Structural Biology Core Facility

Facilities and equipment

The facility hosts a temperature controlled laboratory (22°C) with a cold room dedicated for protein crystallization. The protein crystallization laboratory is equipped with TTP Lab Tech Mosquito LCP nanodispenser and TECAN Freedom EVO liquid handling robot for setting-up crystallization experiments, three Formulatrix Rock Imagers (at 22°C and 4°C) for imaging of the crystallization experiments and several microscopes for inspection of crystallization drops. Dedicated areas, equipment and tools are available for crystal storage, handling, shipment to synchrotrons and in house data collection.

Sample requirements

Protein sample intended for crystallization has to be pure and preferably not in phosphate buffer. The buffer concentration should be around 25 mM without any additional salt. Protein concentration should be approximately 5 mg/ml. The minimum required quantity for one screen with 96 crystallization conditions with 50 nl drop volume is 8 μL (40 µg protein in total).

Screening initial crystallization conditions

As a standard procedure, the initial crystallization conditions are screened with a sitting-drop vapor-diffusion method in a 96-well plate using crystallization screens made in-house or using commercial screens. The protein solution is mixed in different ratios with the crystallization solutions in a 100-200 nl -drop volume using a TTP Lab Tech Mosquito LCP nanodispenser available at the protein crystallization laboratory (F028). Hanging-drop vapor-diffusion as well as microbatch under oil method in 96-well format can also be used for initial screening. It is recommended to setup the crystallization experiments at room temperature as well as at 4°C.

Optimization

If there are crystals or “promising” precipitate the crystallization conditions are optimized varying the pH and the precipitant concentration around the condition of the hit. The optimization screen is prepared with the CrysScreen software and pipetted with the TECAN Freedom EVO liquid handling robot. Stock solutions are available for the in-house screens. If a suitable crystallization condition has been found, it is also worthwhile to optimize the condition using the hanging-drop (or sitting-drop) vapor diffusion method in 24-well plates and bigger drops (1 μL + 1 μL) from which the crystal mounting is easier and the crystals tend to grow to a larger size. Macro and micro seeding methods can also be used.

Crystallization plates

Crystallization experiments are set-up into a 96-well or a 24-well crystallization plates depending on the experiment (screening, optimization, soaking, hanging drop, sitting drop). For the sitting-drop vapor diffusion set-ups we are using the TTP Lab Tech Triple Sitting Drop plate, Corning 3556 plate and the Greiner 609101 CrystalQuick Crystallization plate. The microbatch under oil screen, where a small volume of protein solution and the crystallization solution are pipetted underneath an oil layer, are either carried out using the Douglas microbatch plate using the Tecan robot (0.5-1 µl) or Swissci 3 well plate (Hampton Research) using Mosquito pipetting robot (50-1200 nl).

Imaging

The crystallization experiments on the 96-well plates are imaged with Formulatrix Rock Imagers; RI54 at room temperature, and RI54 and RI2 imagers in a cold room at +4°C. Both RI54 imagers are equipped with a UV imaging option. Images of the crystallization experiments can be viewed via a web browser using the IceBear software which is being developed locally in collaboration with Diamond Light Source (Didcot, UK), Weizmann Institute of Science (Rehovot, Israel) and Instruct-ULTRA for the viewing and documentation of the crystallization experiments and for data tracking of protein crystal handling.

Contact person:

Ville Ratas, Lab technician

Faculty of Biochemistry and Molecular Medicine F210

University of Oulu

PO Box 5400, 90014 University of Oulu, Finland

tel. +358-(0)294 481174

e-mail: ville.ratas (at) oulu.fi

Structure determination

In order to determine the protein crystal structure the phase problem has to be solved. One commonly used method to solve the phases is the molecular replacement method where an existing protein structure with high sequence (>25%) identity is used as a search model. If there is no homologous protein structure available in the RCSB or PDBe Protein Data Bank the phases can be solved by experimental phasing using heavy atoms such as Pb, Hg, Pt or Au incorporated into the protein crystal by soaking or Se incorporated into the protein by metabolic labeling using Selenomethionine. In the special cases the structure can also be solved by using the anomalous signal from the sulphur atoms present in the protein (Sulphur-SAD).

The crystallographic computing is done in a Linux environment. For structure determination, model building and refinement there are several tools available in the software packages installed on our linux system. The most important software packages are:

• CCP4
• Phenix
• Coot
• HKL2MAP which is a graphical user interface to run SHELXC, SHELXD, SHELXE

For an automated crystal structure determination there is an Auto-Rickshaw web server available at EMBL-Hamburg.

Structure analysis

In the final stages of the protein model building and before the deposition at the RCSB or EBI, the model is validated and the analysis of the structure is carried out. For the structure validation, analysis and visualization there are several tools available in the web as well as in the software packages installed in our Linux computing system.

Validation and analysis tools:

• CCP4
• Phenix
• Coot

A short guide how to validate an existing PDB-entry is provided here.

Visualisation:

• Pymol
• CCP4MG

Important servers for structure validation:

• MolProbity
• Structure deposition

Server for analysis of protein interfaces:

• PISA

Contact person:

Tiila-Riikka Kiema, PhD

Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine F210

University of Oulu

PO Box 5400, 90014 University of Oulu, Finland

tel. +358-(0)294 481174

e-mail: tiila.kiema (at) oulu.fi

For the X-ray diffraction data collection we have the Bruker Microstar X8 Proteum Cu-rotating anode X-ray generator equipped with a Helios MX focusing optics and kappa goniometer (Figure 1 and 2). The wavelength of the X-ray beam is 1.5418Å. The data is collected with a Platinum 135 CCD detector. Data collection is carried out at room temperature or with cryo-cooled crystals. The cryounit is an Oxford Cryostream 700 series. Data collection at cryo temperature is usually done at 100K (30 degrees above the boiling temperature of liquid nitrogen).

The size of the beam at the crystal is 160 micron (FWHM). The optimal size of the crystals is 100-200 micrometer in diameter. The total flux is 0.6x1010 photons/sec. There are three collimators, providing beam divergence of 7, 5, and 3 mrad (10mrad corresponds to a beam divergence of 0.6 degrees). For large cell dimensions and high quality data it is best to use the 3mrad collimator. In that case the total flux is about 3 fold less as compared to the flux obtained with the 7mrad collimator. Data processing is done with the Proteum3 software.

The kappa axis is at 50° with respect to the ω-axis. The crystal can be rotated about Φ and ω, allowing for an efficient collection of a complete and highly redundant dataset. The detector can be rotated about 2θ, allowing for collecting data to high resolution.

Diffraction data is also regularly collected at synchrotrons. Occasionally, the data collection is done remotely, from our laboratory. For these data collection efforts we maintain storage dewars and transport dewars. For the freezing of crystals a range of loops are available, including bar-coded loops, which are in particular important for synchrotron visits. The data processing is done routinely with XDS or iMOSFLM.

For data tracking we use and develop (in collaboration with Diamond Light Source (Didcot, UK), Weizmann Institute of Science (Rehovot, Israel) and Instruct-ULTRA), the IceBear software package.

Contact persons:

Tiila-Riikka Kiema, Ph.D., Data collection

Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine F210

University of Oulu

PO Box 5400, 90014 University of Oulu, Finland

tel. +358-(0)294 481174

e-mail: tiila.kiema (at) oulu.fi



Kristian Koski, Ph.D., Data collection and remote synchrotron data collection

Faculty of Biochemistry and Molecular Medicine F210

University of Oulu

PO Box 5400, 90014 University of Oulu, Finland

tel. +358-(0)294 481174

e-mail: kristian.koski (at) oulu.fi



Ed Daniel, Software-application designer, IceBear development

Faculty of Biochemistry and Molecular Medicine F202C

University of Oulu

PO Box 5400, 90014 University of Oulu, Finland

tel. +358-(0)294 481151

e-mail: edward.daniel (at) oulu.fi

The IceBear (Integrated Crystal-data-tracking Enhancing Biochemistry Education And Research) software, developed and installed at FBMM, is designed to help the FBMM researchers to follow the results of their crystallization experiments from their desk (or from outside the workplace, anywhere in the world). It is also possible to annotate these results.

The FBMM IceBear server can be reached at https://oulu.icebox.icebear.fi. There is a demo account, username and password both demo. Note that the demo account is read-only and has very limited access; you will need a personal IceBear account to view your own plates and to use all the available functionality.

For more information about IceBear, please visit the project website at https://www.icebear.fi/.

Further information on the use and setting up an individual account can be provided by Ed Daniel.

Should you wish to install IceBear in your own lab, it is available under an MIT license. Please get in touch to discuss your requirements.

Contact person:

Ed Daniel, Software developer

Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine F202C

University of Oulu

PO Box 5400, 90014 University of Oulu, Finland

tel. +358-(0)465 218511

e-mail: edward.daniel (at) oulu.fi

To determine the molecular weight, the oligomeric state and the homogeneity (monodispersity) of the protein samples we have the Wyatt MiniDawn multi-angle light scattering (MALS) detector together with a Wyatt Optilab refractive index (RI) –detector (for the determination of the protein concentration) connected to the Shimadzu HPLC purification unit. With this method the absolute molecular weight is obtained without the calibration of the size exclusion-column. A small sample of 30 μL with a protein concentration of 2 mg/ml is sufficient.

Contact persons:

Kristian Koski and Ville Ratas

Faculty of Biochemistry and Molecular Medicine, F210

University of Oulu

PL 3000, 90014 University of Oulu, Finland

tel. +358-(0)294 481174

e-mail: kristian.koski (at) oulu.fi, ville.ratas (at) oulu.fi

Wyatt DynaPro Plate Reader II can be used for example for assessing the quality of purified protein samples; it can analyze whether the sample is monodisperse and free of aggregates. It can also be used for size determination of proteins, for screening optimal buffer/additive conditions for protein solutions, for determining critical micelle forming concentrations etc. What makes the DynaPro plate reader very user friendly, is its ability to read protein samples directly on multiwell (96-1536 well) plates without transferring the sample to a separate measurement cuvette. This minimizes the risk of contamination and makes the measurements fast. In addition of the DLS function the instrument has a camera that can take pictures of the wells for the visual inspection of possible aggregates present in the sample.

Contact persons:

Kristian Koski and Ville Ratas

Faculty of Biochemistry and Molecular Medicine, F210

University of Oulu

PL 3000, 90014 University of Oulu, Finland

tel. +358-(0)294 481174

e-mail: kristian.koski (at) oulu.fi, ville.ratas (at) oulu.fi

The Jasco V-660 spectrophotometer is a double monochromator, double beam instrument that enables high resolution, precise kinetic measurements of enzymes. Due to the cell-holder set-up, 6-8 enzyme samples can be analyzed in one run, making for example Km, kcat as well as Ki measurements fast to perform. The instrument has a water thermostated automatic cell changer which enables the measurement at the temperature range of 10-90 °C. The stirring option using magnetic beads facilitates uniform mixing of the samples.

Contact person:

Tiila-Riikka Kiema, Ph.D.

Faculty of Biochemistry and Molecular Medicine F210

University of Oulu

PO Box 5400, 90014 University of Oulu, Finland

tel. +358-(0)294 481174

e-mail: tiila.kiema (at) oulu.fi

Stopped-flow instrument is a rapid mixing device combined with a spectrofluorometer used for pre-steady state kinetic measurements. We have a TgK Scientific Hi-Tech KinetAsyst Stopped-Flow system, which can be operated in a single and double mixing mode and which can be used to detect absorbance and fluorescence signals. There are three light sources (Xenon, Xenon-Mercury and Tungsten), three photomultipliers, a rapid scanning CCD detector and a set of cut-off filters providing versatile possibilities for measurements. Fluorescence polarisation accessory is available for polarisation/anisotropy detection. With the standard cell, the dead-time of the instrument is 1-2 ms and the minimum shot volume is 40 µl.

Contact person:

Tiila-Riikka Kiema, Ph.D.

Faculty of Biochemistry and Molecular Medicine F210

University of Oulu

PO Box 5400, 90014 University of Oulu, Finland

tel. +358-(0)294 481174

e-mail: tiila.kiema (at) oulu.fi

Some useful characterization for the protein sample before the structural studies, such as CD spectroscopy or ITC, can be done in the Protein biophysical measurements and amino acid analysis facility.

It is assumed that the protein has been well purified and is pure on an overloaded SDS-PAGE analysis. As a rule of thumb, the protein samples obtained from his-tag affinity chromatography are not pure enough. It is best to have at least one ion exchange column in the purification protocol, in order to avoid charge heterogeneity of the protein sample. The last purification step is always a size-exclusion column. The best crystallization results are obtained when using only the central part of the peak fractions. Each characterization technique has different requirements for buffer, buffer concentration and protein concentration. Consultation with the local staff is required to discuss these issues, as they depend also on the properties of the protein to be investigated. Knowledge of the isoelectric point of the protein is important for setting up the ion exchange chromatography.

The Infinite M1000 Pro multimode plate reader is a fast and sensitive instrument that can measure well plates using different techniques including absorbance, fluorescence intensity, fluorescence polarization, time-resolved fluorescence, luminescence and AlphaScreen. It can read plates with up to 1536 wells. The plate reader is based on monochromators enabling flexible wavelength selection between 230-850 nm. The instrument has also dispensers for adding reagents to the wells during the measurement protocols.

Contact persons:

Lari Lehtiö, Ph.D.

Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, F103

University of Oulu

PL 3000, 90014 University of Oulu, Finland

tel. +358-(0)294 481169

e-mail: lari.lehtio (at) oulu.fi

Kristian Koski, Ph.D.

Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine F210

University of Oulu

PO Box 5400, 90014 University of Oulu, Finland

tel. +358-(0)294 481174

e-mail: kristian.koski (at) oulu.fi

Mantis uses microfluidic chips that can accurately dispence fixed volumes of 0.1µL/0.5 µL (low volume chip) or 1 µL/5µL (high volume chip). Positive displacement method makes it possible to dispence accurately solutions to up to 1536-well assay plates and to design simple optimization screens for crystallization.

Contact persons:

Ville Ratas, Lab technician

Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, F210

University of Oulu

PO Box 5400, 90014 University of Oulu, Finland

tel. +358-(0)294 481174

e-mail: ville.ratas (at) oulu.fi

Sven Sowa

Faculty of Biochemistry and Molecular Medicine

University of Oulu

PO Box 5400, 90014 University of Oulu, Finland

tel. +358-(0)294 481167

JANUS liquid handling workstation has 8-channel head and plate gripper. It can be used to prepare source plates to acoustic dispensed and for filling various assay plates up to 384-well format. JANUS can also use disposible tips to avoid washing and to prevent possible carry over.

Contact persons:

Ville Ratas, Lab technician

Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, F210

University of Oulu

PO Box 5400, 90014 University of Oulu, Finland

tel. +358-(0)294 481174

e-mail: ville.ratas (at) oulu.fi

Mirko Maksimainen, Ph.D.

Faculty of Biochemistry and Molecular Medicine

University of Oulu

PO Box 5400, 90014 University of Oulu, Finland

tel. +358-(0)294 481167

e-mail: mirko.maksimainen (at) oulu.fi

The Echo 650 acoustic dispenser can transfer crystallization solutions and protein samples to crystallization plates in 2.5 nl increments contact free using acoustic energy. Echo can be also used for a range of liquid transfer needs including compound libraries to an assay plate. Echo compatible source plates are available through the core facility.

Contact person:

Ville Ratas, Lab technician

Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, F210

University of Oulu

PO Box 5400, 90014 University of Oulu, Finland

tel. +358-(0)294 481174

e-mail: ville.ratas (at) oulu.fi

• The crystallization of one protein is tested in two temperatures. The service setup includes two well-plates with 96 crystallization conditions / plate
• The service is a pretest for a broader service (Screening of crystallization conditions or Optimization of crystallization condition)
• The successful execution of the service requires purified protein solution (1-10 mg/ml), which is needed at least 5,5 µl / well-plate (optimum 40 µl / well-plate)
• The customer receives preliminary information about the crystallizability of the protein (and possibly suitable crystals for the collection of diffraction data)
• The service price is 130 € / protein
• The service can be initiated within one week after protein delivery and completed within one week after initiation

Additional services:

• The quality check of protein with SEC-MALS&QELS technique 150 €
• The optimization of protein sample buffer content with DLS instrument 75 €

• The crystallization of one protein is screened in different conditions. The service setup includes at maximum 576 crystallization conditions / temperature
• The temperatures used are +4 °C and +22 °C
• The progression of the crystallization in every condition is photographed once a day for the first week and once a week after that.
• The customer receives information about the crystallizability of the protein (and possibly suitable crystals for the collection of diffraction data). The customer can follow the progression of the crystallizations through IceBear software.
• The service price is 60 € / well-plate (minimum 4 well-plates)
• The service can be initiated within one week after protein delivery and completed within one month after the initiation

Additional services:

• Optimization of crystallization condition
• Special conditions according to separate offer

• The crystallization of one protein is optimized, or the quality and size of protein crystals identified in earlier conditions are being improved
• The service is a continuation for Crystallization test or Screening of crystallization conditions
• The successful execution of the service requires purified protein solution (1-10 mg/ml), which is needed at least 5,5 µl / well-plate (optimum 40 µl / well-plate)
• The customer receives suitable crystals for the collection of diffraction data or conditions for additional optimization of crystallization
• The service price is 150 € / protein
• The service can be initiated within one week after protein delivery and completed within one month after the initiation

Additional services:

• Collection of diffraction data
• Special conditions according to separate offer (e.g. additives, seeding)

• One crystal is characterized under the x-ray beam
• The crystal emerged in Crystallization test, Screening of crystallization conditions or Optimization of crystallization condition is picked from well-plate and the diffraction is tested with X-ray beam
• The service reveals the quality of the crystal, resolution and possibly the symmetry and size of the unit cell
• The customer receives information about the suitability of the crystal to the structural determination of the protein (if the crystal was protein)
• The service price is 120 €
• The service will be delivered within a week

Additional services:

• Collection and processing of crystal data
• Characterization of an additional crystal (during the same day) 40 € / crystal

• The diffraction data is collected from one protein crystal and the data is processed for structure determination
• The service requires Characterization of a crystal
• The protein crystal is centered to the x-ray beam for test measurements (2 angles, 5 pictures), and the strategy for overnight data collection is determined
• The customer receives a report and processed crystal data for protein structure determination
• The service price is 175 €
• The service will be delivered within a week

Additional services:

• Additional data collection from the same crystal 150 € / 24 h
• Primary survey of the crystal data 100 €

• Protein structure is determined from protein crystal data
• Crystal data can be collected by X-ray diffractometry or synchrotron
• The service includes a kick-off meeting with the customer to agree the content and milestones of the service
• The customer receives the coordinates of the solved protein structure
• The service price depends on the case and is base on the rate 70 € / h
• The service delivery time depends on the case

FBMM has a highly developed infrastructure for the purification of recombinant proteins. The facility hosts several purification systems, with or without cooling cabinets, as well as a dedicated labs for protein purification (F205, F208A), which also includes a Nanodrop 1000 spectrophotometer, gel documentation device ChemiDocTM XRS+ (Bio-Rad) and other devices related to protein purification. Many devices, especially those locating in the protein purification lab, are also available to non-academic users. For the academic users we can provide help for: i) planning your protein purification protocol, ii) running your experiments, and iii) analyzing your results (for a basic price or via collaboration). For regular or large-scale purifications research groups must provide required consumables by themselves. Some consumables, like columns, certain column material and SDS PAGE system, we can only provide for test runs. For the commercial users the devices will be provided using an hour- or daily-based rent depending on the device. Weekly or monthly “package offers” for a set of specific devices including working place in the F208 laboratory are also provided if needed.

Contact persons:

Kristian Koski and Ville Ratas

Faculty of Biochemistry and Molecular Medicine, F210

University of Oulu

PL 3000, 90014 University of Oulu, Finland

tel. +358-(0)294 481174

e-mail: kristian.koski (at) oulu.fi, ville.ratas (at) oulu.fi