The authors would like to thank BESSY II, operated by Helmholtz-Zentrum Berlin for beam time access and support, and the departments of Sample Environment and Technical Design for their help with design and construction and the access to the 3D-printer facilities.

CAUTION: For all subsequent work, it is very important that the yellow-colored polyimide foil must not be touched with unprotected fingers, because of possible contaminations to the sample holder. Also, the usage of protected forceps is highly recommended.
1. The sample holder
<ol>
	<li>Use one of the three types of sample holder. 
	NOTE: Three different versions of the new developed sample holder are shown in Figure 1. All of them contain a black plastic support structure, an airtight COC foil on the outside and a microporous structured polyimide foil on the inside. Type 1 (Figure 1A) contains a fixed outer plastic ring, whereas for types 2 and 3 (Figure 1B,1C) the outer ring can be broken off mechanically at the designated respective break points for use in automated sample transfer systems (see red arrows in Figure 1B). The design of the sample holders allows the setup of multiple crystallization drops on the yellow polyimide foil. It does not compromise the monitoring of the crystallization experiment, as the material is highly transparent for visible light. The 21 &#956;m thick polyimide foil also features 5 &#956;m pores, which allows simple crystal manipulation by soaking later on. Since the transmission of X-rays is close to 1.0 at all commonly used diffraction data collection energies in macromolecular crystallography, the contribution of the foil to the background scattering in a diffraction experiment is negligible28.</li>
</ol>
2. Setting up crystallization drops
<ol>
	<li>Create a clean and dust-free surface using a damp lint-free cloth. Take one sample holder from its box and gently place it, yellow foil facing up, on the cleaned surface to avoid damage or unwanted puncture of the backside COC foil.</li>
	<li>Set up the crystallization drops with a maximum recommended volume of 2 &#181;L on the yellow foil as it would be done on commonly used cover slides. Place the drops gently to avoid any rupture or piercing of the foil using a pipette. On a sample holder of type 1 (Figure 2A) up to three drops can be placed, whereas on sample holders of type 2 and 3 two drops are the recommended maximum (Figure 2C).</li>
	<li>Flip the sample holder over and place it onto a pre-greased cavity of a 24-well Linbro style plate. Use the positioning aids (see red arrows in Figure 1A) of the sample holder to guide it to its optimal position.</li>
	<li>Ensure the correct position of the sample holder in order to avoid unwanted evaporation (Figure 2A).</li>
</ol>
3. Observing crystal growth
<ol>
	<li>By placing the crystallization plate under a transmission light microscope, with or without polarizers, monitor crystal growth without any disturbance of the experiment (Figure 4).</li>
	<li>When using the smaller 18-mm sample holders of type 3 (Figure 1C), which were designed for use on SBS footprint plates, use an imaging robot capable of handling SBS-footprint plates to monitor the crystal growth in a more automated way.</li>
</ol>
4. Crystal manipulation
NOTE: It is recommended to perform all subsequent steps under a transmission light microscope.
<ol>
	<li>Cryo-protection
	<ol>
		<li>Gently pierce the outer COC foil using a fine cannula. Make sure the inner yellow foil remains untouched. The puncture should be right next to the drop that is to be manipulated (Figure 3A,3C).</li>
		<li>Use a fine paper wick and insert it in the poked hole. Carefully push the wick forward until it touches the yellow polyimide foil. Keep the wick in contact with the perforated foil. The wick will suck away all excess solution. The time required for complete liquid removal depends on the viscosity of the solutions and the mother liquor composition (Figure 3B).</li>
		<li>After all liquid is sucked away, gently retract the paper wick. Remember the position of the drop, since it may not be visible after removal of the mother liquor.</li>
		<li>Take a standard pipette to apply a small volume of cryo-protectant solution, max. 3 &#181;L, using an extruded tip (e.g., a gel loading tip) through the same hole. Once the liquid is dispensed, retract the tip. The porosity of the yellow foil allows for diffusion across the foil. The time to attain cryo-protection of your crystals highly depends on the employed solution and its components.</li>
		<li>To reseal the self-healing COC foil, gently place a protected finger on the hole for about 1 s and slide it across the puncture. The slight pressure in combination with the elevated temperature will promote the resealing of punctures, which are not too large.</li>
	</ol>
	</li>
	<li>Ligand soaking 
	 
	NOTE: Excess mother liquor may be removed before ligand soaking. To do so, follow the steps described in 4.1.1 to 4.1.3.
	<ol>
		<li>Dissolve the ligand in mother liquor in the desired concentration in a reaction tube.</li>
		<li>Spin the solution for 10 minutes at 12,000 x g in order to remove insoluble particles. Use a temperature-controlled centrifuge if needed.</li>
		<li>Gently place a volume of max. 3 &#181;L of ligand containing solution in the gap between the COC foil and the polyimide film using a long, extruded pipet tip. Retract the tip.</li>
		<li>To reseal the self-healing COC foil, gently place a protected finger on the hole for about 1 s and slide it across the puncture (see also 4.1.5).</li>
		<li>Incubate the experiment for some time in order to allow for diffusion across the membrane. The soaking time highly depends on the viscosity of the diffusing solution and its components29.</li>
		<li>Repeat steps 4.2.1 to 4.2.5 multiple times to subsequently soak different ligands.</li>
	</ol>
	</li>
</ol>
5. In situ diffraction data collection at ambient temperature
NOTE: In order to minimize solvent scattering, remove excess solution before data collection.
<ol>
	<li>Ensure a stable humidity controlled beamline environment with pre-established conditions30.</li>
	<li>Gently lift the transparent COC foil at the designated point using forceps and peel it off like one would remove the lid from a yogurt cup ( Figure 6B).</li>
	<li>Gently lift the sample holder from its cavity and insert it immediately into a pre-prepared magnetic sample holder base. No glue is necessary for this step (Figure 6B).</li>
	<li>Apply gentle pressure to ensure the correct positioning of the sample holder within the base.</li>
	<li>Mount the sample holder on a beamline goniometer and ensure correct positioning of the holder. Depending on goniometer geometry the sample holder can be rotated by up to 160&#176; without causing any shadowing during the diffraction experiment.</li>
	<li>Use a paper wick and gently touch the yellow polyimide foil from the backside to remove excess mother liquor. Please note, that at that stage ligand soaking or cryo-protection may be performed just as well. The sample is now ready for centering and diffraction data collection.</li>
	<li>When using a sample holder with removable outer ring, apply gentle pressure by holding on to the outer ring and break it off at the designated break points (Figure 6C). The sample is now ready for centering and diffraction data collection.</li>
</ol>
6. In situ diffraction data collection at cryogenic temperature
NOTE: It is recommended to remove residual mother liquor from the sample by performing the steps 4.1.1. to 4.1.3. before continuing with the next steps to minimize solvent scattering. Most samples may be transferred to liquid nitrogen without prior cryo-protection31. If cryo-protection is needed, see steps 4.1.1. to 4.1.5.
<ol>
	<li>Gently lift the COC foil at the designated point using a forceps and peel it off (see step 5.1.2) (Figure 6A).</li>
	<li>Take the sample holder off the cavity and mount it on a magnetic sample holder base. Gentle pressure may be applied in order to ensure correct and tight fitting (see step 5.1.5, Figure 6B). 
	NOTE: The symmetrically arranged designated break points allow for simple removal of the outer ring of the sample holder by applying gentle pressure (see step 5.1.8., Figure 6C). Now, the sample holder is ready and can be plunged into liquid nitrogen. The geometry of the sample holder types 2 and 3 (Figure 1B,1C) allows their transfer into standard SPINE sample vials, which can be used for robot assisted sample mounting (Figure 6D).</li>
</ol>

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</ol>

Patent applications regarding the reported sample holder have been filed by Helmholtz-Zentrum Berlin with the following registration numbers and registration dates with the German Patent and Trademark Office: DE 10 2018 129 125.6, registration date November 20th, 2018; DE 10 2018 125 129.7, registration date October 11th, 2018; DE 10 2017 129 761.8, registration date December 13th, 2017. A subsequent international patent application via the PCT route, using the priority of DE 10 2017 129 761.8 has been filed, PCT/DE2018/101007. A registration of a utility model with the number DE 20 2018 106 955.1 was filed on December 6th, 2018. The sample holder has been made commercially available under the trade names XtalTool and XtalTool/HT by Jena Bioscience, Jena, Germany.

Suitability for crystallization experiments. The new sample holders can be used for standard hanging drop crystallization experiments using either 24-well Linbro type plates (types 1 and 2), or 24-well SBS footprint plates in which each well has a diameter of 18 mm (type 3). They can be used instead of the standard microscope cover slips. The amorphous COC foil ensures the airtightness of the system. The monitoring of the crystallization experiment is possible using a transmission light microscope, because of the use of high clarity foils. To the best of our knowledge, no other sample holders exist for 24-well crystallization plates, which would allow crystal manipulation or diffraction experiments, without mechanically removing the crystal from the drop, in which it is grown. This is of particular importance, since many researchers in the field still rely on such plates for crystal optimization, due to the fact that larger drop volumes can be used compared to 96-well sitting-drop plates. With these larger drop volumes, larger crystals may be obtained.
Suitability for crystal manipulation. Due to the self-healing properties of the outer COC foil and the microporous structure of the inner yellow polyimide foil, the crystal environment is accessible and the crystals can be manipulated without mechanically transferring them to other containers. This makes the sample holders very convenient. The only other system we know of, which allows this indirect and gentle access to the crystal, is the CrystalDirect system26. However, CrystalDirect is less flexible since special 96-well crystallization plates have to be used. The foil, on which the crystals are growing, is the same that seals the crystallization experiment and it is not self-healing. This means that an aperture that has been pierced into the foil by laser ablation for ligand or cryo-protectant delivery to the crystals will remain open, increasing the chance for liquid evaporation. This is&#160;in contrast to our design, where&#160;crystals will not be directly exposed to the environment even if the COC foil gets pierced a number of times.
Suitability for in situ diffraction experiments at ambient temperature. The sample holder can be removed from the crystallization plate in a straight-forward manner, stuck onto a magnetic base and put on a beamline goniometer. For a diffraction experiment at room temperature, it is advisable to put the sample into an air stream of defined humidity33. The mother liquor around the crystal may be removed prior to putting the sample holder on the goniometer in order to reduce the background scattering. Such a set-up is stable for hours.
Suitability of the used material for operation and storage at 100 K. Neither the material used for the production of the sample holder nor the polyimide film are adversely affected by cooling them down to low temperatures34. Hence, working with the sample holder at low temperature (e.g., 100 K) does not pose a serious problem.
Suitability for in situ diffraction experiments at 100 K. For data collection at 100 K in a nitrogen stream, the sample holder needs to be removed from the crystallization plate as in the previous paragraph, stuck onto a magnetic base and put into a gaseous nitrogen stream at 100 K on a beamline goniometer. If desired, the sample may also be cryo-protected, although it is likely that for naked samples this may not be necessary in most cases31. For experiments at 100 K, the sample holders type 2 and 3 are better suited because the outer plastic ring can be removed. Hence, they are of smaller size and should therefore be less prone to icing. However, even a sample holder of type 1 may be used. Given a not too high humidity in the experimental hutch and a properly aligned cryo-system icing up of the holder is not really a problem.
Limitations. The sample holder&#39;s geometry permits unobstructed diffraction data collection by the rotation method over a total rotation range of 160&#176;. This is sufficient so that complete diffraction data sets can be obtained for most crystal systems. In cases where this is not possible, data from more than crystal need to be merged together. When crystals are grown together, it may be possible to adjust the size of the incident X-ray beam so that only parts of individual crystals are exposed. In extreme cases, one may need to resort to a data collection strategy similar to the MeshAndCollect approach35. In summary, while there are certain limitations associated with the sample holders, these can be overcome in most cases. Of course, it is always possible that situations are encountered, in which none of this is possible. In such cases, one may need to resort to other crystal mounting methods.
We have described a novel type of sample holder for macromolecular crystallography and we have demonstrated the suitability of the sample holders for various applications. Taking into account the simple and reproducible handling of protein crystals, as well as the unique properties of the sample holders, we believe that these sample holders will prove to be a valuable addition to the arsenal of sample holders for macromolecular crystallography.

The knowledge of the three-dimensional structure of biological macromolecules constitutes an important cornerstone in all basic biological, biochemical and biomedical research. This even extends to certain translational aspects of such research, such as for instance drug discovery. Among all methods for obtaining such three-dimensional information at atomic resolution X-ray crystallography is the most powerful and the most prominent one as is evidenced by the fact that 90% of all available structural information is contributed by X-ray crystallography1. The major prerequisite of X-ray crystallography, which is at the same time its major limitation, is that diffraction-quality crystals have to be produced and prepared for the diffraction experiment. This step still constitutes one of the major bottlenecks of the method.
Historically, diffraction data from protein crystals were collected at ambient temperature. Individual crystals were carefully transferred into glass or quartz capillaries prior to data collection, mother liquor was added to the capillaries so that the crystals would not dry out and the capillaries were sealed2,3,4. Since the 1980s, it became more and more apparent that due to the ionizing properties of X-radiation and the imminent radiation sensitivity of macromolecular crystals, data collection at ambient temperature poses severe limitations on the method. Consequently, approaches were developed to mitigate radiation damage effects by cooling macromolecular crystals down to 100 K and to collect diffraction data at such low temperature5,6. For working at low temperatures, the mounting of the samples in capillaries became impractical due to the low rate of heat transfer. In spite of this, there are ongoing efforts to also use capillaries, in particular from counter-diffusion crystallization experiments, for low-temperature diffraction work7,8, but, irrespective of that, it became the standard approach in macromolecular crystallography to mount macromolecular crystals held by a thin film of mother liquor inside a thin wired loop9,10. Even though a number of improvements (e.g., the introduction of lithographic loops and similar structures11) have been made over time to this loop-based mounting, the basic principles that were developed in the early 1990s are still in use today. It may be safely stated that most diffraction data collections on macromolecular crystals nowadays still rely on this approach5.
Over time, there were some interesting new developments and modifications of the loop-based mounting method, but these approaches have so far not been widely adopted in the community. One is the so-called loop-less mounting of crystals, which was developed to achieve lower background scattering12,13,14. Another one is the use of graphene sheaths to wrap the crystalline samples and to protect them from drying out. Graphene is a well-suited material in that respect because of its very low X-ray scattering background15.
More recently, developments in the field of sample mounts were mainly focused on standardizing the mounts with the aim of increasing sample throughput16 or on designing mounts, which can hold more than one sample17, such as for instance patterned membranes on a silicon frame, which are capable of holding hundreds of small crystals mostly in the field of serial crystallography18,19,20,21,22.
All of the sample mounting methods discussed so far still require some degree of manual intervention, which means that there is an inherent danger of causing mechanical damage to the sample. Therefore, novel approaches are being sought by engineering the sample environment such that diffraction data of crystals can be collected within their growth environment. One such method is termed in situ or plate-screening23,24 and it is already implemented at a number of macromolecular crystallography beamlines at various synchrotron sources worldwide25. However, the use of this method is limited by the geometrical parameters of the crystal plate and the space available around the sample point of the instrument.
Yet another approach is realized in the so-called CrystalDirect system26. Here, entire crystallization drops are harvested automatically. The foils on which the crystals have been grown are custom-cut using a laser and directly used as the sample holder27.
In the work described here, the aim was to develop a sample holder, which would allow a user to move the crystalline sample from its growth chamber to the data collection device without touching it and which would enable the user to manipulate the sample easily. Since many researchers in the field of macromolecular crystallography are still using the 24-well crystallization format for optimizing crystal growth by modifying conditions identified in large screening campaigns, the new sample holder was designed to be compatible with this format. In the following, the design of the new sample holder will be described and the handling and the performance of the sample holder for in situ data collection and ligand soaking will be demonstrated. Finally, the suitability of this new sample holder as well as its limitations for the various work steps will be discussed.

<table><tbody><tr><td>AF Satetiss</td><td>RS Components</td><td>101-5738</td><td>lint-free paper, multiple retailer</td></tr><tr><td>Cannula</td><td>Dispomed Neoject</td><td>25 G 5/8&quot; 0.5 x 16, Ref:10026</td><td>multiple retailer</td></tr><tr><td>COC foil</td><td>HJ-Bioanalytik GmbH</td><td>900360</td><td></td></tr><tr><td>ComboPlate</td><td>Greiner Bio-one / Jena Bioscience</td><td>662050 / CPL-131</td><td>pre-greased plate, multiple retailer</td></tr><tr><td>Cryo Vials</td><td>Jena Bioscience</td><td>CV-100</td><td></td></tr><tr><td>Eppendorf Research Plus&amp;nbsp;</td><td>Eppendorf</td><td>3123000012</td><td>0.1 - 2.5 &amp;micro;L volume</td></tr><tr><td>Eppendorf Tubes</td><td>Eppendorf</td><td>30125150</td><td>1.5 mL g-Safe Eppendorf Quality, manufacturer reference number</td></tr><tr><td>Forceps Usbeck</td><td>FisherScientific</td><td>10750313</td><td></td></tr><tr><td>GELoader Eppendorf Quality</td><td>Eppendorf</td><td>30001222</td><td>extruded&amp;nbsp; tips (0.2 - 20 &amp;micro;L), manufacturer reference number</td></tr><tr><td>Magnetic CryoVials</td><td>Molecular Dimension</td><td>MD7-402</td><td></td></tr><tr><td>Microfuge Thermo</td><td>ThermoFisher Scientific</td><td>R21</td><td></td></tr><tr><td>Paper wicks</td><td>dental2000</td><td>64460</td><td>Set of paper wicks, multiple retailer</td></tr><tr><td>Rotiprotect Nitril-eco&amp;nbsp;</td><td>Carl Roth</td><td>TC14.1</td><td>powder free, multiple retailer</td></tr><tr><td>SuperClear Plates</td><td>Jena Bioscience</td><td>CPL-132</td><td>pre-greased plate</td></tr><tr><td>UHU super glue</td><td>UHU GmbH &amp;amp; Co KG</td><td>45545</td><td>manufacturer reference number, multiple retailer</td></tr><tr><td>VeroBlackPlus</td><td>Alphacam</td><td>OBJ-40963</td><td>manufacturer reference number</td></tr><tr><td>XtalTool&amp;nbsp;</td><td>Jena Bioscience</td><td>X-XT-101</td><td>sample holder set</td></tr><tr><td>XtalTool HT</td><td>Jena Bioscience</td><td>X-XT-103 / X-XT-104</td><td>SPINE compatible sample holder set</td></tr><tr><td>XtalToolBases</td><td>Jena Bioscience</td><td>X-XT-105</td><td>Magnetic sample holder bases set</td></tr></tbody></table>

an all-in-one sample holder for macromolecular x-ray crystallography with minimal background scattering

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

The sample holder type 1 has been designed so that it fits onto a well of a 24-well Linbro style plate. Each individual sample holder contains positioning aids on either side of the outer rim in order to ensure optimal positioning on the rim of the well (Figure 1A, Figure 2A). Up to three individual crystallization drops of maximum volume 2 &#181;L each can be placed onto the yellow polyimide foil (Figure 2B). For sample holders of types 2 and 3, it is recommended to set a maximum of two drops of maximum volume 2 &#181;L each. 24 sample holders can be fitted onto one 24-well Linbro plate (Figure 3D).
A crystallization experiment on a 24-well Linbro plate using sample holder type 1 was set up. 1 &#181;L of hen egg-white lysozyme solution (15 mg/mL) was mixed with 1 &#181;L of mother-liquor comprising 50 mM NaAc pH 4.7, 500 mM NaCl and 25% (w/v) PEG-6000 on the yellow polyimide foil on the sample holder (Table 1). The drop was equilibrated at 293 K against 500 &#181;L of mother-liquor and crystals of the size 40-50 &#181;m were observed after 5 hours (Figure 4). Crystal growth can be observed using a transmission light microscope (Figure 4) with or without a polarizer. High transparency films ensure best observation and monitoring of crystal growing conditions using both, a conventional light microscope or an automated crystal imaging system. Crystal growth observation using UV-light was not tested.
After removing the mother liquor from around the crystals, a sample holder with hen egg-white lysozyme crystals was taken from the crystallization plate and placed in a humidity-controlled airstream on HZB-MX beamline 14.332. Diffraction data were collected at ambient temperature in 1&#176;-increments using a 150 &#956;m beam at 13.8 keV energy with 4 x 1010 photons/s and an exposure time of 5 s per image. A typical diffraction image is shown in Figure 5. No elevated background scattering on the diffraction image can be detected. Further experimental details as well as associated data processing statistics are listed in Table 2.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59722/59722fig1.jpg" /> 
Figure 1: Schematic view of the new sample holders. The sample holders consist of a black plastic support, which is covered on the outer side with an amorphous cyclic olefin copolymer (COC) foil. This foil (colored in blue) is highly transparent and self-healing. It also ensures gas tightness of the experiment. The inner foil (colored in yellow) is made of bio-inert polyimide, which is highly transparent for X-rays. On this foil, the crystallization drops can be placed.The outer rim of the sample holder contains two positioning aids indicated by the red arrow (panel A), which allows accurate placement of the sample holder on the individual pre-greased cavity of the crystallization plate. (A) Sample holder (type 1) with 22 mm diameter with a fixed external support ring. (B) Sample holder (type 2) with 22 mm diameter with removable external support ring. (C) Sample holder (type 3) with 18 mm diameter with removable external support ring. The latter two have been developed for using them in a high-throughput fashion with automated sample mounting robots using SPINE standard. The designated break points are highlighted by the red arrows in panel B. The black arrow in panel C indicates the positioning marker. The protruding pins at the outer perimeter of the yellow foil are necessary to align the polyimide foil during the production process. <a href="https://www.jove.com/files/ftp_upload/59722/59722fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59722/59722fig2.jpg" /> 
Figure 2: The sample holder may be used on a 24-well Linbro plate in the same way as the commonly used microscope cover slips. It seals the cavity airtight. Positioning aids ensure the correct positioning of the sample holder on the cavity (red arrows in panel A). Up to three individual drops may be placed onto a type 1 sample holder (panel B), whereas the recommended maximum number of drops placed on a type 2 or 3 sample holder is two. The maximum recommended volume for each drop is 2 &#181;L. <a href="https://www.jove.com/files/ftp_upload/59722/59722fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59722/59722fig3.jpg" /> 
Figure 3: 24 type 1 sample holders fit on a 24-well plate. The sample holders can be placed in two orientations on the 24-well plate as indicated (panel D). A cannula is used to pierce the back COC foil in order to remove excess liquor from a crystallization drop (panels A and C) by using a paper wick gently inserted in the same hole (panel B). <a href="https://www.jove.com/files/ftp_upload/59722/59722fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59722/59722fig4.jpg" /> 
Figure 4:&#160;Image of hen egg-white lysozyme crystals observed through a transmission microscope equipped with a polarizer. Individual crystals are easily discriminated from precipitated protein solution. The crystals in this image are of an average size of 40 &#181;m x 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59722/59722fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59722/59722fig5.jpg" /> 
Figure 5: A typical X-ray diffraction image of a lysozyme crystal grown on the sample holder. Prior to exposure to X-rays all excess mother liquor was removed from around the crystal. Diffraction data were collected at ambient temperature on BL14.3 at the electron storage ring BESSY II32 using a humidity controlled sample environment with 97.5% relative humidity. No elevated background due to the sample holders can be observed. The dashed lines in the image indicate the resolution rings. <a href="https://www.jove.com/files/ftp_upload/59722/59722fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59722/59722fig6.jpg" /> 
Figure 6:&#160;The sample holder is prepared for diffraction data collection. First, the COC film is lifted gently by using a forceps and then peeled off (panel A). Subsequently, the sample holder is removed from the cavity and inserted into the central hole of a magnetic base until indicated by the marker (panel B). By holding on to the central part, gentle pressure is applied to the outer ring to free the central part using the symmetrically arranged designated break points (panel C). After the removal, the sample holder can be plunged into liquid nitrogen and transferred into standard SPINE vials. Placed, for instance, in pucks they can be transported to synchrotron sites where automated sample-mounting robots recognize them as regular samples (panel D). <a href="https://www.jove.com/files/ftp_upload/59722/59722fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Crystallization details</td>
			<td></td>
		</tr>
		<tr>
			<td>Method</td>
			<td>Hanging drop, vapor diffusion method</td>
		</tr>
		<tr>
			<td>Plate type</td>
			<td>SuperClear Plates</td>
		</tr>
		<tr>
			<td>Temperature (K)</td>
			<td>293</td>
		</tr>
		<tr>
			<td>Protein concentration (mg mL-1)</td>
			<td>15</td>
		</tr>
		<tr>
			<td>Composition of reservoir solution</td>
			<td>50 mM NaAc pH 4.7, 500 mM NaCl, 25 % (w/v) PEG-6000</td>
		</tr>
		<tr>
			<td>Volume and ratio of drop</td>
			<td>2 &#181;L total, 1:1 ratio (protein : mother liquor</td>
		</tr>
		<tr>
			<td>Volume of reservoir</td>
			<td>500 &#181;L</td>
		</tr>
		<tr>
			<td>Incubation time</td>
			<td>12 hours</td>
		</tr>
	</tbody>
</table>
Table 1: Experimental details of the described crystallization experiment. 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Data collection and processing</td>
			<td></td>
		</tr>
		<tr>
			<td>Wavelength (&#197;)</td>
			<td>0.89429</td>
		</tr>
		<tr>
			<td>Temperature (K)</td>
			<td>293</td>
		</tr>
		<tr>
			<td>Detector</td>
			<td>Rayonix MX225 CCD&#160;</td>
		</tr>
		<tr>
			<td>Crystal-detector distance (mm)</td>
			<td>120</td>
		</tr>
		<tr>
			<td>Rotation range per image (&#176;)</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>Total rotation range (&#176;)</td>
			<td>120</td>
		</tr>
		<tr>
			<td>Exposure time per image (s)</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Space group&#160;</td>
			<td>P43212</td>
		</tr>
		<tr>
			<td>Unit-cell parameters (&#197;)</td>
			<td>a = 79.01, b = 79.01, c = 37.95</td>
		</tr>
		<tr>
			<td>Mosacity (&#176;)</td>
			<td>0.07</td>
		</tr>
		<tr>
			<td>Resolution range (&#197;)</td>
			<td>39.50 - 1.35 (1.37 - 1.35)</td>
		</tr>
		<tr>
			<td>Total number of reflections</td>
			<td>191940 (8932)</td>
		</tr>
		<tr>
			<td>Number of unique reflections</td>
			<td>27020 (1292)</td>
		</tr>
		<tr>
			<td>Completeness (%)</td>
			<td>99.88 (99.20)</td>
		</tr>
		<tr>
			<td>Multiplicity</td>
			<td>7.1 (6.9)</td>
		</tr>
		<tr>
			<td>Mean I/&#963;(I)</td>
			<td>15.0 (1.9)</td>
		</tr>
		<tr>
			<td>Rmeas35 (%)</td>
			<td>6.3 (107.0)</td>
		</tr>
		<tr>
			<td>Rpim36 (%)</td>
			<td>2.4 (40.4)</td>
		</tr>
		<tr>
			<td>CC1/237&#160;</td>
			<td>99.9 (68.5)</td>
		</tr>
		<tr>
			<td>ISa38</td>
			<td>&#160;16.1</td>
		</tr>
		<tr>
			<td>Wilson B-factor (&#197;2)</td>
			<td>17.0</td>
		</tr>
	</tbody>
</table>
Table 2: Diffraction data collection and processing statistics.

Watch this Scientific Journal Video about An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering at JoVE.com

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

an-all-one-sample-holder-for-macromolecular-x-ray-crystallography

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Biochemistry

13.1K Views.  Helmholtz-Zentrum Berlin. A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

Video: An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Microcrystallography of Protein Crystals and In Cellulo Diffraction

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in their largest dimension, which used to represent a challenge. We present two alternative workflows for the structure determination of protein microcrystals by X-ray crystallography with a particular focus on crystals grown in vivo. The microcrystals are either extracted from cells by sonication and purified by differential centrifugation, or analyzed in cellulo after cell sorting by flow cytometry of crystal-containing cells. Optionally, purified crystals or crystal-containing cells are soaked in heavy atom solutions for experimental phasing. These samples are then prepared for diffraction experiments in a similar way by application onto a micromesh support and flash cooling in liquid nitrogen. We briefly describe and compare serial diffraction experiments of isolated microcrystals and crystal-containing cells using a microfocus synchrotron beamline to produce datasets suitable for phasing, model building and refinement.
These workflows are exemplified with crystals of the Bombyx mori cypovirus 1 (BmCPV1) polyhedrin produced by infection of insect cells with a recombinant baculovirus. In this case study, in cellulo analysis is more efficient than analysis of purified crystals and yields a structure in ~8 days from expression to refinement.

Microcrystallography of Protein Crystals and In Cellulo Diffraction

A protocol is presented for X-ray crystallography using protein microcrystals. Two examples analyzing in vivo-grown microcrystals after purification or in cellulo are compared.

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological macromolecules. Until recently, a major requirement has been the availability of relatively large, well diffracting crystals, which are often challenging to obtain. However, the advent of serial crystallography and a renaissance in multi-crystal data collection methods has meant that the availability of large crystals need no longer be a limiting factor. Here, we illustrate the use of the automated MeshAndCollect protocol, which first identifies the positions of many small crystals mounted on the same sample holder and then directs the collection from the crystals of a series of partial diffraction data sets for subsequent merging and use in structure determination. MeshAndCollect can be applied to any type of micro-crystals, even if weakly diffracting. As an example, we present here the use of the technique to solve the crystal structure of the Cyan Fluorescent Protein (CFP) Cerulean.

We present the use of the MeshAndCollect protocol to obtain a complete diffraction data set, for use in subsequent structure determination, composed of partial diffraction data sets collected from many small crystals of the fluorescent protein Cerulean.

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Chemistry

Synchrotron X-ray Microdiffraction and Fluorescence Imaging of Mineral and Rock Samples

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder microdiffraction two-dimensional (2D) maps at beamline 12.3.2 of the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. Measurements can be performed on any sample that is less than 10 cm x 10 cm x 5 cm, with a flat exposed surface. The experimental geometry is calibrated using standard materials (elemental standards for XRF, and crystalline samples such as Si, quartz, or Al2O3 for diffraction). Samples are aligned to the focal point of the x-ray microbeam, and raster scans are performed, where each pixel of a map corresponds to one measurement, e.g., one XRF spectrum or one diffraction pattern. The data are then processed using the in-house developed software XMAS, which outputs text files, where each row corresponds to a pixel position. Representative data from moissanite and an olive snail shell are presented to demonstrate data quality, collection, and analysis strategies.

We describe a beamline setup meant to carry out rapid two-dimensional x-ray fluorescence and x-ray microdiffraction mapping of single crystal or powder samples using either Laue (polychromatic radiation) or powder (monochromatic radiation) diffraction. The resulting maps give information about strain, orientation, phase distribution, and plastic deformation.

In this report, we describe a detailed procedure for acquiring and processing&#160;x-ray microfluorescence (&#956;XRF), and Laue and powder ...

Engineering

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source1. Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise.
Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. 
We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

The signal-to-noise ratio of data is one of the most important considerations in performing X-ray diffraction measurements from microcrystals. The VMXm beamline provides a low-noise environment and microbeam for such experiments. Here, we describe sample preparation methods for mounting and cooling microcrystals for VMXm and other microfocus macromolecular crystallography beamlines.

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality ...

Sample Preparation and Transfer Protocol for In-Vacuum Long-Wavelength Crystallography on Beamline I23 at Diamond Light Source

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 &#197;. 
To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools. 
Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.

Here, we present a protocol for cryogenic sample preparation and transfer of crystals into the vacuum endstation on beamline I23 at Diamond Light Source, for long-wavelength macromolecular X-ray crystallography experiments.

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, ...

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (&#60;1 &#181;L) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Summary

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