Protein Crystallography Platform
PSF protein crystallography harbors robotics for liquid handling (ml-scale with TECAN robot) and crystallization (sub-microliter drop dispensing with the mosquito-LCP dispenser), imaging systems (Formulatrix) and microscopes for crystal visualization, tools for crystal handling and mounting, Diamond and Bessy beamtime access and crystallographic software and super-computer HPC access through a collaboration with the Swedish National Infrastructure for Computing (SNIC).
Contact - MX manager
Get started at PSF protein crystallography
All users are required to undergo training before access to the individual instruments is granted. We provide crystal screens and consumables to our users.
Users are encouraged to send crystals for data collection at synchrotron sources such as BESSY and DIAMOND according to the beamtime schedule listed below. The protein crystallography software setup for data processing, structure determination, refinement and visualization are available to academic researchers both physically at PSF and remotely accessible at the National Supercomputer Center (NSC) in Linköping from whatever computer the user might have. Get started at NSC today!
More than 100 researchers have already been working with the PSF protein crystallography setup and around 50 researchers are active today (March 2015) producing a large set of PSF related publications.
PSF Protein Crystallography offers
- Mosquito-LCP for sitting drop & Lipidic Cubic Phase protein crystallization in 100 nL scale where Phoenix is used for 96 well liquid transfer
- Tecan liquid handler for 24, 48 and 96 well sitting/hanging drop crystallization condition optimization
- RockMaker/Imager/Web for manual and statistical design, visualization and scoring of crystallization experiments monitored by visual and polarized light
- CrystalLight 100 for protein material detection by UV fluorescence under the microscope
- Synchrotron access to BESSY and DIAMOND beamlines for diffraction data collection
- Computer and software access for protein structure determination and visualization
PSF X-ray User training
Session 1: Mosquito, Phoenix and basic RockMaker training
Requires a purified protein batch for crystallization.
Suitable to PSF Xray newcomers.
After first training session a fresh user should be able to
- handle the Phoenix robot for 96 well liquid transfer prior to running Mosquito
- handle Phoenix washing and refill of cleaning water with Benzalkonium chloride.
- handle the Mosquito robot for standard crystallization experiments in 48 and 96 well format
- handle the plate sealer for sealing of deep well blocks containing crystal screens available at PSF
- setup experiments and edit in their home made protein batches in RockMaker for imaging in RockImager
- know how to get crystallization plates in and out of RockImager
- know how to perform scoring, zoom in pictures, saving images and edit searches in RockMaker
The first training session should also contain information regarding:
- How to perform additive screening and robot seeding using Phoenix and Mosquito
- How to make edit changes into their own Mosquito protocols OBS! Never edit the standard protocol
- How to contact the PSF Xray for questions instead of making guesses when running the robots
Session 2: Tecan and more RockMaker training
Once the user obtained his/her first crystals to be optimized its time for the Tecan training - session 2.
RockMaker is controlling the two RockImagers or 4C and 20C imaging as well as the Tecan liquid handler for dispensing wells of 24 well crystallization plates or 96 and 48 well containers in shape of Greiner deep well blocks. The goal of the Tecan training is making each user able to dispense:
A) The wells of 24 well crystallization plates:
- 24 well hanging drop plates (24 Well Hampton - X drop) where X is 1-3
- 24 well sitting drop plates (24 Well IntelliPlate - 4 drop)
The crystallization drops are manually made, however both 24-well plates can be imaged in RockImager 4C & 20C.
B) Liquid containers such as:
- 96 Well Greiner Deep Well
- 96 Well Greiner Deep Well 8x6 (only six first columns used of 96 well block to save protein)
- PCR tubes in CoolBlock (to make cryo solutions)
for subsequent liquid transfer using Phoenix 96-well head into the wells of Mosquito crystallization plates
Dispensing ingredients from the 144 tubes and 3 troughs into these items require more RockMaker knowledge than given during Mosquito training. The use of Tecan troughs for user ingredients is recommended when users bring their own solutions to the Tecan robot since dispensing speed increases dramatically when all eight needles enter the trough simultaneously. Use of RackEditor for adding and deleting user made ingredients into Tecan troughs is discussed. After use deleting ingredients from RackEditor in RockMaker is very important to avoid problems for subsequent users.
Training session 2 gives a brief orientation regarding:
- Ingredient, stocks and liquid classes for Tecan dispensing
- Various items in use at PSF Xray
New trainees are adviced to contact the PSF Xray manager when
- adding their first new ingredients into RockMaker
- wanting to run other protocols than the standard protocol
- wanting to run alternative crystallization plates than Corning 3550
Use of Tecan for crystal screen reformatting from 10mL tubes into Greiner deep well blocks is not included and instructed separately when required.
PSF Protein Crystallization robotics
For protein crystallization screening and optimization, a Mosquito LCP crystallization robot is available through a donation from Stiftelsen Olle Engkvist Byggmästare.
96 well sitting, hanging and LCP screening and optimization
- We recommend the sitting drop Corning 3550 plates due to its conical drop wells that are guiding the mosquito needles towards its narrow and flat bottoms to enable high zoom when pictures are taken in RockImager.
- The iQ plate can be used for sitting drop optimization to ease crystal mounting.
- Crystal screening can also be done in 96-well hanging drop format however with twice the screen consumption due to the larger wells of the sitting drop plates.
- LCP screening under a humidity chamber and automated bech-top monoolein & membrane protein mixer using SwissCI and Laminex plates are possible.
Plates, tape and other consumables required for crystal screening and optimization can be found here.
Tecan liquid handler
The Tecan robot consists of eight dispensing needles and 144 tubes with septa with chemicals for long term storage. Chemicals can also be placed in the three troughs for fast dispensing using all eight needles simultaneously. Plates available for Tecan dispensing are listed here.
- 24 well 1-3 drop VDXm pre-greased plates (HR3-306)
- 24 well 4 drop IntelliPlate (HR3-114)
- 96 well Greiner Deep Well Blocks
- 48 well Greiner Deep Well (column 1-6 above)
- Reformat 96 x 10 ml tubes, ten 96 well Greiner
- PCR tubes in CoolBlock
Today the Phoenix robot is mainly used for 96 well liquid transfer from the Greiner Deep Well Blocks filled at the Tecan into the wells of the crystallization plates to be used at the Mosquito robot. Protein crystallization experiments can be dispensed at the Phoenix robot.
RockImager and RockMaker
RockImager is taking five images of each drop of each calibrated 24 or 96 well crystallization plate between day 0 to 56 where the upper limit of five is used to avoid queues and preserve disk space. PSF has two imagers running at 4ºC and 20ºC respectively. The plates calibrated for imaging are listed here.
Crystallization experiments are typically edited into RockMaker that keeps track of the experimental details regarding protein batches and well solutions, images taken and scores given. In essence RockMaker is a database where 24 and 96 well optimizations as well as cryo solutions can be designed to be subsequently dispensed by the Tecan robot and imaged by the 4ºC and 20ºC imagers. From the home laboratory RockMaker is accessed via RockMakerWeb. RockMakerWeb allows scoring and viewing of the ongoing experiments. Database tools for analysing scoring results and images with well solutions and protein batches content should be developed in the future.
Beamtime schedule and refunding
PSF submit reports and application for beamtime based on the results by its user community from previous beamtime allocations. Diamond create a beamtime schedule with fixed dates while Bessy enable manual bookings. Beamtime experimentalists are decided on regular PSF user meetings.
|Bessy||BL14-2||12 Nov 2016||24||10:00||X-ray User Meeting #39|
|Bessy||BL14-2||1 Dec 2016||24||10:00||X-ray User Meeting #39|
|Diamond||i04-1||19 Dec 2016||24||10:00||X-ray User Meeting #39|
|Diamond||i24||28 Jan 2016||24||10:00||To be determined|
|Bessy||BL14-2||2 Feb 2017||24||10:00||To be determined|
|Diamond||i04||24 Feb 2017||24||10:00||To be determined|
|Diamond||i03||5 Mar 2017||24||10:00||To be determined|
Protein crystallography software
PSF Linux software environment was are currently in upgrade into Scientific Linux 6 (June 2015) and a new list of software and settings files available below. In addition PSF are migrating its entire Protein Crystallography setup into National Supercomputer Centre in Linköping since their computers run faster and are accessible from any mac/PC/Linux computer with an internet connection. Since we are new to working in a HPC environment a startup guide directed to our pilot project named "A pilot macromolecular 3D structure determination project" has been developed
- Software Available and user settings at PSF Linux computers
- Getting started using National Supercomputer Center (NSC)
PSF crystal screens
We purchase Crystal Screens from commercial vendors that are reformatted from 10 ml tubes into 10 x 1 ml 96 Deep Well plates. The data sheets for each screen is linked below and Qiagen publish online production reports of interest during optimization.
|48_AmSO4+MPD||Prepared from 10 ml HR2-211/HR2-215|
|48_PEG-Ion||Prepared from 10 ml Peg/Ion HR2-126|
|Additive Screen HT||Available in floor 4 freezer|
|Crystal Screen HT||Phoenix splashes frozen tert-butanol at 20C. From 10ml HR2-110/HR2-112|
|Index HT||Prepared from 10 ml Index HR2-144|
|Peg/Ion HT||Prepared from 10 ml Peg/Ion&Peg/Ion2|
|MembFac HT||Consist of MembFac (1-48) and Crystal Screen Lite (49-96)|
|Salt Rx HT||Salt-Rx 1 (HR2-107) and Salt-Rx 2 (HR2-109)|
|MemStartSys||Prepared from 10 ml MD1-21/MD1-25|
|Qiagen||Generic online recipe: NeXtal 10 ml tubes/Lot ID any number|
PSF Xray Newsletter
Additional information and links