Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

The automated MeshAndCollect protocol was developed to combine serial crystallography with standard rotation data collection to measure small crystals off from puck samples. The method first identifies the position of crystals mounted on the same sample holder to then direct the collection of partial data set that will be subsequently merged and used for the solution of the structure. MeshAndCollect is compelling as it allows you to do experiments rapidly and with the same setup, structure solution, ligand screening, and all that with tiny crystals.

The method is compatible with synchrotron beamline's VMX ideally equipped with high photon flex, a small beam diameter, and a fast reload detector. First, connect to the extended information system for protein crystallography beamline's database and choose MX.Log in with the experiment number and the password from the A form. Next, select Shipment Add New and fill in the requested information.

Select Add Partial and fill in the relevant data. Select Add Container, choose an SC3 puck, and fill in the information required including the positions of the sample holders in the puck. In the experimental hutch, load the puck into the sample changer doer and note its position.

Next, log in to the information system for protein crystallography beamline's database. Select Prepare Experiment, find the Shipment, select Next, and indicate the beamline and puck position in the sample changer. Log in to the beamline control software with the experimental number and password provided on the A form.

Press Sync to synchronize the beamline control software with the information system for protein crystallography beamline's database. Use the beamline control software to mount the sample holder onto the goniometer. Then right-click a position in the sample changer area and select Mount Sample.

Select the center button and then three clicks center on the middle of the edge of the tip of the loop. Save the centered position by selecting Save. Under Advanced, add the workflow Visual Reorientation to the data collection queue.

Then launch the workflow by clicking on Collect Queue. Next, select one of the saved center positions by clicking on it. Click again on the center button, then a three-click center on the middle of the start of the stem of the loop.

Save the second position by clicking on Save. And then click on Continue. After the workflow aligns the plane of the sample holder with the rotation axis of the goniometer, center the sample holder again somewhere in the middle of the mesh.

Orient the sample holder so that the face of the mesh is perpendicular to the x-ray beam direction by rotating the omega axis using the beamline control software. In the beamline control software, click on the Aperture dropdown menu and select a value. Then click on the mesh tool icon to bring up the mesh tool window.

In the sample view of the beamline control software, draw the mesh by left-clicking and dragging the mouse over the area containing crystals on the sample holder. To save the mesh, click on the plus button in the mesh tool window. In the resolution field of the beamline control software, enter the resolution at which diffraction images should be collected.

If no prior information is known about the diffraction quality of the crystals, a value between two and and 2.5 is recommended. Select MeshAndCollect in the Advanced data collection tab. Add it to the queue and click Collect the Queue.

In the parameter window, use the beamline dependent default parameters. In the experiment described here, default parameters are 0.037 second exposure time per mesh scan point, 100%transmission, and one degree oscillation per mesh scan line. Click Continue.

The mesh scan runs and the diffraction images collected at each grid point are analyzed and ranked according to diffraction strength with the software Dozer. After the Dozer analysis, a heat map is generated and the order for subsequent partial data collections is assigned automatically based on diffraction strength. Finally, click Continue to launch the partial data collections.

MeshAndCollect as implemented in MXCuBE was used for the collection of partial diffraction data sets from small crystals of Cerulean located on the same sample holder in which visual identification of crystals was difficult. To screen the sample holder, a grid was drawn over the center of the mesh loop and based on the Dozer score heat map, 85 partial diffraction data sets were automatically collected. These were individually integrated then merged to produce a data set with 99.8%completeness at a resolution of 1.7 angstroms.

As expected, the crystal structure of Cerulean could be solved by molecular replacement using the data set generated. After refinement, an R work of 22.8%and an R free of 25.4%were obtained. Super position of the previously determined structure shows a global RMSD on c-alpha positions of 0.1 angstroms.

For projects where the optimization of the crystal growth phase, MeshAndCollect provides the possibility to obtain a complete data set based on the combination of these amorphous partial data sets obtained from smaller crystals. This technique paved the way for structural biologists to solve a structure from parts sample where only a few tenths of micro crystals couldn't be produced.