein in the present invention.

The present invention provides a polyacrylamide (PAM) delivery material that does not interact with protein crystal samples and has no X-ray background scattering. PAM is a non-toxic polymer (-CH2CHCONH2-) composed of acrylamide subunits. Cross-linked PAM is insoluble in water and has gel properties. PAM is widely used in electrophoresis to separate proteins, nucleic acids and other biomolecules, and exhibits stability over a wide range of pH intervals (pH, 3-11). In particular, since PAM does not have non-specific or specific binding interactions with proteins, it is possible in native gel electrophoresis. As a result, in the present invention, it is confirmed that SFX analysis can be performed using the newly developed PAM delivery material.

Since protein samples do not interact with PAM, as a result, protein crystal samples also do not undergo chemical reaction with PAM. This means that when conducting SFX studies, all protein crystal samples can use PAM as a shipping material. In addition, since there is no X-ray background scattering from the material, it can increase the experimental success rate by providing an excellent signal / noise ratio (SNR) in the data for which phasing is to be found in crystallography. As a result, it is possible to apply a variety of crystal samples compared to previously reported delivery materials, and to ensure excellent data quality.

Accordingly, the present invention comprises the steps of preparing a polyacrylamide injection matrix by mixing a composition for X-ray crystallography analysis, including polyacrylamide, polyacrylamide, and a sample to be analyzed (S1); delivering the polyacrylamide injection matrix to X-rays (S2); and analyzing the crystal structure of the sample to be analyzed by collecting the diffraction image generated after the X-ray transmission (S3).

The polyacrylamide of the present invention is utilized as a shipping material in crystallographic analysis, and the crystallographic analysis method is specifically continuous crystallographic analysis. However, it will be apparent to those skilled in the art that polyacrylamide is not limited to the above method as long as it is a crystallographic analysis used as a delivery material for a sample to be analyzed.

The polyacrylamide of the present invention may be used at a concentration of 10 to 50% (w/v), but is not limited thereto.

The polyacrylamide of the present invention may be mixed with a sample to be analyzed to form a polyacrylamide injection matrix. As in step S2, the polyacrylamide injection matrix may be delivered to the X-ray pulse through a sprayer equipped with a nozzle. The spraying may be performed by electrospray or the like, but is not limited thereto.

In the present invention, the polyacrylamide is characterized in that it is in the form of a fragment. The polyacrylamide of the present invention has a fragment form, thereby preventing physical damage to the sample to be analyzed. In order for the polyacrylamide of the present invention to have a fragment form, the PAM solution before polymerization is put into a syringe and left for about 10 minutes to remove heat, and then an empty syringe is connected to the syringe through a coupler, and then the plunger of the syringe is removed by 30 It was made into fragments by pressing back and forth more than once.

In the present invention, the sample to be analyzed may be a protein sample. The protein sample may be lysozyme or thrmolysin, but the protein sample is not limited thereto and can be used for SFX analysis.

In the present invention, the polyacrylamide may be mixed with at least one delivery material selected from the group consisting of monoolein, grease, and agarose, and the type of the delivery material is not limited.

Step S3 of the present invention is a step of analyzing the crystal structure of the sample to be analyzed by collecting the diffraction image generated after the X-ray transmission. A method of collecting and analyzing the scattering signal data generated after the X-ray transmission can be used without limitation through conventionally known methods.

In an embodiment of the present invention, SFX analysis was performed on a protein sample using polyacrylamide as a delivery material, and as a result of the analysis, it was confirmed that a clear structural analysis was possible without radiation damage.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

[Example]

Example 1. Preparation of Polyacrylamide Implanted Matrix for Continuous Femtosecond Crystallography

In the conventional PAM manufacturing process, ammonium persulfate and N,N,N',N'-tetramethylethylenediamine (TEMED) are added, and heat is generated during this polymerization process. The heat generated can potentially have a physical effect on the protein crystal lattice. In order to solve this problem, the PAM solution before polymerization was put in a syringe and left for 10 minutes to remove the heat from the polymerization home, as shown in FIG. 1A.

For the SFX experiment, a final 10% (w/v) PAM injection matrix was prepared and used by mixing 20% (w/v) PAM and a protein crystal solution 1:1.

20% (w/v) PAM has high strength, and when the crystal sample is directly mixed, it may physically damage the crystal, which may increase the mosiity of the crystal sample or decrease the diffraction intensity. Therefore, in order to prevent physical damage to the crystal sample during the delivery process, a PAM piece was first produced by repeating it 30 times or more in a syringe (FIG. 1b). Thereafter, the PAM fragment was transferred to one syringe and a crystal solution was put into the other syringe, and then the two syringes were connected using a coupler ( FIG. 1C ).

The PAM and the crystal solution were gently distributed over 20 times to evenly distribute the crystal sample within the PAM pieces (FIG. 1d). Thereafter, the PAM fragment including the crystal sample was used for the SFX experiment.

Example 2. Continuous Femtosecond Crystallography Using a Polyacrylamide Implanted Matrix

To prove the application of the developed PAM delivery material, an SFX study was performed using lysozyme and thermolysin as model samples. The lysozyme crystal size was 30 × 30 × 30 μm ³ and the crystal density was about 6.7 × 10 ⁷ /ml. A total of 25,603 images of lysozyme were collected at room temperature during the SFX experiment. During diffraction image collection, the total crystal volume used was 10 μl and 0.5 mg of lysozyme was used. An image containing 20 or more Bragg peaks was filtered using the peakfinder8 algorithm by Cheetah program to obtain 17,675 diffraction images (hit rate=69%). The diffraction images were indexed by CrystFEL, which converged the input cell parameters to default values with tolerances. A total of 11,936 indexed images (indexing rate = 67.52%) were obtained and are shown in FIG. 2 . The number of unique reflections was 14,007. The overall signal-to-noise ratio (SNR) was 4.32, indicating 100% completeness. The overall R _split and Pearson correlation efficiencies (CC*) were 15.31% and 99.2%, respectively (Table 1, data collection and structural analysis statistics).

Collected data Lysozyme Thermolysin Energy (eV) 9700 9692 Photons/pulse ~ 2 x 10 ¹¹ ~ 2 x 10 ¹¹ Pulse width 20 fs 20 fs space group P4 ₃ 2 ₁ 2 P6 ₁ 22 Cell dimensions a , b , c (Å) 78.61, 90.55, 73.42 92.66, 92.66, 128.59 No. collected diffraction images 25603 221595 No. of hits 17676 47865 No. of indexed images 11936 22213 No. of unique reflections 14007 (1353) 30878 (3028) Resolution (Å) 80.0-1.70 (1.76-1.70) 131.57-1.80 (1.86-1.80) completeness 100.0 (100.0) 100.0 (100.0) Redundancy 965.3 (676.7) 663.8 (471.0) I/σ(I) 4.32 (1.39) 4.67 (1.16) R _split 15.31 (73.37) 14.63 (84.09) CC 0.970 (0.644) 0.975 (0.617) CC* 0.992 (0.885) 0.993 (0.874) Wilson B factor (Å ² ) 38.88 33.18 Refinement statistics Resolution (Å) 26.04 - 1.70 38.3 - 1.80 R _factor / R _free (%) 19.61 / 21.74 17.27 / 20.24 B-factor (Averaged) Protein 45.32 37.66 Metal 45.80 29.57 Water 46.20 41.00 Product 40.22 R.m.s. deviations Bond lengths (Å) 0.008 0.011 Bond angles (°) 0.94 1.01 Ramachandran plot (%) favored 97.64 96.82 allowed 2.36 3.18

The resolution of the lysozyme structure was 1.7Å, and the R _work and R _free were 19.61% and 21.74%, respectively. The electron density map was very clear when interpreting amino acids from Lys1 to Leu129. Catalytic amino acids (Glu35 and Asp52) in the active site of lysozyme were clearly observed (Fig. 3 below).

In addition, as the disulfide bond of the crystal structure was clearly observed, it was confirmed that the structure was observed without radiation damage. Comparing the structures using PAM with the lysoyzme structures previously elucidated using a GDVN liquid jet injector (PDB code 4ET8) and a drip syringe (5DM9) at room temperature, the r.m.s.deviation was <0.1691 Å for all Cα atoms.

4 shows the crystal structures of lysozyme, thermolysin and broken thermolysin. The thermolysin crystal size was about 8 × 8 × 200 μm ³ and the crystal density was about 3.6 × 10 ⁷ /ml (Fig. 4(b)). Therefore, using a pipette tip in a microcentrifuge tube, the size of the crystal was made to be about 8 × 8 × 8 μm ³ and used in the experiment. The data processing progress of thermolysin was the same as that of lysozyme. A total of 228,304 thermolysin images were collected at room temperature. During diffraction image collection, 1.0 mg of themolysin was used. A total of 47,865 diffraction images (hit rate=20.96%) were collected (FIG. 5). The number of unique reflections was 30,878. The overall signal-to-noise ratio (SNR) was 4.67, indicating 100% completeness. The overall R _split and Pearson correlation efficiencies (CC*) were 14.63% and 99.3%, respectively (Table 1). The resolution of the thermolysin structure was 1.8 Å, and R _work and R _free were 17.27% and 20.24%, respectively. The electron density map was very clear in the interpretations from Ile1 to Lys316. Zinc ions in the active site of Thermolysin were coordinated by His142, His146 and Glu166 (Fig. 6). Calcium ions directly involved in the activity were observed without radiation damage. Previously, a structure using PAM with a nano flow injector (PDB code 4OW3) at room temperature, an acoustic injector for drop-on-demand (5HQD), and thermlysin structure using synthetic grease super lubricant (5WR2) and hydroxyethyl cellulose (5WR3) were described. By comparison, the rmsdeviation was 0.1902-0.3439 Å for all Cα atoms.

Example 3. Background Scattering Measurements

The X-ray scattering signal is generated from the transmission medium as well as the diffraction signal from the crystal sample. Minimizing the X-ray scattering generated from the transmission medium plays an important role in improving the SNR of the diffraction pattern during data processing. Transmission media for SFX have so far provided unique X-ray scattering signals for specific angles. Analysis of this background scattering provides important information for the selection of shipping materials taking into account the diffraction limits of the crystal sample. X-ray scattering measurements of monoolein and PAM, which are widely used in SFX studies, were compared. 10% (w/v) PAM and 60% (v/v) monoolein were analyzed by extracting only diffraction images using X-rays of about 2 × 10 ¹¹ photon/pulse. The average scattering intensity was obtained from 1,000 images considering the intensity fluctuation of XFEL, and the scattering signals from PAM and monoolein were analyzed as 1.6 Å at the beam center (Fig. 7).

The PAM showed diffuse scattering at a resolution of 3.2 Å, which is considered to originate from the solvent containing water and not from the PAM matrix (Fig. 7a). The average maximum scattering intensity from the solvent was about 275 ADU. On the other hand, monoolein showed diffuse scattering at a resolution of 4.5 Å, which is believed to be derived from the lipid chain (Fig. 7b). The average maximum scattering intensity from monoolein was about 510 ADU. Therefore, PAM in the maximum scattering region showed lower background scattering than monoolein (Fig. 7c).

Materials and methods used in Examples of the present invention are described in more detail below.

<Materials and Methods>

Lysozyme and thermolysin preparation

Lysozyme was purchased from Hampton Research (Cat No. HR7-110). Protein powder was dissolved in a buffer containing 10 mM Tris-HCl, pH 8.0 and 200 mM NaCl. Lysozyme solution (200 μl; 50 mg/ml) was mixed with a crystallization solution (200 μl) containing 0.1 M sodium acetate, pH 4.0, 6 % (w/v) polyethylene glycol 8000 and 10 % (w/v) mM NaCl (200 μl) and 1.5 mL Mix using a vortex machine at 3000 rpm for 10 seconds in a microcentrifuge tube. After incubation at 20° C. for 4 hours, the supernatant of the mixture solution was removed to avoid crystal size growth. In addition, crystal samples were stored in a solution containing 0.1M sodium acetate (pH 4.0) and 0.2M NaCl.

Thermolysin from Bacillus thermoproteolyticus was also purchased from Hampton Research (Cat No. HR7-098). Protein powder was stored in deionized water for 20 min. NaOH (0.1M) was added, and a final thrmolysin (25 mg/mL) solution was prepared as a 50 mM NaOM solution. The protein solution was exchanged with a solution containing 10 mM Tris-HCl (pH 8.0) and 200 mM NaCl using a Centricon-30 YM filter (Millipore). The protein solution (100 mg/mL) showed microcrystals 2 months after incubation at 4 °C.

Manufacture of polyacrylamide

Acrylamide/bis-acrylamide [500 μl; 20% (v/v)] solution, ammonium persulphate (5 μl; 15%), and TEMED (0.5 μl) were mixed in a microcentrifuge tube, and PAM solution (50 μl) was transferred with a Hamilton syringe (81065). -1710RNR). After polymerizing the PAM solution in the syringe, the empty syringe was connected to the syringe using a coupler. The syringe plunger was moved back and forth 30 times to make the PAM piece, causing the PAM to break through the narrow inner diameter of the coupler. The PAM piece was transferred to one syringe, and the opposite syringe containing the crystal suspension (50 μl) was prepared. The PAM pieces and crystal suspensions were mixed by gently moving the plunger back and forth. The final proportion of crystal pellets was about 20-25% of the total injection matrix. The crystal sample mixed with the PAM pieces was transferred to a carrier matrix deliver (CMD) injector for SFX analysis.

data collection

X-ray crystal diffraction data using a PAM implanted matrix were collected at the Nano Crystallography and Coherence Imaging (NCI) laboratory of PAL-XFEL. The X-ray energy was 9.7 keV (1.2782 Å) with a pulse energy of about 500 μJ, provided at 30 Hz. Each X-ray pulse contained 1-2 × 10 ¹¹ photons, and the interval between the X-ray pulses was 20 fs. X-ray pulses were focused at 5 × 5 μm ² (FWHM) by a Kirkpatrick-Baez mirror. Samples were subjected to data collection in an atmospheric (lysozyme) or helium (thermolysine) environment at room temperature in a MICOSS (various injection chamber for molecular structure study) system. Diffraction data were collected in the pixel of the MX225-HS (Rayonix, LLC) detector in 4×4 binning mode (pixel size: 156 × μm × × 156).

Data processing and structure determination

The diffraction pattern was monitored in real time by the OnDA program. The hit image was filtered by Cheetah program. The diffraction images were indexed, scaled and merged by the CrystFEL program. The phase problem of lysozyme was solved by molecular substitution using PHENIX's Phaser-MR as a search model for lysozyme (PDB code 4ET8). The phase of thermolysin was obtained using PHENIX's Phaser-MR using thermolysin (PDB code 5DM9) as a search model. Model building and refinement were performed using PHENIX's Coot and Phenix.refinement, and geometrical analysis was performed using MolProbity. Figures were generated by PyMOL (https://pymol.org/).

Chemical compatibility test

Chemical compatibility studies between PAM and crystallization precipitants [(NH ₄ ) ₂ SO ₄ , NaCl, PEG 400, PEG 1000, PEG 4000, 2-methyl-2,4-pentanediol (MPD)] were performed at room temperature. A solution containing 0.2 M Tris-HCl (pH 7.5) was used to produce a final 10% (w/v) or 15% (w/v) PAM. PAM (50 μl) and each precipitant (50 μl) were mixed using two combined Hamilton syringes. The viscosity and injection stream properties of the mixture were visually judged based on the extruded behavior. The mixture was manually extruded from a Hamilton syringe through a needle with a 168 μm inner diameter (ID).

access code

Coordinates and structural factors have been deposited with the Protein Data Bank under the accession codes 6IG6 (lysozyme) and 6IG7 (thermolysin). Diffraction images were stored in CXIDB as ID84 (lysozyme) and 85 (thermolysin).

Although preferred embodiments of the present invention have been described in detail above, the rights of the present invention are not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims are also provided. is within the scope of the right.

Claims (9)

containing polyacrylamide;
The polyacrylamide is included in a concentration of 10 to 50% (w/v), and the polyacrylamide is to deliver the sample to be analyzed to X-rays, the composition for X-ray crystallography analysis.
delete According to claim 1,
The polyacrylamide is in the form of a fragment (fragment), the composition for X-ray crystallography analysis.
According to claim 1,
The X-ray crystallography analysis is a continuous crystallographic analysis, the composition for X-ray crystallography analysis.
According to claim 1,
The sample to be analyzed is a protein sample, wherein the protein sample is lysozyme or thrmolysin, the composition for X-ray crystallography analysis.
An X-ray crystallography analysis method using polyacrylamide as a shipping material, comprising the steps of:
preparing a polyacrylamide injection matrix by mixing polyacrylamide and a sample to be analyzed (S1); and
delivering the polyacrylamide injection matrix to X-rays (S2); and
Analyzing the crystal structure of the sample to be analyzed by collecting the diffraction image after the X-ray transmission (S3).
7. The method of claim 6,
Wherein the X-ray crystallography analysis is a continuous crystallographic analysis, X-ray crystallography analysis method.
7. The method of claim 6,
The polyacrylamide is in the form of a fragment (fragment), X-ray crystallography analysis method.
7. The method of claim 6,
The sample to be analyzed is a protein sample, wherein the protein sample is lysozyme or thrmolysin, X-ray crystallography analysis method.

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