PSF Macromolecular X-ray crystallography is an infrastructure for structural biology including Mosquito-LCP, Tecan Freedom Evo and Formulatrix RockImager 1000 systems for imaging of crystallization experiments. PSF coordinates synchrotron beamtime block allocation groups at BESSY, DIAMOND and MAXIV and a software installation for Macromolecular X-ray crystallography at High Performance Compute resources of Swedish National Infrastructure of computing (SNIC) for our structural biology community.
Welcome to MX at PSF
All MX users are required to undergo training before access to the individual HTP crystallization instruments is granted and we also provide crystal screens and consumables for use at PSF protein crystallography.
Users are encouraged to send crystals for data collection at synchrotron sources such as BESSY, DIAMOND and MAX IV according to the beamtime schedule listed below. The protein crystallography software setup for data processing, structure determination, refinement and visualization are remotely accessible either at the National Supercomputer Center (NSC) in Linköping or at LUNARC Aurora from whatever computer the user might have - Welcome to PReSTO!
The PSF protein crystallography infrastructure have attracted more than 100 researchers since 2012 producing a large set of PSF related publications and around 20 researchers are active today (May 2019).
PSF MX 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, polarized and UV light
- Synchrotron access to BESSY, DIAMOND and MAX IV beamlines for diffraction data collection
- Computer and software access for protein structure determination and visualization
PSF HTP MX training
Session 1: Mosquito, Phoenix and basic RockMaker/Imager/Web training
Requires a purified protein batch for crystallization and is 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
- know how to make "hit reports" 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 MX manager 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 these 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.
HTP MX robots
For protein crystallization screening and optimization, a Mosquito LCP crystallization robot is available through a donation from Stiftelsen Olle Engkvist Byggmästare in 2011.
96 well sitting, hanging and LCP screening and optimization
- We recommend the sitting drop Corning 3550 plates due to A) 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 and B) Corning 3550 and Clear Seal Film are very UV compatible consumables.
- 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.
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.
- 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 into the wells of the crystallization plates to be used at the Mosquito robot. The Greiner Deep Well blocks are filled with the Tecan robot.
RockImager and RockMaker
RockImager is taking six images of each drop of each calibrated 24 or 96 well crystallization plate between day 0 to 56 where the upper limit of six 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 are in development.
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 and MAXIV allow for manual beamtime bookings. Beamtime experimentalists are decided on regular PSF user meetings and please find dewar shipping instructions for Diamond online and in PDF-format and online for BESSY. Covid19 is impacting on our MX beamtime schedules and more info at Diamond homepage and Bessy homepage.
New procedures for "unattended" and "remote" experiments at Diamond Light Source.
Welcome to HPC-MX
Together with Swedish National Infrastructure of Computing (SNIC) and MAX IV, PSF has installed MX software for use in a High Performance Compute environment available at SNIC resources NSC Tetralith in Linköping, LUNARC Aurora in Lund, and the BioMAX beamline in Lund. We call this installation PReSTO and it is accessible through terminals or a remote Linux desktop solution called Thinlinc. Thinlinc enables graphics software to run remotely which is an essential part of the macromolecular X-ray crystallography workflow. Please join PReSTO available to Swedish academic researchers
Crystal Screens @ PSF
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)
- MemMeso - MD1-86
- MemGold - MD1-39
- MemGold2 - MD1-63
- MemStartSys - Prepared from 10 ml MD1-21/MD1-25
- Morpheus - MD1-46
- ProPlex - MD1-38
- AmSO4 - 130705
- ClassicsLite - 130702
- JCSG+ - 130720
- Nucleix - 130719
- PACT - 130718
- PEGs suite - 130704
- pH-Clear - 130709
HTP crystallization consumables provided by PSF: for use at PSF-MX platform only