The MRC-LMB crystallization facility is kindly supported by the Medical Research Council (UK). We thank members of the LMB for their support: Olga Perisic (PNAC), Tony Warne, Fusinita Van den Ent and Pat Edwards (Structural Studies), Steve Scotcher and the other members of the Mechanical Workshop, Neil Grant and Jo Westmoreland (Visual Aids), Paul Hart and Tom Pratt (IT). We also would like to thank to Steve Elliot (Tecan, UK), Mitchell Stuart and Heather Ringrose (Hamilton Robotics, UK), Paul Thaw, Robert Lewis and Joby Jenkins (TTP Labtech, UK), Paul Reardon (Swissci AG, Switzerland), George Stephens and Donald Ogg (Alphabiotech, UK), Neil Williams (Markem Imaje, UK) and Graham Harris (The Cleveland Agency) for technical help.

1. The two fully automated systems (initial screening)
<ol>
	<li>System 1: preparation of 72 96-well crystallization plates filled with screening kits (LMB plates)
	<ol>
		<li>Prior to performing the procedure, ensure that the system&#39;s robots are initialized and their controlling software is open. Turn on the chiller of the tube-cooling carrier about 30 minutes before the main program will be started.</li>
		<li>Place a test plate on the motorized SBS carrier of the plate sealer. Run the plate sealer and check that the film is properly applied. Repeat this test twice more to verify that the plate sealer is ready.</li>
		<li>Then, add 20 L of deionized water to the main container of the liquid handler. Disconnect the coupling inserts from the small container (20% ethanol rinsing solution) and connect the inserts to the main container.</li>
		<li>Enter the appropriate screen name (Table 1) on the inkjet printer touchscreen. Then, open and close the carousel door to trigger the carousel rotation. Open the door when the first stack presents itself.</li>
		<li>Fully load the first stack with 22 crystallization plates, each with column 1 facing out. Fully load the next two stacks in the same way.</li>
		<li>Load the remaining 6 plates in the highest positions of the fourth stack. Then, close the carousel door.</li>
		<li>Verify that the chiller display indicates 14 &#176;C. Gently and repeatedly invert the selected screening kits for 1 minute. Then, open the front panel of the liquid handler.</li>
		<li>Open the tubes and place them in the cooling carrier according to the standard 96-well layout (A1, A2, etc.). Upon placing each tube in the carrier, place its lid on a tray in the same 96-well layout.</li>
		<li>Cross-check the tube positions and ensure that all tubes are level and settled in the carrier. Then, close the front panel.</li>
		<li>In the &#39;Startup&#39; window of the liquid handler software, select &#39;Run maintenance&#39; and open the flushing program. Run the program to flush the 20% ethanol from the system and to wash the outsides of the liquid-dispensing tips.</li>
		<li>When flushing is complete, click &#39;Cancel&#39; to return to &#39;Startup&#39;. Select &#39;Run an existing process&#39; and click &#39;Start your selection&#39;. Open the &#39;MRC kit dispensing&#39; program. Fill in &#39;18&#39; for &#39;Instances&#39; in the configuration screen and run the program.</li>
		<li>Monitor the system as the first four plates are labeled, filled, and sealed.</li>
		<li>Once all 72 plates have been prepared and placed back in the carousel, switch the coupling inserts from the main container to the small container. Run the &#39;Start up flush&#39; program (see step 1.1.10).</li>
		<li>Turn off the chiller and discard the empty screening kit tubes. Carefully remove the 72 prepared plates from the carousel. Discard incorrectly-filled or poorly-sealed plates. Store the ready-to-use plates at 10 &#176;C.</li>
	</ol>
	</li>
	<li>System 2: setting up crystallization droplets (100 nL protein + 100 nL condition) from a single sample in 20 LMB plates
	<ol>
		<li>First, ensure that the system&#39;s robots are on, the nanoliter dispenser is initialized with its software open, and the liquid handler&#39;s method manager is open. Turn on the microtube-cooling carrier about 15 minutes before the main program will be started.</li>
		<li>Place a test plate on the motorized SBS carrier of the plate sealer. Run the plate sealer and verify that the plate is properly sealed. Test the plate sealer three times.</li>
		<li>Then, run the nanoliter dispenser to set 100-nL droplets of test solution in a test plate. Check under a microscope that the droplets are set correctly. Close the nanoliter dispenser software and remove the strip-holder block from the deck.</li>
		<li>Next, insert in the custom-designed plate holder (see Representative Results: Crystallization Devices Developed at the LMB) the LMB plate with the highest relative quantity of volatile reagents in its conditions (Table 1). Remove the adhesive film from the plate.</li>
		<li>Open the liquid handler front panel and place the unsealed plate on the deck, at the back of the first sliding carrier (Sliding carriers may be pulled out for ease of access).</li>
		<li>Cover the plate with an SBS aluminum lid. Settle the lid towards the rear left corner of the carrier by applying gentle pressure to the opposite corner of the lid.</li>
		<li>Unseal, load into the sliding carriers, and cover the remaining 19 plates in the same way, working from most to least volatile conditions. Once the microtube-cooling carrier is at 4 &#176;C, as indicated by a green light, remove its cover.</li>
		<li>Cut off the lid of a microtube containing (at least) 440 &#181;L of protein sample (Table 2). Ensure that the sample has no foam above the meniscus, as this will interfere with the liquid detection system. Place the tube in Position 1 of the microtube-cooling carrier.</li>
		<li>Place a PCR plate on the liquid handler deck in front of the plate-moving adapter carrier. Then, close the front panel.</li>
		<li>After loading the deck, ensure that the nanoliter dispenser deck is clear of the strip-holder block and that the carriers at the back of the 50-&#181;L tip stacks are clear of the aluminum SBS lids.</li>
		<li>In the liquid handler &#39;Method Management&#39; interface, select &#39;Setup plates&#39;. Monitor the initialization of both systems and fill in the run parameters. Follow the guidelines of the Method Management interface (prompts, figures and a tip-management system help with preparation).</li>
		<li>Double-check that all required components are ready, and then start the process.</li>
		<li>Monitor the system as the nanoliter dispenser sets the drops in the first plate and the plate sealer subsequently seals the plate.</li>
		<li>Once all 20 plates have been prepared with crystallization droplets and automatically returned to the sliding carriers, open the front panel and gently remove the plates. Check that the plates are correctly sealed before storing them for crystallization.</li>
		<li>Clean the SBS lids with a 20% ethanol solution before stacking them on the left-hand side of the liquid handler for storage. Discard the PCR plate and the microtube.</li>
		<li>Turn off the microtube-cooling carrier and wipe away the condensation. Leave a paper towel on top of the cooler surface to absorb further condensation. Then, replace the carrier cover and close the front panel.</li>
	</ol>
	</li>
</ol>
2. Optimization of the conditions
<ol>
	<li>Syringe-based liquid handler: producing two linear gradients of concentrations into the reservoirs of a crystallization plate (the 4-corner method).
	<ol>
		<li>First, ensure that the liquid handler is on and initialized with its software open.</li>
		<li>On the &#39;GRADIENT&#39; tab: Open the required program, select the crystallization plate type and the final volume in the reservoirs (Table 3). The advanced setting for &#39;max shot vol&#39; should be lowered from 6,000 to 3,000 when using solutions containing [isopropanol] &#62; 10% v/v and [MPD] &#62; 20% v/v.</li>
		<li>Prepare the syringes. Place a piston in each syringe (pointed ends down) and insert the back of the syringes into the designated grooves underneath the robot head. Twist a syringe clockwise to lock it in position (The program will start only with all the required syringes attached correctly).</li>
		<li>Prepare the troughs. Remove the stainless-steel frame and insert the troughs. The 4 positions on the left correspond to the 4 corners A, B, C, D. Switch to &#39;SET UP&#39; tab which displays the volumes of solutions required in each syringe (on Table 3, 0.5 mL dead volume was added to the volumes displayed). Pour the corner solutions into their respective troughs and place the frame back on the deck (the frame holds in position with 2 small magnets located at the front of the deck). An alternative way to proceed with this step is to pour the solutions into the troughs when they are already placed on the deck.</li>
		<li>Place the crystallization plate on the motorized SBS carrier.</li>
		<li>Click &#39;ASPIRATE&#39; and wait for this step to be completed (when pistons stopped on their way up).</li>
		<li>Switch to the &#39;RUN&#39; tab and run program.</li>
		<li>Upon completion of the program, go back to the SET UP tab and click &#39;REMOVE&#39;: the system purges the syringes from leftover solutions, and then lifts the pistons all the way up. &#39;PURGE&#39; may be requested instead of &#39;REMOVE&#39;; this will leave the pistons at the bottom position, ready to aspirate more solutions with the same syringes.</li>
		<li>Remove the syringes by twisting them anticlockwise.</li>
		<li>Discard syringes and troughs in the appropriate bin (or rinse them with deionized water and then 20% v/v ethanol solution for reuse).</li>
		<li>Seal the plate and place it onto the microplate mixer for 3 min at 1,000 rpm, or 10 min when highly viscous solutions are being mixed. The plate is ready for setting up crystallization droplets on the nanoliter dispenser.</li>
	</ol>
	</li>
	<li>Additive screening protocol 1 (setting up crystallization droplets in a 96-well crystallization plate pre-filled with additive screen)
	<ol>
		<li>First, prepare the condition with initial concentrations of reagents increased by 10% (min. vol. 15 mL when transferring the condition from a container onto the additive screen with the liquid handler).</li>
		<li>Ensure that the liquid handler is ready to operate. Open the program &#39;Add screen to additives&#39; for a single plate (enter volume in reservoirs: &#39;72 &#181;L&#39;). Prompts, figures and a tip-management system (12 x 1,000 &#181;L tips required) help with making sure the robot is ready to operate according to the selections.</li>
		<li>Retrieve the additive screen from the -20 &#176;C incubator and remove its aluminum seal immediately (use the plate holder), then place the plate on the deck of the liquid handler. The program operates according to a portrait layout (the plate is placed with the A1-corner located in the front-left of the carrier). Also place also the container filled with the condition. Run the program.</li>
		<li>Once the reservoirs of the plate have been filled, place it onto the microplate shaker and run the program. Rinse the container of condition with deionized water and 20 % ethanol for reuse.</li>
		<li>Set up the droplets on the nanoliter dispenser. First, unseal the crystallization plate (use the plate holder). Then, place the plate and the 8-well protein strip in the first position of the strip-holder block. The &#39;Setup&#39; tab on the controlling software displays the actual positions of each component on the deck. Load each well of the strip with protein sample according to the drop size required (Table 4). Run the program to prepare droplets.</li>
		<li>Upon completion of the program, remove the plate from the deck and seal it immediately (use the plate sealer, 3-inch wide adhesive tape). Discard the strip in the appropriate bin.</li>
		<li>Assess the size, shape, and centering of the droplets under the microscope before storage.</li>
	</ol>
	</li>
	<li>Additive screening protocol 2 (setting up crystallization droplets in a 96-well crystallization plate with a re-usable additive screen)
	<ol>
		<li>First, prepare the additive screen: leave the corresponding frozen 96-well cell culture plate to thaw at room temperature for 40 min. Then, centrifuge the additive screen at 1,000 x g for 2 min.</li>
		<li>Prepare the condition (min. vol. 15 mL when transferring the condition from a container onto the additive screen with the liquid handler).</li>
		<li>Fill the reservoirs of a 96-well crystallization plate with the condition. On the liquid-handler, proceed the same way as protocol 1, step 2.2.2, but enter &#39;80 &#181;L&#39; for the volume in reservoirs.</li>
		<li>Set up the droplets on the nanoliter dispenser with 3 components on the deck (the plate containing the additive screen, along with the crystallization plate and the 8-well protein strip in the first position of the strip-holder block, Table 4).</li>
		<li>Seal the cell culture plate containing the screen with an aluminum sheet and place it back in the -20 &#176;C incubator.</li>
		<li>Assess the size, shape, and centering of the droplets under the microscope before storage.</li>
	</ol>
	</li>
</ol>

We hereby state a conflicting commercial interest since LifeArc commercializes the following items: the 96- and 48-well MRC plates, the MRC hanging drop seal, the MORPHEUS, Pi, ANGSTROM and LMB crystallization screens.

1- Preparation and use of initial screens stored in plates
Screening kits should be mixed before being dispensed into plates because light precipitation or phase separation occurs in some tubes during storage. When a screen is composed of two kits (2 x 48 tubes), the first tube of the second kit is placed in in location E1 of the cooling carrier. When a screen is composed of 4 kits (4 x 24 tubes), the first tube of the second kit is placed in in location C1, the first tube of the third kit is placed in location E1 and the first tube of the fourth kit is placed in location G1. While inserting the tubes in their cooling carrier, lids are placed on a tray following the standard 96-well landscape layout. Since well numbers are indicated on top of the lids by the manufacturers, this enables cross-checking if all tubes have been placed in the right order. This also helps to replace the correct lids on the tubes when filling a reduced number of plates.
We store the pre-filled plates at 10 &#176;C, a compromise to avoid freezing and storage at 4 &#176;C that may cause deterioration of conditions and issues with sealing. Plates are stored for up to several months with normally no noticeable condensation on the inner face of the seal. This is less true for LMB05, LMB06, LMB09 and LMB10 plates as these contain conditions with relatively high concentrations of volatile reagents (Table 1). Small amount of condensation on the inner side of the seal reduces sealing efficiency and can cause cross-contamination between wells while unsealing the plates. To help with preventing condensation during initial cooling, plates can be first transferred from the carousel into an insulated picnic cooler which is stored in a 4 &#176;C cold room overnight. The very slow cooling minimizes the development of temperature gradients within the sealed wells and hence reduces condensation overall15. In addition, once the plates are stored in the 10 &#176;C incubator, an in-house custom SBS polystyrene lid is placed on the plate at the top of each stack (not shown).
The entire set of our pre-filled plates can be used as a large initial screen against a novel, water-soluble, protein sample. Alternatively, fewer plates may be selected to match specific requirements. For example, LMB15 and LMB19 are screens formulated specifically for membrane protein samples26,27, or LMB20 is a screen formulated with heavy-atoms to facilitate the experimental phasing of diffraction data28 (see also: <a href="https://www.youtube.com/watch?v=Gpb4SypWnQY&amp;t=11s">Formulation of the MORPHEUS protein crystallization screens</a>).
2. Setting up crystallization droplets
When using the system 2, screening kits with significant amounts of volatile reagents should be processed first. This avoids condensation forming on the rubber of the SBS lids, which could affect lid handling and plate sealing. An SBS lid has a bit of clearance when on the top of a plate, which is why they need to be aligned initially (see Protocol, step 1.2.6). The protein dead volumes in the wells of the PCR plate are relatively generous (0.8 &#181;L, see legend of Table 2). Note that equally generous dead volumes are employed when using the nanoliter dispenser individually with protein in 8-well strips (Table 4). Smaller dead volumes may work, however some samples adhere to the tips, calibration of a robot may become slightly inaccurate, the room may be warmer than usual, etc. All lead to sample losses covered by the generous dead volumes in order to consolidate the approach.
Recent developments enabled further miniaturization of experiments and hence the volume of sample required for screening crystallization conditions can be significantly reduced by integrating the corresponding technology29,30. However, some aspects of further miniaturization need careful consideration, such as the evaporation of droplets31 and the manipulation of microcrystals32.
Finally, centrifugation of the plate (2,000 rpm, 1 min) could be integrated as a routine final step when setting up crystallization droplets (in spherical upper-wells). A more consistent size and shape of droplets resulting from centrifugation may reduce reproducibility issues33,34. Surely, centered drops will ease the later assessment of experiments using a microscope as the required focal length will be similar across the entire plate.
3. Advantages of the 4-corner method
The most significant advantage of the 4-corner method is its simplicity, which minimizes errors and facilitates straightforward automated protocols. For example, the 4 corner solutions will always be placed on the deck of a liquid handler following the same layout. Also, all programs are based on fixed ratios between the solutions (Figure 3C). 
Manual preparation of the 4 corner solutions is preferred to automated handling of solutions at high concentrations which can be highly viscous. Relatively fast and accurate aspiration/dispensing is then possible on most types of liquid handlers with minimum requirements for optimization of liquid classes. Nevertheless, some corner solutions may still be too viscous for a robot operating with a liquid-system to operate efficiently. This is why we opted for a liquid handler operating with positive displacement (Figure 3B).
In addition to the 2 linear gradients of concentrations, a third component (i.e., a set of buffers/additives) can be tested at a constant concentration in a convenient way. For this, a relatively large volume of a core set of corner solutions at a suitably higher concentration, excluding the component to be varied, is prepared first. Then, stock solutions including this component is added to adjust the final concentrations. For example, 50 mL of a set of 4 corner solutions are prepared at 10 % higher concentrations than initially. This core set is then split into 5 smaller subsets of 4. Finally, 10% in volume of different buffer-pH solutions is added to each subset.
4. Formats and types of additive screens
The screens are normally stored at -20 &#176;C (Figure 4) since they are not used regularly and contain volatile/unstable compounds. The use of a frozen additive screen stored in a deep well block (1 mL in wells) must be planned early because it will take 12-24 hr for all the additive solutions to thaw completely at room temperature. Also, a multitude of users share the same additive screen, potentially causing problems with cross-contamination. Finally, the height of deep well blocks makes them unsuitable for most nanoliter dispensers. As a convenient solution to circumvent these issues, the screen should be transferred from the deep well block to low-profile plates (Figure 4).
Historically, additive screens which include a broad variety of single reagents (with single concentrations) have been very popular35,36. However, other types of additive screens have been developed that integrate mixes of additives37 or a reduced number of single additives found at different concentrations38. Finally, a complementary approach is to investigate the effect of additives on the samples prior to crystallization39,40.
5. More considerations
Good practice: Most screens contain harmful or even toxic substances and hence adequate personal protection must be employed during the protocols. Equally, moving parts of the robots may lead to injuries, especially when trying to manually interfere while a program is running (although most of the robots have emergency stop button/system). Because of the technical complexities involved, regular checking of robots, screens and programs with previously characterized test samples are important for sustained high levels of reproducibility.
Throughput: As an indication, between 4,000 to 8,000 LMB plates are produced yearly with the system 1 (and subsequently employed by users for initial screening). It is not adapted to stock a large amount of pre-filled plates at 10 &#176;C when the expected turnover is much lower, as after 4-5 months, some conditions will start to deteriorate and evaporate. Different approaches to automation protocols have been implemented for small- to medium-size laboratories41.
Storing and assessing experiments: After preparing the droplets, plates are stored on low-vibration shelves in a room at 4 or 18 &#176;C with tightly controlled temperature (+/- 0.5 &#176;C maximum deviation). Experiments are assessed using cold light source microscopes. Various automated imaging systems are commercially available, however one should carefully consider all aspects: Will the speed required to scan a plate be sufficient for high throughput? Will objects other than crystals interfere with autofocus? Will the resulting quality of images be sufficient to spot very small crystals (especially around the edge of the droplets)?42,43,44
Comparison of crystallization conditions: After careful investigations about the nature of the initially-obtained crystals, one can analyze trends and similarities across conditions using the <a href="https://search.mrc-lmb.cam.ac.uk/screens2/search.html">LMB screen database</a> or the C6 <a href="http://c6.csiro.au/">Web Tool</a>45.

X-ray crystallography is extensively applied to further advance our understanding of biological and disease mechanisms at the atomic level and to subsequently assist rational approaches to drug discovery1. For this, purified and concentrated (2-50 mg/mL) macromolecular samples of protein, DNA, RNA, other ligands, and their complexes are trialed for their propensity to form ordered three-dimensional lattices through crystallization2,3,4. When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution5,6. Crucially, the conditions to crystallize a novel sample cannot be predicted and the yield of high quality crystals is usually very low. An underlying reason is that many samples of interest have challenging biochemical properties, which make them unstable on the corresponding timescale for crystallization (typically a few days). Finally, the process is compounded by the time required to produce samples and sample variants, and to optimize their purification and crystallization7,8.
A crystallization condition is a solution with a precipitant that reduces sample solubility, and conditions often also contain buffers and additives. Hundreds of such reagents are well suited to alter the parameters of the crystallization experiments as they have low propensity to interfere with sample integrity (such as protein or nucleic acid unfolding). While testing millions of combinations of crystallization reagents is not feasible, testing several to many screening kits - formulated with various strategies9,10 - is possible with miniaturized trials and automated protocols. In this perspective, the most amenable technique is probably vapor diffusion with 100-200 nL droplets sitting on a small well above a reservoir containing the crystallization condition (25-250 &#181;L), implemented in specialized crystallization plates11,12. The protein sample and condition are often combined in a 1:1 ratio for a total volume of 200 nL when setting up the droplets in the upper-wells. Robotic nanoliter protein crystallization can be implemented with alternative techniques and plates such as the under-oil batch13 and the Lipidic Cubic Phase14 (the latest one being applied specifically to trans-membrane proteins that are very poorly soluble in water).
<a href="http://www2.mrc-lmb.cam.ac.uk/groups/JYL/WWWrobots/robot.html">The crystallization facility at the MRC-LMB</a> was started in the early 2000s and an early summary of our automated protocols was presented in 200515. A historical introduction to protein crystallization was presented and also an outline of the advantages of robotic nanoliter approach (then a novel approach to routine experimentation). Since macromolecular crystallization is essentially a stochastic process with very little or no useful prior information, employing a broad variety of (suitable) initial conditions increase the yield of quality diffraction crystals16. Besides, an often-overlooked advantage of a large initial screen is to significantly reduce the need for optimization of samples and crystals in many cases. Of course, one may still need to proceed with optimization of some initial conditions later. Typically, the concentration of reagents and the pH are then systematically investigated. More reagents can also be introduced into the optimized condition(s) to further alter parameters of crystallization. Certainly, one should attempt crystallization with a sample freshly prepared, hence the corresponding protocols must be straightforward and available any time.
Here, two fully automated systems designed at the MRC-LMB (systems 1 and 2) and the corresponding protocols are fully described. The main application of these two systems is initial screening by vapor diffusion in sitting drop crystallization plates. System 1 integrates a liquid handler, an automated carousel to stock plates, an inkjet printer for plate labeling and an adhesive plate sealer. On the system 1, 72 96-well plates are filled with commercially available screening kits (80 &#181;L of condition transferred to reservoir from a starting volume of 10 mL in test tubes), labeled and sealed. The plates are then stored in a 10 &#176;C incubator where they are available to users anytime (as initial screens called &#39;LMB plates&#39;).
System 2 integrates a liquid handler, a nanoliter dispenser, and an adhesive plate sealer. On the system 2, sitting droplets (100-1,000 nL) for vapor diffusion experiments are produced by combining conditions and the sample in the upper-wells of 20 48- or 96-well plates pre-filled with conditions. This means 1,920 initial screening conditions are trialed when using 20 LMB plates on the system 2.
Robots are also used individually for the optimization of selected conditions, and the corresponding semi-automated protocols are also described. The 4-corner method17 is employed routinely to produce optimization screens. The corresponding protocol first requires the manual preparation of 4 solutions (&#39;A, B, C, and D&#39;). Two linear gradients of concentrations (for two main crystallization agents) are then automatically generated directly into the reservoirs of a crystallization plate. For this, a syringe-based liquid handler dispenses the 4 corner solutions at different ratios.
To further optimize a condition, one can employ additive screens that potentially enhance the properties of resulting crystals18. Two approaches are available for additive screening: a protocol starting with additives dispensed into the reservoirs of crystallization plates before setting up the droplets (protocol 1) and another protocol where the additive screen is dispensed directly onto the droplets (protocol 2).
Other useful developments that were initiated at the MRC-LMB to facilitate automated macromolecular crystallization, are also presented. Essentially, crystallization plates and associated devices such as a stackable Society of Biomolecular Screening (SBS) lid that minimizes evaporation of conditions when using the system 2.
For brevity, it is assumed that users are familiar with the basic functions and maintenance of the nanoliter dispenser, the inkjet printer, and the adhesive plate sealer. Unless stated otherwise, plates on the deck of the robots are positioned such that the well A1 (&#39;A1-corner&#39;) is towards the back-left corner of a plate carrier.

<table><tbody><tr><td>&lt;strong&gt;Robots&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Freedom EVO&amp;reg;</td><td>Tecan</td><td>n/a</td><td>Liquid handler (System 1). Aspiration/Dispense based on system liquid. Integrates an automated carousel. EVOware plus controlling software v.2.4.12.0.&amp;nbsp;</td></tr><tr><td>Microlab&amp;reg; STAR&amp;trade;</td><td>Hamilton</td><td>n/a</td><td>Liquid handler (System 2). Aspiration/Dispense based on positive displacement (CO-RE&amp;trade; technology). Hamilton STAR controlling software v.4.3.5.4785 with method management interface.</td></tr><tr><td>Mosquito&amp;reg;</td><td>TTP Labtech</td><td>n/a</td><td>Microsyringe-based nanoliter dispenser used to set up droplets (System 2 and stand-alone), 3-position deck. Controlling software v.3.11.0.1422.&amp;nbsp;</td></tr><tr><td>Dragonfly&amp;reg;</td><td>TTP Labtech</td><td>n/a</td><td>Syringe-based liquid handler used to produce optimization screens (4-corner method). Controlling software v.1.2.1.10196.&amp;nbsp;</td></tr><tr><td>Adhesive plate sealer</td><td>Brandel</td><td>n/a</td><td>Integrated to Systems 1 and 2 (also used as stand-alone robot).</td></tr><tr><td>Inkjet printer 9232</td><td>Markem-Imaje</td><td>n/a</td><td>Integrated to System 1. Touchscreen interface.</td></tr><tr><td>&lt;strong&gt;Crystallization screens&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Crystal Screen&amp;trade; 1</td><td>Hampton Research</td><td>HR2-110</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB01</td></tr><tr><td>Crystal Screen 2&amp;trade;</td><td>Hampton Research</td><td>HR2-112</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB01</td></tr><tr><td>Wizard&amp;trade; Classic 1</td><td>Rigaku</td><td>1009530</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB02</td></tr><tr><td>Wizard&amp;trade; Classic 2</td><td>Rigaku</td><td>1009531</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB02</td></tr><tr><td>Grid Screen&amp;trade; Ammonium Sulfate</td><td>Hampton Research</td><td>HR2-211</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB03</td></tr><tr><td>Grid Screen&amp;trade; PEG/LiCl</td><td>Hampton Research</td><td>HR2-217</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB03</td></tr><tr><td>Quick Screen&amp;trade;</td><td>Hampton Research</td><td>HR2-221</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB03</td></tr><tr><td>Grid Screen&amp;trade; Sodium Chloride</td><td>Hampton Research</td><td>HR2-219</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB03</td></tr><tr><td>Grid Screen&amp;trade; PEG 6000</td><td>Hampton Research</td><td>HR2-213</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB04</td></tr><tr><td>Grid Screen&amp;trade; MPD</td><td>Hampton Research</td><td>HR2-215</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB04</td></tr><tr><td>MemFac&amp;trade;</td><td>Hampton Research</td><td>HR2-114</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB04</td></tr><tr><td>PEG/Ion&amp;trade;</td><td>Hampton Research</td><td>HR2-126</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB05</td></tr><tr><td>Natrix&amp;trade;</td><td>Hampton Research</td><td>HR2-116</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB05</td></tr><tr><td>Crystal Screen Lite&amp;trade;</td><td>Hampton Research</td><td>HR2-128</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB06</td></tr><tr><td>Custom Lite screen</td><td>Molecular Dimensions Ltd</td><td>n/a</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB06</td></tr><tr><td>Wizard&amp;trade; Cryo 1</td><td>Rigaku</td><td>1009536</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB07</td></tr><tr><td>Wizard&amp;trade; Cryo 2</td><td>Rigaku</td><td>1009537</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB07</td></tr><tr><td>JBS1</td><td>JenaBioScience</td><td>CS-101L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB08</td></tr><tr><td>JBS2</td><td>JenaBioScience</td><td>CS-102L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB08</td></tr><tr><td>JBS3</td><td>JenaBioScience</td><td>CS-103L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB08</td></tr><tr><td>JBS4</td><td>JenaBioScience</td><td>CS-104L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB08</td></tr><tr><td>JBS5</td><td>JenaBioScience</td><td>CS-105L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB09</td></tr><tr><td>JBS6</td><td>JenaBioScience</td><td>CS-106L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB09</td></tr><tr><td>JBS7</td><td>JenaBioScience</td><td>CS-107L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB09</td></tr><tr><td>JBS8</td><td>JenaBioScience</td><td>CS-108L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB09</td></tr><tr><td>JBS9</td><td>JenaBioScience</td><td>CS-109L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB10</td></tr><tr><td>JBS10</td><td>JenaBioScience</td><td>CS-110L</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB10</td></tr><tr><td>Clear Strategy&amp;trade; Screen 1 pH 4.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB10</td></tr><tr><td>Clear Strategy&amp;trade; Screen 1 pH 5.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB10</td></tr><tr><td>Clear Strategy&amp;trade; Screen 1 pH 6.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB11</td></tr><tr><td>Clear Strategy&amp;trade; Screen 1 pH 7.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB11</td></tr><tr><td>Clear Strategy&amp;trade; Screen 1 pH 8.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB11</td></tr><tr><td>Clear Strategy&amp;trade; Screen 2 pH 4.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB11</td></tr><tr><td>Clear Strategy&amp;trade; Screen 2 pH 5.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB12</td></tr><tr><td>Clear Strategy&amp;trade; Screen 2 pH 6.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB12</td></tr><tr><td>Clear Strategy&amp;trade; Screen 2 pH 7.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB12</td></tr><tr><td>Clear Strategy&amp;trade; Screen 2 pH 8.5</td><td>Molecular Dimensions Ltd</td><td>MD1-16LMB</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB12</td></tr><tr><td>Index&amp;trade;</td><td>Hampton Research</td><td>HR2-144</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB13</td></tr><tr><td>SaltRX&amp;trade; 1</td><td>Hampton Research</td><td>HR2-107</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB14</td></tr><tr><td>SaltRX&amp;trade; 2</td><td>Hampton Research</td><td>HR2-109</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB14</td></tr><tr><td>MemStar&amp;trade;</td><td>Molecular Dimensions Ltd</td><td>MD1-21</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB15</td></tr><tr><td>MemSys&amp;trade;</td><td>Molecular Dimensions Ltd</td><td>MD1-25</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB15</td></tr><tr><td>JCSG-plus&amp;trade; Suite</td><td>Qiagen</td><td>130720</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB16</td></tr><tr><td>MORPHEUS&amp;reg; screen</td><td>Molecular Dimensions Ltd</td><td>MD1-46</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB17</td></tr><tr><td>Pi minimal screen</td><td>JenaBioScience</td><td>CS-127</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB18</td></tr><tr><td>Pi-PEG screen</td><td>JenaBioScience</td><td>CS-128</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB19</td></tr><tr><td>MORPHEUS&amp;reg; II screen</td><td>Molecular Dimensions Ltd</td><td>MD1-91</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB20</td></tr><tr><td>LMB crystallization screen&amp;trade;</td><td>Molecular Dimensions Ltd</td><td>MD1-98</td><td>Crystallization kit (test tubes, 10mL per condition) used in LMB21</td></tr><tr><td>&lt;strong&gt;Additive screens&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HT additive screen</td><td>Hampton Research</td><td>HR2-138</td><td>Frozen in 96-well deepwell block (1 mL per well).</td></tr><tr><td>MORPHEUS&amp;reg; additive screen</td><td>Molecular Dimensions Ltd</td><td>MD1-93-500</td><td>Frozen in 96-well deepwell block (500 &amp;micro;L per well).</td></tr><tr><td>ANGSTROM additive screen&amp;trade;&amp;nbsp;</td><td>Molecular Dimensions Ltd</td><td>MD1-100</td><td>Frozen in 96-well deepwell block (1 mL per well).</td></tr><tr><td>MORPHEUS&amp;reg; additive screen</td><td>Molecular Dimensions Ltd</td><td>MD1-93</td><td>Frozen in 96-well cell culture Costar&amp;reg; plate with V-shaped wells (100 &amp;micro;L per well).</td></tr><tr><td>ANGSTROM additive screen&amp;trade;&amp;nbsp;</td><td>Molecular Dimensions Ltd</td><td>MD1-100-FX</td><td>Frozen in 96-well cell culture Costar&amp;reg; plate with V-shaped well (100 &amp;micro;L per well).</td></tr><tr><td>HIPPOCRATES additive screen</td><td>Molecular Dimensions Ltd</td><td>n/a</td><td>48 single additives (drug compounds found in MORPHEUS&amp;reg; III).</td></tr><tr><td>&lt;strong&gt;Other consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96-well MRC 2-drop plate</td><td>Swissci</td><td>MRC 96T-UVP</td><td>Sitting-drop, vapor diffusion plate. Reservoir recommended volume: 80&amp;nbsp; &amp;micro;L. Range of useful droplet volumes: 10-1000 nL. UV transmissible.</td></tr><tr><td>48-well MRC 1-drop plate&amp;nbsp; (&#039;MAXI plate&#039;)</td><td>Swissci</td><td>MMX01-UVP&amp;nbsp;</td><td>Sitting-drop, vapor diffusion plate for scale-up/optimization. Reservoir recommended volume: 200&amp;nbsp; &amp;micro;L. Range of useful droplet volumes: 0.1-10 &amp;micro;L. UV transmissible.</td></tr><tr><td>MRC hanging drop seal</td><td>Swissci</td><td>n/a</td><td>Hanging-drop, compatible with both MRC vapor diffusion plates (MRC 96T-UVP and MMX01-UVP ). UV and X-ray transmissible.</td></tr><tr><td>Adhesive sealing tape</td><td>Hampton Research</td><td>HR4-50</td><td>3-inch wide Duck&amp;reg; HD Clear&amp;trade; for sealer and manual sealing.</td></tr><tr><td>Adhesive aluminium sheet</td><td>Beckman Coulter</td><td>538619</td><td>Used to reseal additive screens.</td></tr><tr><td>Ink cartridge</td><td>Markem-Imaje</td><td>9651</td><td>System 1 (inkjet printer).</td></tr><tr><td>Solvent cartridge</td><td>Markem-Imaje</td><td>8652</td><td>System 1 (inkjet printer).</td></tr><tr><td>50 &amp;micro;L tips&amp;nbsp;</td><td>Hamilton</td><td>235947</td><td>System 2 (STAR&amp;trade; liquid handler). Box of 6 sets with 1920 x CO-RE&amp;trade; tips in disposable stacks.</td></tr><tr><td>Reagent container</td><td>Hamilton</td><td>194052</td><td>Used to dispense a condition into plate(s) during additive screening protocols.&amp;nbsp;</td></tr><tr><td>PCR plate</td><td>Thermo Scientific&amp;trade;</td><td>AB-2150</td><td>System 2 (contains protein to be transfer to the Mosquito&amp;reg;). Abgene Diamond ultra, 384 V-shaped wells.</td></tr><tr><td>microsyringes</td><td>TTP Labtech</td><td>4150-03020</td><td>Spool of 26,000 microsyringes for the Mosquito&amp;reg; nanoliter dispenser (9mm spacing).</td></tr><tr><td>strip-holder block</td><td>TTP Labtech</td><td>3019-05013</td><td>SSB device for the Mosquito&amp;reg; strips, aka &#039;4-way Reagent Holder&#039;.</td></tr><tr><td>2 &amp;micro;L 8-well strip</td><td>TTP Labtech</td><td>4150-03110</td><td>Contains protein on the deck of the Mosquito&amp;reg;. Box of 40 strips, max. vol. in well is 3.2 &amp;micro;L.</td></tr><tr><td>5 &amp;micro;L 8-well strip</td><td>TTP Labtech</td><td>4150-03100</td><td>Contains protein on the deck of the Mosquito&amp;reg;. Box of 40 strips, max. vol. in well is 7.5 &amp;micro;L.</td></tr><tr><td>5 mL syringes</td><td>TTP Labtech</td><td>4150-07100</td><td>Syringe body and piston for the Dragonfly&amp;reg; liquid handler. Pack of 100.</td></tr><tr><td>Troughs/Reservoirs</td><td>TTP Labtech</td><td>4150-07103</td><td>Contains stock solutions on the deck of the Dragonfly&amp;reg;. Pack of 50.</td></tr><tr><td>Orbital microplate shaker</td><td>CamLab Limited</td><td>n/a</td><td>Variomag&amp;reg; for mixing conditions in a single plate (0-2000 rpm).</td></tr><tr><td>Microplate mixer</td><td>TTP Labtech</td><td>3121-01015</td><td>MxOne. Mixing condition in a single plate with 96 vibrating pins.</td></tr></tbody></table>

automated protocols for macromolecular crystallization at the mrc laboratory of molecular biology

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize proteins, DNAs, RNAs, and their complexes can however not be predicted. Employing a broad variety of conditions is a way to increase the yield of quality diffraction crystals. Two fully automated systems have been developed at the MRC Laboratory of Molecular Biology (Cambridge, England, MRC-LMB) that facilitate crystallization screening against 1,920 initial conditions by vapor diffusion in nanoliter droplets. Semi-automated protocols have also been developed to optimize conditions by changing the concentrations of reagents, the pH, or by introducing additives that potentially enhance properties of the resulting crystals. All the corresponding protocols will be described in detail and briefly discussed. Taken together, they enable convenient and highly efficient macromolecular crystallization in a multi-user facility, while giving the users control over key parameters of their experiments.

Watch this Scientific Journal Video about Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology at JoVE.com

Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

Automated systems and protocols for the routine preparation of a large number of screens and nanoliter crystallization droplets for vapor diffusion experiments are described and discussed.

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize ...

automated-protocols-for-macromolecular-crystallization-at-mrc

Research

JoVE Journal

Biochemistry

16.3K Views.  Medical Research Council. Automated systems and protocols for the routine preparation of a large number of screens and nanoliter crystallization droplets for vapor diffusion experiments are described and discussed.

Video: Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Genetics

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

Behavior

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice of detergent is critical, because it affects both the stability and monodispersity of the complex. We recently determined the crystal structure of an active state of bovine rhodopsin coupled to an engineered G protein, mini-Go, at 3.1 &#197; resolution. Here, we detail the procedure for optimizing the preparation of the rhodopsin&#8211;mini-Go complex. Dark-state rhodopsin was prepared in classical and neopentyl glycol (NPG) detergents, followed by complex formation with mini-Go under light exposure. The stability of the rhodopsin was assessed by ultraviolet-visible (UV-VIS) spectroscopy, which monitors the reconstitution into rhodopsin of the light-sensitive ligand, 9-cis retinal. Automated size-exclusion chromatography (SEC) was used to characterize the monodispersity of rhodopsin and the rhodopsin&#8211;mini-Go complex. SDS-polyacrylamide electrophoresis (SDS-PAGE) confirmed the formation of the complex by identifying a 1:1 molar ratio between rhodopsin and mini-Go after staining the gel with Coomassie blue. After cross-validating all this analytical data, we eliminated unsuitable detergents and continued with the best candidate detergent for large-scale preparation and crystallization. An additional problem arose from the heterogeneity of N-glycosylation. Heterologously-expressed rhodopsin was observed on SDS-PAGE to have two different N-glycosylated populations, which would probably have hindered crystallogenesis. Therefore, different deglycosylation enzymes were tested, and endoglycosidase F1 (EndoF1) produced rhodopsin with a single species of N-glycosylation. With this strategic pipeline for characterizing protein quality, preparation of the rhodopsin&#8211;mini-Go complex was optimized to deliver the crystal structure. This was only the third crystal structure of a GPCR&#8211;G protein signaling complex. This approach can also be generalized for other membrane proteins and their complexes to facilitate sample preparation and structure determination.

This report describes screening of different detergents for preparing the visual GPCR, rhodopsin, and its complex with mini-Go. Biochemical methods characterizing the quality of the complex at different stages during purification are demonstrated. This protocol can be generalized to other membrane protein complexes for their future structural studies.

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Biology

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

Investigating ATG9A Localization in Cells

Imaging ATG9A, a Multi-Spanning Membrane Protein

Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive pathological conditions. A set of proteins, called ATG proteins, comprise the core autophagy machinery and work together in a defined hierarchy. Studies in recent years have improved our knowledge of the autophagy pathway. Most recently, it has been proposed that ATG9A vesicles are at the heart of autophagy, as they control the rapid de novo synthesis of an organelle called the phagophore. The study of ATG9A has proven challenging, since ATG9A is a transmembrane protein, and it is present in different membrane compartments. As such, understanding its trafficking is an important element for understanding autophagy. Here, detailed methods are presented that can be used to study ATG9A and, in particular, its localization using immunofluorescence techniques, which can be assessed and quantified. The pitfalls of transient overexpression are also addressed. The correct characterization of ATG9A function and the standardization of techniques to analyze its trafficking are crucial to further characterize the events governing autophagy initiation.

Author Spotlight: Imaging ATG9A, a Multi-Spanning Membrane Protein  

This protocol describes various methods that can help in the study of ATG9A biology, including immunofluorescence followed by image analysis, transient overexpression considerations, and investigating the ATG9A glycosylation status using western blot.

Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive ...

Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

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Chapters in this video

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Imaging ATG9A, a Multi-Spanning Membrane Protein