Norstruct Offers Different Service Packages

Service package 2: Structure determination

The Norwegian Structural Biology Centre (NorStruct) offers three service packages:

SP1: Protein production SP2: Structure determination SP3: Drug discovery and design

NorStruct's SP2 “Structure determination” services include protein sample evaluation tasks of quality control and stability/solubility optimization, crystallization trials, data collection, using either the home X-ray source or synchrotron radiation, followed by structure determination and refinement.

Prizes and more details about our standard commissioning service packages can be found at the NorStruct web page http://uit.no/norstruct/.

Table 1 Standard commissioning in SP2 Structure determination. Service Task Specification Package (SP) SP2.1 Quality control Dynamic light scattering (DLS) spectra at 4-37°C, SDS- PAGE, Mass spectroscopy (MALDI-TOF).

SP2.2 Initial crystallization Set-up of 96 robotic nanolitre crystallization conditions at 3 screening different mixing ratios to estimate an optimum protein concentration for subsequent crystallization trials.

SP2.3 Robotic nanolitre Set-up of a minimum of 480 robotic nanolitre crystallization crystallization conditions with subsequent grid optimization of promising screening crystallization conditions identified.

SP2.4 Buffer optimization Search for improved solubility and homogeneity 24 different buffer-conditions (pH-range from 4-9), followed by testing of 12 different additives. Thermofluor methods will be used as a standard.

SP2.5 X-ray data collection “ Standard” data collection and processing, phasing, using and structure molecular replacement (MR) or single anomalous dispersion determination (SAD) techniques, and refinement. More challenging projects can be customized.

The Norwegian Structural Biology Centre University of Tromsø, N-9037 Tromsø, Telephone: (+47) 77 64 40 00, Fax (+47) 77 64 47 65 Direct telephone: (+47) 77 64 40 70, E-mail: [email protected] http://uit.no/norstruct/ Preliminary order form for: Structure determination services Please place tick marks to indicate which tasks are desired. For simplicity this icon  can be copied and replaced by the empty boxes. Return this electronic form by E-mail to NorStruct, and you will receive in response a tailored contract for placing the final order.

Order:

Date: ………

Title (and NorStruct project number if availablea):

Customer: Institution: Adress:

Contact person: E-mail: Phone: a Provided by NorStruct

Order: SP Task Specification (Tick) SP2.1 Quality control SDS-PAGE From the customer (mandatory) Estimated purity: ……. %

Mass Provided by the customer spectroscopy: OR To be done by NorStruct SP2.2 Initial crystallization screening

SP2.3 Robotic nanolitre . crystallization screening

SP2.4 Buffer optimization

SP2.5 X-ray data collection and structure determination

The Norwegian Structural Biology Centre University of Tromsø, N-9037 Tromsø, Telephone (+47) 77 64 40 00, Fax (+47) 77 64 47 65 Direct telephone (+47) 77 64 40 70, E-mail: [email protected] http://uit.no/norstruct/

- 2 - General information:

Details Information

Protein name:

Batch identification:

Original source: .

Expression system and vector: Tag (His, GST, MPB; etc.)

Total amount protein (mg):

Total volume (l)b:

Protein concentration (mg/ml): Indicate Method: OD280, Bradford assay or other Recommended storage - 80 °C temperature at NorStruct: - 20 °C 4°C b When shipping, frozen samples protein should be sent in aliquots of 25-50 l (consult NorStruct).

Protein properties

Protein details Information

Molecular weight (kDa): Number of amino acids (including the tag): pI (theoretical and/or measured):

Amino acid sequence of this Please paste the amino acid sequence here (please construct: mark tag and linker regions):

GenBank accession number:

- 3 - Success in crystallization and structure determination requires detailed knowledge of the biochemical and biophysical properties of the protein, including knowledge of all chemicals that may have been carried over from fermentation and purification. Trace chemicals and especially potential ligands may be of particular importance. A brief description of the protocols may be useful to us.

Protein purification details

Protein purification steps Details

Purification steps Please list all steps here, including buffers Ni2+ column; ion exchange, gel filtration, etc. Storage buffer of the protein: Salt concentration, buffer, pH, additives: etc.

Protein stability and solubility details (if available)

Stability Question Information details pH 1) pH-range where the protein is known to be 1) stable? 2) pH optimum for stability? 2) Temperature 1) Is the protein particularly unstable at low or 1) high temperatures? 2) Can or must it be frozen at -20 or -80 °C? 2) 3) Temperature optimum for stability? 3) Additives 1) Are there known buffers or other additives 1) that destabilize the protein? 2) Are there metals or ions that stabilize the 2) 2+ 2+ - 2- protein (Mg , Zn , Mn, Cl , SO4 , etc)

Stabilizers 1) Should stabilizers, inhibitors, co-factors, etc. 1) be added to the protein solution? Detergents/ 1) Is addition of salt or detergent essential for 1) salt keeping the sample soluble?

Additional information

Factors Question Information

Glycosylation 1) Is glycosylation known for this protein sample? 2) Is it at all something to consider? Cystine/Oxidation 1) Are there free cysteines in this protein? 1) 2) Can/should we use reducing agents? 2) 3) Should we use oxygen free 3) atmosphere? Polypeptide chains 1) Is there more than one polypeptide 1) chain forming the quaternary structure? The Norwegian Structural Biology Centre University of Tromsø, N-9037 Tromsø, Telephone (+47) 77 64 40 00, Fax (+47) 77 64 47 65 Direct telephone (+47) 77 64 40 70, E-mail: [email protected] http://uit.no/norstruct/

- 4 - Membrane 1) Is this a membrane or membrane 1) associated protein? Micelles? Domains 1) Are there multiple domains? 1) 2) Are domains separated by flexible 2) hinge regions? Oligomerisation 1) Is non-specific oligomerisation a known 1) property? 2) Is the protein a well defined multimer 2) (dimer, trimer etc.)? Other details

Contact person(s)

Please provide NorStruct with contact information in case more information regarding the protein samples, especially with respect to the details of protein purification, is needed:

Name E-mail Phone