6.6K Views
•
07:11 min
•
March 22nd, 2019
DOI :
March 22nd, 2019
•0:04
Title
0:45
Creation of a Diffraction Plan in ISPyB
3:55
Data Collection, Viewing, and Retrieval
5:02
Results: X-ray Diffraction Analysis of Human Mitochondrial Glycine Cleavage System protein H (GCSH)
6:38
Conclusion
필기록
X-Ray crystallography is the dominant technique for determining 3D structural information from proteins. Synchrotron beam lines are needed to obtain data from the small, weakly diffracting crystals the proteins produce. Massive one is a unique beam line because it fully automates the process of data collection from biological macromolecules.
From sample mounting, location, and final data collection. Full automation of this kind is very important as it allows researchers to spend their time in the lab, instead of collecting data on the beam line. The beam line is also intelligent and on average, can collect better data than when the beam line is operated by humans.
To begin this protocol, perform crystal mounting and request beam time on Massive one as described in the text protocol. Navigate to the information system for protein crystallography beam lines, or ISPyB website. Choose MX experiments, login with the experiment number and password.
Click on shipment, add new, and provide the necessary information. Then click save. Click add parcel, and fill in the information requested.
Click save, then click add container. Give the puck barcode as the name, and choose spine puck, again click save. Click on the container symbol and edit, and fill in the necessary information concerning the samples such as protein name, preferred work flow, and crystal position, in the puck.
Choose the protein that has been approved by the ESRF safety group. Enter a unique sample name to identify each individual sample. Optionally, scan the pin barcode.
The rest of the information to include is also optional. For each individual sample, enter the experiment type. This defines which automatic workflow will be used to process each crystal.
Given that the GCSH crystals are needles, choose MX press P.Next, enter a space group. If present, this will be used for data collection strategy calculations, and by the automatic data processing pipe lines available. Enter the desired resolution.
This defines the crystal to detector distance from the initial mesh scans, characterization, and default data collection. Now, set the desired threshold resolution to prevent the collection of full data sets from crystals that do not diffract to this limit. This can save data storage space and analyses time.
Set the required completeness. Then set the required multiplicity. If more than one crystal is contained on the sample support, set the maximum number of crystals to be analyzed.
The default value is one or five for MX press P.The appropriate beam size can also be selected. Put in the space group, if known, in the force space group column. Then set the radiation sensitivity of the crystals.
If desired, set the total rotation angle to be collected for the full data set collection. Once all information has been input into the system, save the values. Click on, return to shipments and press send shipment to ESRF.
Print the shipping label and send the samples. Users should arrange a pickup with a courier using the ESRF account details. On the day of the experiment, samples are transferred to the Massive one high capacity dew-er.
Beam line scientists then launch the data collection, which can be followed by users remotely. For each different sample type, users receive an email informing them that the data collection has started. The execution of all steps in all workflows can be followed online and in real time.
This is accessible to the user by logging into ISPyB. The results can also be viewed and downloaded. For each sample analyzed, examine the results of the automatic experiment on ISPyB.
Click on the desired experimental session at ID30A1. Select the preferred auto processing pipeline. Download the data written out in the correct space group with the highest completeness and highest resolution.
By clicking on the last collect results and then download. The MX press P workflow was used by the ESRF beam line Massive one to fully automatically mount, center in the x-ray beam, characterize, and collect full diffraction data sets from a series of crystals of human GCSH. The samples were mounted and the loop analyzed for an area to scan.
After the diffraction analyses, four points were selected within the crystal for data collection. Manual structure determination by molecular replacement yielded a high quality electron density map after a single automated refinement cycle. For this data set, the automated pipeline cut the data at a one point three two angstrom resolution.
However, users can decide to cut the data at a lower resolution. Continuous electron density is visible for the entire amino acid chain apart from eterminal histidine tag. Of the four substitutions that distinguish human and bovine GCSH, three are readily identifiable in the electron density.
This is less clear for the aspartic acid to lysine 125 substitution. For which the electron density of the side chain is only partially resolved due to flexibility. The currently obtained model has an R work value of 20 point four percent and an R free value of 23 point eight percent and can be further optimized by additional cycles of automated and manual model building and refinement.
It's important to tailor the requirements to your system. For example, setting the already observed resolution to a known value can save time and ensure better data collection. Fully automatic data collection has democratized crystallography and now even small labs can embark on famous project when this was feasible before for only with enormous man power.
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—all without human intervention.
JoVE 소개
Copyright © 2024 MyJoVE Corporation. 판권 소유