PSF MX

Welcome to PSF MX

PSF MX offers 3D structure determination as a service to academic research groups. Please contact Martin Moche for questions. For researchers that want to work independently learning MX techniques, we also offers access to our crystallization infrastructure after suitable training sessions and provides crystal screening chemicals and consumables for use at PSF protein crystallography.  The crystallization plates calibrated for RockImager are listed here.

PSF MX also coordinates synchrotron beamtime in shape of block allocation groups (BAGs) that enable local researchers to send crystals for data collection at MAX IV and DIAMOND. We alre also involved in creating a remotely accessible software stack for structural biology available at NSC Tetralith in Linköping, LUNARC Cosmos in Lund and at the MAX IV cluster. The GPU resource NSC Berzelius is ideal for cryo-EM data processing so welcome to the PReSTO project! 

Since 2012, the PSF MX infrastructure have attracted 100+ researchers producing a large set of PSF related publications and around 15 researchers are actively using the setup today in 2023.

Crystallization infrastructure

• 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 MARCO autoscoring of crystallization experiments monitored by visual, polarized and UV light
• Synchrotron access to MAX IV and DIAMOND beamlines for diffraction data collection
• Access to software for protein structure determination and visualization in a high performance compute environment via PReSTO

Prices

Starting in 2021 all Karolinska Institutet core facilities use a web-based software solution known as iLab where clients and customers can request services and book instruments after registration. iLab are also used for invoicing and Protein Science Facility entry pages are:

1. PSF services in iLab
2. PSF instruments bookings in iLab

Crystallization infrastructure user 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 
• handle the plate sealer for sealing of deep well blocks containing crystal screens 
• setup experiments and add home made protein batches in RockMaker 
• basic RockImager handling

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 controls the two RockImagers (4C & 20C) and the Tecan liquid handler for dispensing Greiner deep well blocks or 24-well plates. 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)

IN 24-well format the crystallization drops are manually made, however our 24-well plates above can be imaged in RockImager 4C & 20C.

B) Liquid containers such as:

• 96 Well Greiner Deep Well blocks
• PCR tubes in CoolBlock to make crystal cryo solutions

for subsequent liquid transfer using Phoenix 96-well head into the wells of Mosquito 96-well 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 MX

New trainees are adviced to contact the PSF MX 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 BAGs for beamtime

PSF organises block allocation groups (BAGs) at MAX IV and DIAMOND and together we assign experimentalists at regular PSF MX user meetings.

MAX IV introduced remote visits as a result of Covid-19 and DIAMOND introduced "unattended" data collection while remote experiments are common since long time at Diamond. 

• Unattended Data Collection at Diamond Light Source

• MX Responsive Beamtime at Diamond Light Source

Please find dewar shipping instructions for DIAMOND and MAX IV.

DIAMOND opportunities

On the 16^th of December 2021 staff from Diamond Light Source presented “News from Diamond Light Source” covering modern use of their excellent structural biology facilities.

Dave Hall presented an overview of MX operation updates, access modes and unattended data collection status along with a few practicals regarding shipping tool maintenance, Brexit consequences, early puck definition for improved scheduling, ERA admin opportunities and use of Alpha Fold models in iSpyB for swift MR phasing after data collection. 

Armin Wagner presented long-wavelength crystallography at I23 for phasing and element identification discriminating calcium, potassium, magnesium etc. and we can contact the i23 team and use I23 without a dedicated application.

Ralf Flaig demonstrated the multi-axis goniometer called “SmarGon” and procedures for optimized data collection strategy including beamsize and dose for i04 SAD/MAD data collection.  

Halina Mikolajek gave an overview of the Diamond crystallization facility and possibilities for automated crystal harvesting or X-ray data collection from crystal plates using VMXi beamline.

Daren Fearon described how to access and perform efficient fragment screening at XChem.   Development of XChem ready crystal systems and EU partnerships for progressing hits to chemical probes was presented.  Quality criterion for academic labs interested in submitting BAG applications for XChem access was presented.

Andrew Quigley started by introducing the membrane protein laboratory accepting samples or visitors bringing clones, cells or purified proteins for crystallization, or EM grid preparation, prior to diffraction or imaging experiments.

Finally, Robin Owen shared the new i24 monochromator, undulator and CdTe Eiger detector enabling shorter 17 keV wavelengths to be used for diffraction experiments leading to higher resolution datasets for each crystal exposed by the i24 beam.  Multiple datasets collected from hundreds of small crystals mounted at thin films can be merged by multiplex DIALS automatically rejecting outlier crystals.  Modification of sample environments for serial crystallography and reaction triggering by lasers or chemicals delivered by micro drop ejectors were highlighted.

Beamtime schedule

Crystal Screens @ PSF

We purchase Crystal Screens from commercial vendors and reformat these from 10 ml tubes into 10 x 1 ml 96 Deep Well plates.  The data sheets for each screen is linked below.

Hampton research

1. 48_AmSO4+MPD - Prepared from 10 ml HR2-211/HR2-215
2. 48_PEG-Ion - Prepared from 10 ml Peg/Ion HR2-126
3. Additive Screen HT - Available in floor 4 freezer
4. Crystal Screen HT - Phoenix splashes frozen tert-butanol at 20C. From 10ml HR2-110/HR2-112
5. Index HT - Prepared from 10 ml Index HR2-144
6. Peg/Ion HT - Prepared from 10 ml Peg/Ion&Peg/Ion2
7. MembFac HT - Consist of MembFac (1-48) and Crystal Screen Lite (49-96)
8. Salt Rx HT - Salt-Rx 1 (HR2-107) and Salt-Rx 2 (HR2-109)
9. Natrix HT - Natrix (HR2-116) and Natrix2 (HR2-117) 

Jena Bioscience

1. Pentaerythritol_HTS

Molecular Dimensions

1. MemMeso - MD1-86
2. MemGold - MD1-39
3. MemGold2 - MD1-63
4. MemStartSys - Prepared from 10 ml MD1-21/MD1-25
5. Morpheus - MD1-46
6. ProPlex - MD1-38
7. BCS screen - MD1-104

Qiagen

1. AmSO4 - 130705
2. ClassicsLite - 130702
3. JCSG+ - 130720
4. Nucleix - 130719
5. PACT - 130718
6. PEGs suite - 130704
7. pH-Clear - 130709
8. Cubic_Phase_II_Suite
9. Procomplex 

Links to all Crystal Sceens from Hampton, Jena, and Molecular Dimensions.

HTP crystallization consumables provided by PSF: for use at PSF-MX platform only

PSF MX User Meetings

Please find protocols from PSF MX User Meetings