TEST OF THE GRANADA CRYSTALLISATION BOX IN SPACEeea.spaceflight.esa.int/attachments/spacestations/... · Final Report (Part II of II) Test of the Granada Crystallisation Box in the

Final Report (Part II of II) Test of the Granada Crystallisation Box in the ISS

TEST OF THE GRANADA CRYSTALLISATION BOX

IN SPACE

Final Report Part II

Date: August, 2003 Written by: Prof. Juan Ma Garcia-Ruiz, Dr. Luigi Carotenuto and Dr. Eva Mañas

This document is the last and final report of the work performed under ESTEC Contract entitled "Granada Crystallisation Box".



APPENDIX 1

Invitation letter


APPENDIX 2

Proposal form


QUESTIONNAIRE TO BE FILLED OUT FOR EACH MOLECULE PROPOSED

Data of the Research Group Proposing scientist: Institution: Address:

Phone: Fax: Email: Data of the proposed macromolecule Name of the macromolecule Source: Biological function Molecular weight Iso-electric point Thermal stability Influence of ageing on the stability of the solution.

Buffer Precipitating agent Additives (if any) Protein net charge versus pH

Crystallisation pH Storage temperature Crystallisation temperature Crystallisation method PLEASE, FILL BELOW IN THE

APPROPRIATE BOXES 1) Hanging or sitting drop Components and starting concentration in the drop

Components and starting concentration in the well

Waiting time for crystals 2) Batch method Components and starting concentration in the batch

Waiting time for crystals

The submission of this questionnaire to ESA implies the acceptance by the proposing scientist of the conditions and objective of the overall project as outlined in the invitation letter to which it responds, and his/her commitment to contribute as required to the analysis of the results of the flight-test.


3) Free Interface diffusion Components and starting concentration of the protein solution

Components and starting concentration of the precipitating agent solution

Waiting time for crystals 4) Dyalisis Components and starting concentration in the solution inside the bag or button

Components and starting concentration in the solution outside the button

Waiting time for crystals (Best) X-ray data collection details of the proposed macromolecule Crystal size (mm) X-ray source Wavelength (Å) Oscillation angle Detector distance (mm) Space group Unit cell Resolution range (Å) Unique reflections Completeness Reflections with I/σ(I) > 2

Rmerge (%) Outer resolution shell Resolution range (Å) Unique reflections Completeness Reflections with I/σ(I) > 2


APPENDIX 3

Previous results report


Granada, July 25, 2001

Dear colleagues, Thank you very much for your contribution to the pre-flight scientific and technical activities related to the test of the Granada Crystallization Facility. As you know, our experimental test will fly on board of the French-Russian Mission Andromede. The launch date has been advanced and right now is scheduled for August 21, the exact time to be defined.

As seems to be typical of the experimentation in microgravity, we can tell you that the logistic aspect concerning the flight activities is by no means an easy task. We do not want to bore you with the details, the only important matter is that we do not know yet if we will assemble the experiment either in Baikonur (the Russian cosmodrome in Kazajstan) or in Moscow. This will change a little the configuration for each crystallization box but we are ready to fulfill either scenario. To become familiar with the technical aspects of this research, you can download from our web page a PowerPoint file (http://lec.ugr.es/GCF.ppt) which is a presentation of the whole assembly process of the Granada Crystallization Facility to be done before the flight. Depending on where we do this assembly, the waiting time to have the box orbiting in the ISS will change. We have to decide then, using the

computer simulation performed in cooperation with MARS center in Napoli, the final penetration length of the capillaries, which right now is 10 mm. Figure on the left, shows a typical output of the simulation. The first run of experiments has just finished last Friday 20 of July with the visit of the last participants to set-up their first experimental proofs in our lab. We have now a better understanding of the crystallization behavior of the 24 proteins we were considering for this test. As the flight will be launched 21 of August, we have no time to optimize the crystallization conditions but we have enough

information to tune the set-up of the experiments. In a few days we will send you a report of the experimental setup of your protein/s as well as the results till now (if any) and our comments. We have finally selected your protein/s to be included in this flight experiment and we need from you now to receive the protein solution, the buffer and the additives for both the flight and the on-ground test. We would like to receive it NOT LATER THAN AUGUST 5, in order to prepare properly the logistic of the trip to Baikonur. How much protein? It depends on its availability and the total number of capillaries you want to try. As you know each GCB can be loaded with six capillaries. One of the main objectives of the test is to find the influence of the capillary diameter on the convective flow versus the diffusive flow and sedimentation. The following table shows the volume of the capillaries as a function of the capillary diameter (for a height of the solution of 60 mm).


Diameter (mm)

Volume (µl)

0.2 4 0.3 10 0.4 15 0.5 20 0.6 25 0.7 30 1.0 50 1.5 70

According with the values of this table and your personal decision about which capillaries you want to test the crystallization of your protein/s, you should send us at least:

Volume (µµµµL) = ∑∑∑∑(Nº capillaries x Vol. Capillary) x 2 x 1.4 Obviously, the factor 2 is because we must perform the on-ground counterpart and the term 1.4 is a factor we estimate for volume loosen during manipulation. Example: if you want to use the following combination of capillaries to crystallize your

protein: 1 capillary of 0.2 mm, 2 capillaries of 0.3 mm, 1 capillary of 0.6 mm, 1 capillary of 0.7 mm and 1 capillary of 1.0 mm

you should send us AT LEAST:

Volume = {(1x4) + (2x10) + (1x25) + (1x30) + (1x50) } x 2 x 1.4 = 361.2 µµµµL

Please, keep in mind that this is just an example. Feel free to choose the capillaries you want to use. Do not hesitate to contact us if you have any question. Looking forward to receiving your samples, Yours sincerely, Prof. J.M. Garcia-Ruiz Laboratorio de Estudios Cristalográficos Instituto Andaluz de Ciencias de la Tierra CSIC – Universidad de Granada Av. Fuentenueva s/n 18002 – Granada Tfn.: + 34 958 243 360 Fax: +34 958 243 38


APPENDIX 4

After launch letter


Granada, August 31, 2001

Dear colleague,

The first part of the project is done. We settled the experiments in Baikonur, the Russian space launch facility area in Kazakhstan. RKK-Energia provided an air-conditioned room of the hotel in Baikonur where the temperature was measured to be 20ºC (±1.5) during the four days we were working with the preparation of the experiment (see attached figures in a PowerPoint presentation “Short report.ppt”). Only two members of the LEC were allowed to fly to Baikonur and therefore the work was hard, with an average sleeping time of three hours per day. We did our best. The precipitating agent was poured 20 hours before the launch, i.e., the box was under microgravity conditions much earlier (about three days, according with our calculations) than the precipitating agent reaches the protein. The ground model is now in a room at controlled temperature in RKK-Energia and the flight model is now at the International Space Station, where the temperature is not expected to be out of the range between 18 and 23 Celsius degrees. The Flight model is expected to return by October 31. Both the flight and on-ground model will be transported to Granada by November 1. After a first micro photographic study in our lab, we will send you your corresponding crystallization boxes by the end of November.

We are sending you herewith attached the crystallization conditions used for your particular protein. With the best wishes JuanMa Garcia-Ruiz


APPENDIX 5

After landing letter


Granada, November 8, 2001

Dear colleagues,

Last October 31st at 08:00 the Soyouz TM32 successfully landed in North

Kazakhstan. This spacecraft brought back our crystallization experiment, which had been for 72 days in space. The GCF flight model was immediately placed into a cooler box to keep the samples at a temperature of 18 degrees Celsius. We recieved the cooler box at 17:30 in Moscow. A couple of hours later we arrived at the Flight Control Centre in Korolev (Moscow Region) where we received the on-ground parallel experiment which had been in the RKK buildings in Korolev these 72 days. We opened there the flight and ground models to make a first visual inspection of the GCBs, and we placed them again in the cooler box.

After a flight back with plenty of bureaucracy, we finally arrived in Granada last Saturday November 3rd and since then we have been working to get the first results of the experiments. We have taken pictures of the boxes and carefully collected some data such as: - None of the GCBs has any damage - All the capillaries have remained in their positions - None of the gels are broken - No significant leakage has been observed that affects the physicochemical conditions of the experiment. - There are crystals in 62% of the experiments performed. When there are no crystalls from space there are none in the on-ground experiment, either. This means that for these samples the crystallization conditions were not the right ones and that they should be improved. We realise that there was just one experiment per protein and that many of you had no time to perform enough pre-flight experiments to search for the right crystallization conditions. There will probably be another opportunity to do a similar experiment on ground versus in space, particularly because there are already companies interested in this simple and inexpensive device. Then, certainly we have to look in detail at the crystallization behaviour of each protein.

We are very happy with these first results, which definitively validate the GCB for flying. The next step will be to perform the X-ray diffraction of the crystals for comparative studies to know if there are significant differences between the ground and space crystals obtained.

Once we finish this first inspection study, we will send you your samples. With best wishes JuanMa Garcia-Ruiz


APPENDIX 6

After landing reports


Appendix 6.1 PROTEIN: Low Density Lipoprotein (LDL)

Please, note that labeling of the capillaries is made when the box is oriented with the written text GCB-XX up.

GCB1-G Capillary

Ref. Ø Comments Photo ref.

1 2 0.3 mm Amorphous precipitation in the upper part 3 4 5 0.4 mm Amorphous precipitation in the upper part 6

GCB1-S Capillary


1 2 0.3 mm Amorphous precipitation in the upper part 3 4 0.4 mm Amorphous precipitation in the upper part 5 6

1 2 3 4 5 6

1 2 3 4 5 6


First analysis of the results Dear Dr. Baumstark, We are sending you herewith enclosed the first report of the results of the crystallisation experiments of your protein LDL. As you can see no crystals were obtained in these experiments, neither in space nor on-ground with gelled protein. Certainly, there was a separation by buoyancy in the protein solution. This can be observed in the microgravity experiment but also in the gelled on-ground experiment too. Amorphous precipitation appeared in the upper part of the capillaries. Such a phase separation occurs immediately after the set-up of the experiment, and it occurs before the experiment was in space. I was reading that in your hanging drop experiments you also have a separation. Interestingly, the on-ground gelled experiment behaves also in the same way. Note that, in order to make the on-ground experiment as close as possible to the space one, we gelled all the proteins in this Andromede mission with only 0.1% agarose. At that concentration, the agarose is just a viscous fluid or a gel with very large pores. It seems that the large pore size of the gels we used allows the two phases of different density to separate by buoyancy. The case of your protein is very interesting to me because it is a challenge for crystal growth. I would like, if possible to continue with this experiment. I think that if we increases the concentration of gels to create a small pore size network, it will be possible to avoid the phase separation. I suggest two lines of research one with agarose and the other one with silica. We have demonstrated that protein crystals can be grown at gel concentration of up to 20% of silica (J.M. García-Ruiz, J.A. Gavira, F. Otálora, A. Guasch y M. Coll, Reinforced Protein Single crystals. Materials Research Bulletin 33 (1998) 1593-1598). In addition, there is a nother possibility: to use proteins directly into electroforesis gels just after running the electroforesis experiment. We also demonstrated that this is a working method (J.M.Garcia-Ruiz, A. Hernández-Hernández, J. López-Jaramillo, and B. Thomas, Crystallization screening directly in electrophoresis gels. Journal Crystal Growth 232 (2001) 596-602.

Please, let me know if you agree in this cooperation I propose to you and how we can proceed. We do not need financial support for this investigation but obviously we need from you the protein. All the best wishes JuanMa Garcia-Ruiz


Appendix 6.2

The report from Moscow Catalase GCB-2G . There are crystals in 3 capillaries from 6 GCB-2S . There are crystals in all 6 capillaries Crystal size of about 0.1-0.3 mm. See picture in the attachment file PVC.gif SAICAR-synthase GCB-3G . There are crystals in all 4 capillaries GCB-3S . There are crystals in all 4 capillaries Crystal size of about 0.3-0.6 mm See pictures in the attachment files SS_1.gif, SS_2.gif (and the same crystal with more amplification SS_3.gif) CAF_M GCB-4G . There are no crystals in all 6 capillaries GCB-4S . There are no crystals in all 6 capillaries Leghemoglobin GCB-16S There are crystals in all 4 capillaries Crystal size of about 0.01x0.1 mm See picture in the attachment file LEG.gif All pictures are taken from space crystallization in capillaries of 0.4 mm diameter.

Answers on your question GCB-

2G 2S 3G 3S 4G 4S 16S

Is there any contamination?

No No No No No No No

Is there any liquid in the upper part of the GCB (the lid)

Yes Yes Yes Yes Yes Yes Yes

Is there any liquid outside of the GCB?


Have the capillaries remained at their position?


Is there any crystal in any capillary?

Yes Yes Yes Yes No No Yes

Have the crystals remained at their position?

Yes ? Yes ? Yes Yes - - Yes

The height of the two layers Gel /Liquid in mm

25/45 24/45 20/60 20/45 27/47 25/39 25/42


Appendix 6.3 PROTEIN: DHQ-h , 1st batch ; 20 mg/mL


GCB5-G Capillary


1 0.2 mm Amorphous precipitation - 2 0.2 mm Amorphous precipitation - 3 0.3 mm Amorphous precipitation - 4 0.3 mm Amorphous precipitation - 5 0.4 mm Amorphous precipitation - 6 0.5 mm Amorphous precipitation -

GCB5-S Capillary


1 0.2 mm Amorphous precipitation - 2 0.2 mm Amorphous precipitation - 3 0.3 mm Amorphous precipitation - 4 0.3 mm Amorphous precipitation - 5 0.4 mm Amorphous precipitation - 6 0.5 mm Amorphous precipitation GCB5S-6(0.5).jpg

First analysis of the results It is evident that the protein used in this experiment was of poor quality. In fact, it was OK in July because we used this slot for the preflight experiment and we obtained excellent crystals. Therefore, it seems that the protein later degrades.

1 2 3 4 5 6

1 2 3 4 5 6


PROTEIN: DHQ-h , 2nd batch ; 20 mg/mL


GCB6-G Capillary


1 0.2 mm Perfect C-D trend. Excellent crystals and rods. GCB6G-1(0.2).jpg

2 0.3 mm Perfect C-D trend. Excellent crystals.

3 0.5 mm Perfect C-D trend. Excellent crystals. GCB6G-3(0.5).jpg

4 0.6 mm Perfect C-D trend. Excellent crystals. GCB6G-4(0.6).jpg

5 0.7 mm Perfect C-D trend. Excellent crystals.

6 1.0 mm Perfect C-D trend. Excellent crystals. GCB6G-6(1.0)-2.jpg

GCB6-S Capillary


1 0.2 mm Perfect C-D trend. Excellent crystals. GCB6S-1(0.2).jpg 2 0.3 mm Perfect C-D trend. Excellent crystals. 3 0.5 mm Perfect C-D trend. Excellent crystals. 4 0.6 mm Perfect C-D trend. Excellent crystals. GCB6S-4(0.6).jpg 5 0.7 mm Perfect C-D trend. Excellent crystals. GCB6S-5(0.7).jpg

GCB6S-4(0.6)5(0.7)6(1.0)-1.jpg 6 1.0 mm Perfect C-D trend. Excellent crystals. GCB6S-6(1.0).jpg

First analysis of the results Excellent crystallization experiment with lot of crystals arranged in a perfect crystallization trend characteristic of C-D experiments. In the space experiment the crystallization was also excellent and wit the same path. However, during post-flight the crystals move and there are some features characteristic of collapse of crystals and deterioration of quality.

1 2 3 4 5 6

1 2 3 4 5 6


Appendix 6.4 PROTEIN: CabLys3* lysozyme

GCB7-G Capillary Protein

Ref. Ø Cc Comments Photo ref.

1 0.2 mm 6 mg/mL Beautiful single crystal in the middle of the capillary and a large crystal with aggregation in its corner in the upper part of the capillary.

GCB7G-1(0.2)-1.jpg GCB7G-1(0.2)-2.jpg

2 0.2 mm 8 mg/mL Large aggregate in the upper part of the capillary -- 3 0.2 mm 10 mg/mL A single crystal in the upper part of the capillary

and few amorphous spheres. GCB7G-3(0.2)-1.jpg

4 0.3 mm 10 mg/mL Amorphous spherules and a crystal aggregate in the upper part of the capillary

--

5 0.4 mm 10 mg/mL A single crystal in the middle of the capillary and amorphous spherules

GCB7G-5(0.4)-3.jpg

6 0.5 mm 10 mg/mL A large crystal in the lower part of the capillary and many amorphous spherules

GCB7G-6(0.5)-1.jpg

GCB7-S Capillary Protein

Ref. Ø Cc Comments Photo ref.

1 0.2 mm 6 mg/mL A couple of amorphous spherules in the upper part of the capillary

GCB7S-1(0.2)-1.jpg

2 0.2 mm 8 mg/mL Nothing -- 3 0.2 mm 10 mg/mL Several prismatic small crystals in the lower part

of the capillary and a couple of amorphous spherules in the upper part.

GCB7S-3(0.2)-1.jpg GCB7S-3(0.2)-2.jpg

4 0.3 mm 10 mg/mL One single crystal in the middle of the capillary -- 5 0.4 mm 10 mg/mL A couple of crystal aggregates and few

amorphous spherules. --

6 0.5 mm 10 mg/mL A crystal aggregate in the middle of the capillary and few amorphous spherules in the upper part.

GCB7S-6 (0.5)-1.jpg

1 2 3 4 5 6

1 2 3 4 5 6


First analysis of the results Dear Ingrid, Here there is a first description of the results of your box. You have several single crystals to diffract and in addition some large crystal aggregates from which you can separate the larger crystals for diffraction. We have noted the formation of some spherules that do not interfere with light and look like amorphous material. These spherules are not the typical ones formed at high supersaturation near the base of the capillary in counter-diffusion method. Note that they are distributed along the capillary unrelated with the location of the crystals in the capillary. We do not know what they are. Could you tell us something about their origin?. The origin of these aggregates being unknown (if they are made for instance from denaturated protein, it will change the following comment) it seems that we have worked too close to equilibrium. This is clear from the low number of nuclei obtained in the lower part of the capillary. Even in the capillary 3 from the space experiment, there are few crystallites in this part of the capillary. In order to cover a longer path in the phase diagram, it would have been better to try with a higher concentration of precipitating agent or (better) higher protein concentration. But I will wait your comment on the spherulites for further analysis. Let us see what X-ray diffraction deserves. Best wishes and thank you very much. JuanMa Garcia-Ruiz


Appendix 6.5

PROTEIN: EndoVII (inactive mutant)


GCB8-G Capillary


1 0.2 mm Amorphous precipitation -

2 3 0.2 mm Amorphous precipitation - 4 5 0.4 mm Amorphous precipitation GCB8G-5(0.4).jpg

6

GCB8-S

Capillary Ref. Ø

Comments Photo ref.

1

2 0.2 mm No precipitation - 3 0.2 mm No precipitation -

4 0.4 mm Amorphous precipitation in the gel in the lower part of the capillary

-

5 6

1 2 3 4 5 6

1 2 3 4 5 6


PROTEIN: Sm-like protein


GCB9-G Capillary


1

2 0.2 mm No crystals - 3 0.2 mm No crystals - 4 0.4 mm No crystals GCB9G-4(0.4).jpg

5 6

GCB9-S

Capillary Ref. Ø

Comments Photo ref.

1

2 0.2 mm No crystals - 3 0.2 mm No crystals 4 0.4 mm No crystals

GCB9S-3(0.2)4(0.4).jpg

5 6

1 2 3 4 5 6

1 2 3 4 5 6


First analysis of the results Dear Claude, We have no crystals either in the space or in the on-ground gelled experiment for the EndoVII protein. We used exactly the conditions you had suggested. As you can see there are globules (amorphous) precipitate in the case of gelled system but no precipitate in the case of the space experiment. In principle, such a difference could be explained because the actual protein concentration is higher in the case of gelled protein due to the trapping of water by the agarose helixes. However, if that is the case then I would expect single crystals rather amorphous precipitation in the on ground experiment. For the case of SM-like protein we have no precipitation at all, either in space or on-ground The actual difference with your preflight experiment is that you use the test tube configuration where the precipitant is directly in contact with the gelled protein. Therefore, at the very beginning of the experiment we are in the labil zone and we move later to the metastable zone, this is why the route from precipitate to single crystals. With the GCB configuration we used, the precipitant meet the protein at a lower concentration and this is why we have increased the concentration of precipitant. Therefore it should have worked properly. There are three reasons I can see to explain the results:

a) The concentration of protein was lower that the nominal one (we can discard it easily if you used the same batch used for preflight experiments).

b) There was some denaturation of the protein that lower the actual protein concentration (we have not observed a rare behavior of the protein when we handled it at Baikonur)

c) There were some pH changes. Note that we buffered all the three chambers (protein, gel and precipitant). If you did not in the preflight experiments (I mean you only buffered the gelled protein), then when the precipitant diffuse there is a change of pH moving the pH value from the starting value 8.2 which is close to the pI to the pH value of the unbuffered ammonium sulfate solution (which is farther from the pI of the protein and therefore makes it more soluble (see the diagram below). These pH changes may play a very important role and in fact we use it to crystallize several proteins. This could be, in my opinion, the probable explanation at least for the case of SM-like protein. Of course, we need to confirm that you did not buffered the precipitating agent in your preflight experiment.

Let me know, please your comments and let we see what can we do further. We have received several offers from Japan and USA to fly the GCB. Right now, what it is sure is that it will be used again in the Taxi-flight Belgian-Russian next October. This time, we will have months to prepare the experiments properly, i.e., to make preflight experiments to select the best crystallization conditions and perform a full crystal quality comparison. Best wishes JuanMa


SM-like

ENDOVII

pH = 4 pH = 9 pI =8.1

GCB : Initial value andfinal value 4.5

Initial value preflight experiment = 4.5

Final value preflight experiment > 5

pH = 4 pH = 9

GCB: Initial value andfinal value = 8.2

Final value preflightexperiment < 8

pI = 8.8

Initial value preflight experiment = 8.2


Appendix 6.6 PROTEIN: Cytochrome C


GCB10-G Capillary


1 0.2 mm Amorphous precipitation

2 - 3 0.3 mm One single crystal GCB10G-3(0.3)-2.jpg

4 0.5 mm Amorphous precipitation 5 0.7 mm Two large crystals and amorphous precipitation GCB10G-5(0.7)-2.jpg

GCB10G-5(0.7)-3.jpg 6 -

GCB10-S

Capillary Ref. Ø

Comments Photo ref.

1

2 0.2 mm Amorphous precipitation 3 4 0.3 mm Amorphous precipitation 5 0.5 mm One single crystal GCB10S-5(0.5)-2.jpg

6 0.7 mm Four single crystals + aggregate inside the gel GCB10S-6(0.7)-1.jpg GCB10S-6(0.7)-2.jpg

1 2 3 4 5 6

1 2 3 4 5 6


First analysis of the results Dear Francisco: Here by we send you a first description of the obtained results. Something curious happened with this protein: neither in space nor on ground there were crystals when we opened the GCFs in November, just when they when back to our lab. But a few days later, in the meantime that we were taken pictures of every one of the experiments, we realised that it was starting to appear some crystals both on ground and in space experiments. We were observing how this protein was crystallising and this is in fact the reason why we did not send to you all your experiments until yesterday. It is difficult to understand the reason why this protein started to crystallise so late as far the precipitating agent is ammonium sulphate which has a low diffusion coefficient. It could be interesting to check the crystallisation conditions of this protein. The lost of colour that you can observed in the lower part of the capillary could be due to the diffusion of the protein during this long period of time (more than 6 months since they were filled) to the gel layer. Best wishes and thank you very much. JuanMa Garcia-Ruiz


PROTEIN: RNAse II


GCB11-G Capillary


1 0.2 mm No precipitation

2 0.3 mm No precipitation 3 4 0.5 mm No precipitation GCB11G-4(0.5).jpg

5 0.7 mm No precipitation 6

GCB11-S Capillary


1

2 0.2 mm No precipitation GCB11S-2(0.2).jpg 3 0.3 mm No precipitation 4 0.5 mm No precipitation 5 0.7 mm No precipitation 6

1 2 3 4 5 6

1 2 3 4 5 6


PROTEIN: Soluble catechol-O-methyltransferase (S-COMT) (apo protein)


GCB12-G Capillary


1

2 0.2 mm Amorphous precipitation 3 0.4 mm Amorphous precipitation 4 0.6 mm Amorphous precipitation GCB12G-4(0.6).jpg 5 6

GCB12-S Capillary


1

2 0.2 mm Amorphous precipitation 3 0.4 mm Amorphous precipitation 4 0.6 mm Amorphous precipitation

GCB12S-3(0.4)4(0.6).jpg

5 6

1 2 3 4 5 6

1 2 3 4 5 6


Appendix 6.7 PROTEINS: Gamma-E crystalline Nde, 35 mg/ml Gamma C, 39 mg/mL


GCB13-G Capillary

Ref Ø Protein

ref. Comments Photo ref.

1 0.3 mm Gamma E Nde Only amorphous spherules brown in colour with positive sequence of particle size.

--


--


GCB13G-3(1.0). jpg

4 - - - -

5 0.7 mm Gamma C

Large amount of amorphous precipitation in the lower part of the capillary. Then acicular crystals with split ends. Positive sequence of crystal size

GCB13G-5(0.7).jpg

6 1.0 mm Gamma C

Large amount of amorphous precipitation in the lower part of the capillary. Then acicular crystals with split ends. Positive sequence of crystal size

--

1 2 3 4 5 6


GCB13-S Capillary

Ref. Ø Protein


1 0.3 mm Gamma E Nde Brown amorphous precipitation in the gel entrance. Only amorphous spherules in the lower part of the capillary.

2 0.7 mm Gamma E Nde Brown amorphous precipitation in the gel entrance. Only amorphous spherules in the lower part of the capillary

3 1.0 mm Gamma E Nde Brown amorphous precipitation in the gel entrance. Only amorphous spherules (larger diameter in the upper part of the capillary)

GCB13S-3(1.0)-2.jpg

4 0.7 mm Gamma C

Brown amorphous precipitation in the gel entrance. Elongated crystals with split ends and some crystal bundles of about 0.7 mm in size. Sequence of crystal size.

5 1.0 mm Gamma C

Brown amorphous precipitation in the gel entrance. Elongated crystals with split ends and some crystal bundles of about 0.7 mm in size. Sequence of crystal size.

GCB13S-5(1.0)-2.jpg

6 - - - -

1 2 3 4 5 6


First analysis of the results Dear Dean,

Here there is a first description of the results of your box with Gamma E Nde and Gamma C.

Gamma E Nde

The first preflight experiment we used the protein you sent labeled Gamma E-H. We used two initial conditions:

50 % PEG as precipitating agent and 29 mg/ml of protein and,

32 % PEG as precipitating agent and 29 mg/ml of protein

And we got in both cases amorphous precipitation.

For the Andromede experiment we used 40 % PEG as precipitating agent and 35 mg/ml of protein.

And we got amorphous precipitation in space and on-ground. In the on-ground we have amorphous precipitation and spherules brown in color. In the space experiments we have the brown amorphous precipitation only in the part of the capillary where there is the gel. We have to study if there is an interaction of the protein with the gel because this is of maximum importance for us. In any case, a shown by the space experiment, the crystallization conditions must be optimized, particularly, I guess, pH value.

Gamma C

The first preflight experiment we used the protein you sent labeled Gamma C. We used two initial conditions:



And we got a sequence amorphous precipitation in the lower part of the capillary, then some acicular single crystals and large polycrystalline spherulites in the upper part (Photo ref.: GaC-pv2-1(0.3)-2.jpg). The tendency is correct but the existence of these large spherulites made me to think that the protein could be contaminated. Please check the information you can have at hand on that.


In the Andromede mission experiment we used the protein you labeled Gamma C, which I understand is the same protein as above but another purification batch. Is that all right? We used the starting condition:


And we got basically the same pattern but instead large spherulites we obtained large crystal bundle, which is a little better.

Then we have another preflight experiment with the protein you labeled C-H. We used the starting conditions:



And we obtained a sequence of amorphous, the crystalline spherulites and small single crystals with nice equidimensional shapes (very promising). Photo ref.: GaCH-pv1-1(0.3)-4.jpg.

I think that it will be great if you could send me pure samples of all these proteins to start a screening here with 0.2 mm capillaries. I guess we can optimize the crystallization conditions for all them. Best wishes JuanMa Garcia-Ruiz


PROTEIN: Gamma-E crystalline Nco, 18 mg/ml

Please, note that labeling of the capillaries is made when the box is oriented with the

written text GCB-XX up.

GCB14-G Capillary

Ref Ø Protein


1 0.3 mm Gamma E Nco Prismatic microcrystals in the lower part, then no precipitation and later (upper in the capillary) two large crystal aggregates.

2 0.5 mm Gamma E Nco Prismatic crystals along the capillary. No amorphous precipitation, in fact no precipitation in the lower part of the capillary.

3 0.7 mm Gamma E Nco

No amorphous precipitation, in fact no precipitation in the lower part of the capillary. Tabular crystals later and then prismatic crystals along the capillary.





GCB14G-5(1.5)-2.jpg GCB14G-5(1.5)-3.jpg

6 - - - -

1 2 3 4 5 6


GCB14-S Capillary

Ref. Ø Protein



Prismatic microcrystals in the lower part, then no precipitation and later (upper in the capillary) two large crystal aggregates. The population density of crystals does not change noticeably in the capillary.

GCB14S-1(0.3)-3.jpg









GCB14S-4(1.0)5(1.5)-2.jpg

6 - - - -

1 2 3 4 5 6


First analysis of the results Dear Dean,

Here there is a first description of the results of your Gamma E Nco boxes. There is some strange behavior that we should understand and I think that we will do after some information exchange and further experiments.

Till now only two actual experiments were performed. The first one was a preflight experiment in which we used the protein you sent labeled Gamma E. We used two initial conditions:



With them we got just amorphous precipitation and what is surprising that higher the concentration of PEG smaller the amount of amorphous precipitation.

The second experiment was this flight experiment. We used the protein you labeled Gamma E Nco, which I understand is the same protein as above but another purification batch. Is that all right? In one of your text you said that there were some samples with 10% glycerol. Do you know which ones? We got in the flight prismatic crystallization, using basically the same crystallization conditions:


To enhance the crystal quality and size, we should first understand why there is no precipitation at the entrance of the capillary (the region of higher supersaturation) and if the tabular crystals belong to the same polymorph as the prismatic crystals in the upper part of the capillary.

There is one explanation I can see. The protein in the capillary was buffered at pH 6.5 and the precipitating agent was at pH 5.5 because that was the crystallization condition you suggested in the first form for the protein. Therefore, as the pI of the protein is 6.85, when the buffered precipitating agent enters in the capillary it makes the protein more soluble as it moves the protein farther from the pI. This will explain the absence of precipitation at the entrance of the capillary. In the upper part of the capillary, as the concentration of buffered (pH 5.5) is lower, the precipitation of the protein occurs.

To go ahead, it is very important to know the differences in terms of purification

between the two different batches we used in the preflight and flight experiments. If you send us purified protein we can perform a small screening using 0.2 mm capillaries until we got the best starting condition. Best wishes JuanMa Garcia-Ruiz


Appendix 6.8 PROTEIN: Alliinase, 15.8 mg/mL


GCB15- G Capillary


1 - - -

2 0.4 mm Amorphous precipitation along the whole capillary

GCB15G-4(0.4).jpg

3 - - -

4 0.4 mm Amorphous precipitation along the whole capillary

--

5 - - - 6 - - -

GCB15-S Capillary


1 - - - 2 0.2 mm Nothing -- 3 - - - 4 0.4 mm Nothing -- 5 - - -

6 0.6 mm Amorphous precipitation at the lower part of the capillary

GCB15S-6(0.6).jpg

1 2 3 4 5 6

1 2 3 4 5 6


First analysis of the results Dear Rolf,

Here there is a first description of the results of the boxes GCB15G and GCB15S containing your protein Alliinase. As you can see, there are not crystals in the boxes. Here, there is very few to analyze because, as we told you in the after launch report, the protein arrived to Baikonour precipitated and we centrifuged it to try to save the experiment. Therefore, the quality of the protein was not good enough and it is not worth to analyze the experiment having that information.

In addition, and to reinforce what it is said above, crystals were obtained in the preflight experiment (Photo ref. Ali-pv-1(0.3)-2.jpg), which have basically the same, exception made that the in the preflight experiment the protein concentration was lower.

We suggest two experiments (again we will be delighted to do them here if you sent the protein):

a) Repeat the on-ground experiment, i.e., with gelled protein solution in the

capillary. b) Fill the GCB with the precipitating (ammonium sulfate) top solution. Then, fill

the capillary with gelled protein solution at 1% agarose. Then pour the open end of the capillary directly into the precipitating solution.

Best wishes and thank you very much. JuanMa Garcia-Ruiz


PROTEIN: Thermus thermophilus elongation factor (EF-Tu), 13 mg/mL


GCB17-G Capillary


1 - - -

2 0.2 mm Amorphous precipitation in the lower part of the capillary

--

3 0.3 mm Amorphous precipitation along the capillary 4 0.4 mm Amorphous precipitation along the capillary

GCB17G-3(0.3)4(0.4).jpg

5 - - - 6 - - -

GCB17-S Capillary


1 - - - 2 0.2 mm No precipitation -- 3 - - - 4 0.3 mm No precipitation -- 5 - - -

6 0.4 mm Amorphous precipitation in the lower part of the capillary

GCB17S-6(0.4).jpg

1 2 3 4 5 6

1 2 3 4 5 6



Here there is a first description of the results of the boxes GCB17G and GCB17S corresponding to your protein Thermus thermophilus elongation factor (EF-Tu).

Like in the preflight experiment, there are no crystals but just some amorphous precipitation. Certainly the chemical cocktail you use is complex and we should discuss in detail on what role plays each chemical component as well on the crystallization pH value and isoelectric point. I also wonder about the high concentration of ammonium sulfate you use. I hope that in my future visit to Jena we can talk about this interesting case. Best wishes and thank you very much. JuanMa Garcia-Ruiz


PROTEIN: Factor XIII



Ref Ø cc Comments Photo ref.

1 - - - -

2 0.2 mm 2.15 mg/mL

Sequence of small optically perfect crystals. Showing a small variation in size. It is noticeable the absence of precipitation in the lower part of the capillary

--

3 0.4 mm 2.15 mg/mL


--

4 0.2 mm 4.13 mg/mL

Sequence of optically perfect crystals larger in size that for the above capillaries. It is noticeable the absence of precipitation in the lower part of the capillary

--

5 0.4 mm 4.13 mg/mL


GCB18G-5(0.4)-6.jpg

6 - - - -

1 2 3 4 5 6



Ref. Ø cc Comments Photo ref.

1 - - - -

2 0.2 mm 4.13 mg/mL


--

3 0.4 mm 4.13 mg/mL


--

4 - - - -

5 0.2 mm 2.15 mg/mL


--

6 0.4 mm 2.15 mg/mL


GCB18S-6(0.4).jpg

1 2 3 4 5 6



Here there is a first description of the results of the boxes GCB18G and GCB18S containing your protein Factor XIII.

As you can see, we have obtained crystals in all the capillaries. The crystals appear optically perfect and euhedric in shape. There are not large differences between the space and on-ground experiment. The most remarkable observations from the point of view of the crystallization technique are a) the absence of a clear gradient of crystal size and b) the absence of precipitation in the lower part of the capillaries. In fact, the gradient of size exists but it is smaller than expected. Note that absence of gradient of size is not a post-flight effect because the on-ground gelled experiment (where the crystals can not move) also lacks such a gradient. It is also clear that, as expected from simulations, the crystal size is independent of the capillary diameter, while it does increase with protein concentration.

The explanation is clear from the chemical experimental set-up (diagram above). Note that water enters first into the capillary and dilute the protein. Therefore, when MES molecules arrive to the lower part of the capillary, the concentration of protein is very low and therefore, no precipitation occurs. With time, the concentration of MES increases in the upper part of the capillary and the protein crystallize almost like a batch.

In my opinion, the experiment can be enhanced and forces to work as an actual counter-diffusion experiments if you prepare a gel made of agarose with MES 0.2 M and punch the capillary with the protein into this gel. Please, do it or alternatively, send us few microliters of the protein (15 microliters will be enough) to do this experiment.

In addition to the boxes GCB18G and GCB18S, we are sending you the box with the preflight experiment. There are a couple of crystals in these capillaries (Photo ref. Factor XIII-pv-5(0.3)-2.jpg) and also some amorphous precipitation, which is probably due to poorer purification of the first slot, you brought to Granada. Best wishes and thank you very much. JuanMa Garcia-Ruiz

Water pH 6.7 0.2 M MES, pH 6.3

Factor XIII protein


Appendix 6.9 PROTEIN: Lumazine synthase



Ref. Ø cc Photo ref.

1 0.4 mm 10 mg/mL -

2 0.7 mm 10 mg/mL - 3 1.0 mm 10 mg/mL - 4 0.4 mm 20 mg/mL -

5 0.7 mm 20 mg/mL GCB22G-5(0.7)-1.jpg GCB22G-5(0.7)-2.jpg

6 1.0 mm 20 mg/mL GCB22G-6(1.0).jpg


Ref. Ø cc Photo ref.

1 0.4 mm 10 mg/mL GCB22S-1(0.4).jpg

2 0.7 mm 10 mg/mL -

3 1.0 mm 10 mg/mL GCB22S-3(1.0)-1.jpg GCB22S-3(1.0)-2.jpg

4 0.4 mm 20 mg/mL GCB22S-4(0.4).jpg

5 0.7 mm 20 mg/mL - 6 1.0 mm 20 mg/mL -

1 2 3 4 5 6

1 2 3 4 5 6


First analysis of the results

There was a large and intermittent amorphous or polycrystalline precipitation

brown in color. There are beautiful hexagonal crystals of large size. Some of them, grown in the space experiment, fall down towards the lower part of the capillary. It is probably a post-flight effect because the crystallization trend is basically the same.


Appendix 6.10 PROTEIN: HEW Lysozyme, 100 mg/mL


GCB19-G Capillary


1 0.2 mm Perfect C-D trend. Some rods.

2 0.3 mm Perfect C-D trend. Some rods 3 0.5 mm Perfect C-D trend. Large crystals

4 0.7 mm Perfect C-D trend. High nucleation and large crystals. Dissolution of the crystals in the lower part of the capillaries, leaving phantoms.



GCB19-S Capillary


1 0.2 mm Perfect C-D trend. Some rods.

2 0.3 mm Perfect C-D trend. Some rods. 3 0.5 mm Perfect C-D trend. Some rods.

4 0.7 mm Perfect C-D trend. High nucleation and large crystals.



1 2 3 4 5 6

1 2 3 4 5 6



The experiment works as expected with a perfect C-D trend. Clearly, the nucleation density was smaller in the thinner capillaries and larger in the wider capillaries. This is observed in space and on-ground experiments.

The space experiment shows rods in the three thinner capillaries, while in the

on-ground experiment we have rods only in the two thinner capillaries. However, it seems that the larger aspect ratio and the larger crystal size appear in the on-ground experiment.


PROTEIN: Thaumatin, 200 mg/mL


GCB20-G Capillary


1 0.4 mm Excellent rods and crystals

2 0.7 mm Excellent rods and crystals 3 1.0 mm Excellent rods and crystals 4 0.4 mm Excellent rods and crystals 5 0.7 mm Excellent rods and crystals 6 1.0 mm Excellent rods and crystals

GCB20-S Capillary


1 0.4 mm Excellent rods and crystals

2 0.7 mm Excellent rods and crystals 3 1.0 mm Breakage of the C-D symmetry 4 0.4 mm-g Excellent rods and crystals 5 0.7 mm-g Excellent rods and crystals 6 1.0 mm-g Excellent rods and crystals

1 2 3 4 5 6

1 2 3 4 5 6



The C-D trend was perfect. The on ground experiment creates beautiful large crystals and rods. The nucleation density increases with the capillary diameter

The space experiment shows the same trend for the gelled capillaries and for the two thinner ungelled capillaries. However, in the wider capillary of diameter 1.0 mm, the symmetry of the experiment breaks because of the fluid or solid motion of the crystals. This is clear because along the gelled part of the experiment, in the lower part of the ungelled capillary, the C-D trend is perfect.


PROTEIN: Ferritin, 15mg/mL


GCB21-G Capillary


1 0.4 mm Very few crystals in the lower part of the capillary. Follow CD pattern. Non-equilibrated experiment.

sb-GCB21G

2 0.7 mm Dendritic growth following C-D pattern GCB21G-2(0.7)3(1.0) 3 1.0 mm Large dendrites (up to 2 mm long) GCB21G-3(1.0) 4 0.4 mm Very few crystals in the lower part of the

capillary. Follow CD pattern. Non-equilibrated experiment.

-

5 0.7 mm Dendritic growth following C-D pattern GCB21G-5(0.7)6(1.0) 6 1.0 mm Large dendrites (up to 2 mm long) GCB21G-5(0.7)6(1.0)

GCB21-S Capillary


1 0.4 mm Very few crystals in the lower part of the capillary. Follow C-D pattern. Non-equilibrated experiment.

sb-GCB21S

2 0.7 mm Dendritic growth following C-D pattern - 3 1.0 mm Large dendrites and round shaped crystals.

Equilibrated experiment GCB21S-3(1.0)

4 0.4 mm-g Very few crystals in the lower part of the capillary. Follow C-D pattern. Non-equilibrated experiment.

GCB21S-4(0.4)5(0.7)6(1.0)

5 0.7 mm-g Dendritic growth following C-D pattern GCB21S-5(0.7) 6 1.0 mm-g Large dendrites and round shaped crystals GCB21S-

4(0.4)5(0.7)6(1.0)

1 2 3 4 5 6

1 2 3 4 5 6



Ferritin crystals appeared in the six capillaries of the space and on-ground experiments. The experiments were not equilibrated as shown by the colored solution between crystals. Interestingly this is true for both on-ground and space experiments.

The difference between space gelled and ungelled experiment occurs for the

capillary of 1.0 mm in diameter: the ungelled experiment was equilibrated but the gelled experiment was not. Precipitation took place outside the capillaries in both, on ground and space experiments.


PROTEIN: Concanavalin A


GCB23-G Capillary


1 0.4 mm

Perfect C-D trend. Large rod (about 2 mm) in the middle part. The volume of protein in the capillary decreases and we have to seal the capillaries

2 0.7 mm Perfect C-D trend. Large crystals of great optical quality. Some of them show post-flight three dimensional nucleation on the crystal faces.




6 1.0 mm Crystallization of small crystals along the whole capillary.

1 2 3 4 5 6


GCB23-S Capillary


1 0.4 mm Crystallization of small crystals along the whole capillary. They sediment in the lower part of the capillary



4 0.4 mm-g Crystallization of small crystals along the whole capillary. The crystals stay in their nucleation location




The experiments show the possibility to grow large concanavalin A crystals by counter-diffusion. In fact, on ground the trend is typically that of C-D. Unfortunately, the space experiment does not work as expected. By unknown reasons, there were crystals forming along the whole of the capillary without typical C-D trend. There was not a problem related to microgravity because the protein into three of the capillaries for the space box was gelled and also show the same behavior. Clearly, the crystals in the gelled capillaries remain in the location where they nucleated, while the crystals in the ungelled capillaries sediment on the capillary wall. However, no sign of sedimentation was observed along the capillaries.

The capillaries were sealed in Granada before going to Hamburg.

1 2 3 4 5 6


APPENDIX 7

Suggestions for storage and x-ray data collection


SUGGESTIONS FOR STORAGE AND X-RAY DATA COLLECTION

GCB has a passive activation, i.e. the experiment is activated when the

capillaries are punctuated in the gel and it only stops when a equilibrium is reached after the diffusion of the precipitating system along both the gel layer and the capillary. Once you receive the GCB with your samples the counter-diffusion experiment has concluded. However, mechanisms such as ageing of the protein or reorganization due to Ostwald’s ripening may affect to the quality of your crystals. Hence, we recommend to diffract the crystals as soon as possible.

We have written below a few lines to help you to manipulate and store your crystals grown in the GCB but feel free to contact us ([email protected]) in case of any doubt with your particular experiment

X-ray diffraction

The best way to transport your crystals to the x-ray source is in the GCB since they are protected by the capillary, by the gel and by the GCB itself. The diffraction can be carried out a) directly in the same capillary where the crystal grew or b) in a new capillary after extracting the crystal from the one where it grew: a) Directly in the same capillary where the crystal grew: This is possible and recommended when:

- The crystal is isolated (i.e. far enough from any neighboring crystals to ensure that the x-ray beam hits only the specimen that you are interested in).

AND

- The crystal does not slip during the data collection as for the case of rods

(crystals filling the whole diameter of the capillary) for crystals attached to the wall of the capillary, or for experiments where the protein inside the capillary was gelled as on ground controls

The crystal can be diffracted in the same capillary at room temperature or at 100 K:

a. At room temperature. Cut the upper (i.e. wider) end of the capillary (it is heavy and may bend the capillary) and seal both ends. Insert one end in a magnetic base as those used for cryocrystallography (Photo 1) and glue it. Notice that the position of the crystal in the capillary glued to the magnetic base has to allow its alignment in the beam and it depends on your goniometer head.

b. Cryocrystallography inside the capillary. You have to diffuse the

cryoprotectant along the capillary. This implies to replace the precipitating system in the GCB by a cryobuffer consisting of cryoprotectant plus the precipitating system, taking into account the dilution [Top Solution/(Top Solution +Gel Layer)] reached during the experiment.


Keep in mind that the diffusion of the cryoprotectant may be very slow for glycerol. From our experience, glycerol needs about 3 weeks to diffuse along a capillary. For further information look at “Crystallography inside x-ray capillaries. J. Appl. Cryst. (2001) 34: 365-370”.

b) From crystals harvested from the capillary

Pick up the capillary and pull it out gently to extract it from the GCB. Break the upper seal by pricking with a thin needle (Photo 2), then insert the capillary into a rubber tube and blow very gently to push out your crystals (Photo3). Proceed very carefully to ensure that you know where the crystal that you are interested in is. Keep in mind that the best crystals are those grown at lower supersaturation (i.e. those grown further from the end in contact with the gel). Usually we do not extract all crystals from the capillary together, but as small drops as we blow gently. Once you have the crystal in a drop on a cover glass proceed as usual: equilibrate the crystal with the proper buffer (for room temperature data collection the precipitating solution remaining in the GCB is perfect) and mount it.

The above protocol also applies for on ground experiments since the gel inside the capillaries behaves as a viscous liquid and crystals can be extracted from the capillary and manipulated as if they were in solution.

Suggestions for storage

We recommend to diffract the crystals as soon as possible but if for any reason your crystals have to be stored for a long time, we suggest to seal the capillaries:

- Open the GCB and discard the precipitating system (it may be worthy saving it in a Falcon tube as explained above)

- Extract the capillary from the GCB by pulling out from its upper end. - Seal its lower end with glue (epoxy glue is fine). It is very important to

make sure that the sealing is perfect (we usually repeat the operation a couple of times) (Photo 4)

- Punctuate the sealed capillary back into the gel layer, put the lid back (sealing with some vacuum grease), and store it. It may be worthy checking from time to time the crystals to detect any leakage or any sign of drying.

Important remainder: Use the same protocol, x-ray source, detector and software to compare crystals grown on ground and in space.


Photo 1

Photo 2

Photo 3

Photo 4


APPENDIX 8

Guidelines for comparing the quality of protein crystals using X-ray diffraction


Guidelines for comparing the quality of protein crystals using X-ray diffraction

Introduction

Orbiting spacecrafts offer the possibility (in terms of residence time and gravity

scenario) of performing protein crystallisation experiments under reduced gravity values1. Under these conditions, convection due to buoyancy as well as sedimentation of crystals are strongly reduced. This allows the study of nucleation and crystal growth in the absence of the disturbances imposed by convective mass flow that obscure the fundamental processes involved in crystallisation. This possibility justifies by itself the interest of space crystallisation in the framework of fundamental studies.

In addition, it has been claimed that the space scenario may yield macromolecular crystals of higher quality than those obtained on terrestrial laboratories2. Such an enhancement, up to now never unambiguously demonstrated, is based on the higher probability for an ordered attachment of growth units on the crystal surface in the absence of convection and on a impurity filtering process due to the very diffusive mass transport governing space experiments3. Leaving aside considerations on 1 B. Fitton and G. Seibert, in “A world without gravity: Research in space for health and industrial processes”. Edited by Günter Seibert, ESA Publication Division, SP-1251 (2001) 5-34. 2 DeLucas, L. et al., Protein crystal growth in microgravity, Science 246 (1989), 651-654. DeLucas L, et al., Recent results and new hardware developments for protein crystal growth under microgravity, J. Crystal Growth 135 (1994), 183-195; Chayen N.E., Gordon E.J. and Zagalsky P.F., The crystallisation of apocrustacyanin C1 on the International Microgravity Laboratory (IML-2) Mission, Acta Cryst D 52 (1996), 156-159; Wardell M.R., Skinner R., Carter D.C., Twigg P.D. and Abrahams J.P., Improved diffraction of antithrombin crystals grown in microgravity, Acta Cryst. D 53 (1997), 622-625; Declercq J.P., Evrard C., Carter D., Wright B., Etienne G. and Parello J., A crystal of a typical EF-hand protein grown under microgravity diffracts X-rays beyond 0.9Å resolution, J. Crystal Growth 196 (1999), 595-601; Kitano K., Sasaki R., Nogi T., Fukami T.A., Nakagawa A., Miki K. and Tanaka I., Utilization of microgravity to improve the crystal quality of biologically important proteins: chaperonin-60, GrpE, B-subunit of V-type Atpase, and MIF, J. Crystal Growth 210 (2000) 819-823; Eschenburg S., Degenhardt, M., Moore K., DeLucas L.J., Peters P., Fittkau S., Weber W. and Betzel C., Crystallization of proteinase K complexed with substrate analogue peptides on US space missions STS-91 and STS-95, J. Crystal Growth 208 (2000), 657-664; Snell E.H., Weisgerber S., Helliwell J.R., Weckert E., Hölzer K. and Schroer K., Improvements in lysozyme protein crystal perfection through microgravity growth Acta Cryst. D 51 (1995), 1099-1102; Koszelak S., Day J., Leja C., Cudney R. and McPherson A., Protein and virus crystal growth on International Microgravity Laboratory-2, Biophysical Jour. 69 (1995), 13-19; Vaney M.C., Maignan S., Riès-Kautt M. and Ducruix A., High-resolution structure (1.33 Å) of a HEW lysozyme tetragonal crystal grown in the APCF apparatus. Data and structural comparison with a crystal grown under microgravity from SpaceHab-01 mission, Acta Cryst. D 52 (1996), 505-517; Sjölin L. et al., Protein crystals grown on board MASER 3 extend the ribonuclease A structure to 1.06 Å resolution, ESA Special Publication 1132 (1992) 92-103. 3 J.M. Garcia-Ruiz, J. Drenth, M. Riess-Kaut and A. Tardieu, Macromolecular crystallization, in “A

world without gravity: Research in space for health and industrial processes”. Edited by Günter Seibert, ESA Publication Division, SP-1251 (2001) 159-172; Carter D.C., Lim K., Ho J.X., Wright B.S., Twigg P.D., Miller T.Y., Chapman J., Keeling K., Ruble J., Vekilov P.G., Thomas B.R., Rosenberger F. and Chernov A.A., Lower dimer impurity incorporation may result in higher perfection of HEWL crystals grown in microgravity. A case study, J. Crystal Growth 196 (1999), 623-637.


the crystal growth process itself, the path from the crystals to X-ray data quality parameters implies several manipulations/calculations that may be important sources of bias. Thus, the comparison of the quality of different protein crystals is not a trivial matter.

The use of counter-diffusion crystallisation experiments4 promoted in the last years by the European Space Agency implies an additional tour de force. Here, rather than comparing crystals grown in space under reduced convection with crystals grown on earth under convective mass transport, we focus on the comparison between crystals grown in space under reduced gravity with crystals grown on earth in the pure diffusive conditions prevailing in gels5. According to preliminary results from the Andromede mission to the ISS in 2001, the expected differences, if any, will be tenuous, although they might be important for structural studies related to drug design. Therefore, it is obvious that we need of a precise protocol for crystal quality comparison based of x-ray diffraction analysis discarding any artefact during crystal manipulation, data collection and processing that might bias an objective comparison. This issue was raised by the Review Board set-up by the European Science Foundation on the request of ESA to assess the scientific results of several flights of a microgravity protein crystallisation facility.

The present document is a working draft to be discussed by a panel of scientists consulted by the European Space Agency. For writing this draft, I made use of information provided by members of the ESA's Topical Team on Macromolecular Crystallisation, namely: Prof. Jean Paul Declerq, Univ. Louvain (B), Prof. Jan Drenth, Univ. Groningen (NL) Dr. Madeleine Riess-Kaut, Univ. Paris (F) Prof. Adriana Zagari, Univ. Naples (I) Dr. Ingrid Zeggers, Univ. Brussel (B) As well as the following colleagues: Dr. Isabelle Broutin-L'Hermite, Univ. Paris (F) Prof. Santiago García-Granda, University of Oviedo (E) Dr. Javier López Jaramillo, CSIC (E) Prof. Martin Martinez Ripoll, CSIC (E) However, any mistake in what follow is only the responsibility of the writer.

4 J.M. Garcia-Ruiz, Counter-diffusion methods for protein crystallisation. Methods in Enzimology (2002) to be published. 5 Robert M.C., Vidal O., García-Ruiz J.M., and Otálora F., Crystallisation in Gels and related methods in "Crystallization of nucleic acids and proteins: a practical approach" (1999), A. Ducruix and R. Giegé Eds. Oxford: IRL Press. 331 pp. Ch 6 (1999) 149-175; J.M. Garcia-Ruiz, M.L. Novella, R. Moreno, J.A. Gavira, Agarose as crystallization media for proteins. I. Transport processes. Journal Crystal Growth 232 (2001) 165-172.


This protocol is not intended to be mandatory for scientists participating in the

ESA projects on protein crystallisation. However, it is strongly recommended to check all the steps in order to avoid any source of bias when comparing crystal quality.

Juan Manuel García-Ruiz


Guidelines for comparing crystal quality

1. General recommendation GCB operation encompasses passive solute exchange activation, i.e. the

experiment is activated when the capillaries are punctuated in the gel and it only stops when equilibrium is reached after the diffusion of the precipitating system along the whole length of the gel layer and the capillary. At that moment, the concentration of precipitant is homogeneous in the whole GCB.

When you receive the GCBs from space and on-ground counterparts, either the

counter-diffusion experiment is concluded or it is still going on, in which case crystals may continue to grow on earth. That depends on the length of the mission and on the diffusivity of the precipitant you selected. Even in the best case, mechanisms such as ageing of the protein or reorganisation due to Ostwald’s ripening may affect the quality of your crystals. Hence, we strongly recommend 1) to perform diffraction data collection of the crystals as soon as possible, and 2) to compare high magnification pictures of the crystals showing their size and aspect when received back and just before X-ray analysis.

The best way to transport your crystals to the X-ray source is inside the GCB

since they are protected by the capillary, by the gel and by the GCB itself. Remarks:

1) Calculate if your experiment is fully equilibrated. 2) If the experiment is still going on, remove the capillary from the GCB, seal

the lower end and placed them into another empty GCB. 3) Proceed to record the diffraction data as soon as possible 2. Selection of crystals

It must be reminded that counter-diffusion technique yields crystals of different quality along the capillary6. It is expected that the quality of the crystals increases as the precipitation front moves along the capillary, as does the size of crystal. Crystals located at different height in the capillary must be selected to get information about the counter-diffusion technique itself. This will also reduce the number of crystals to be analysed in order to obtain significant information. Therefore, before selecting the crystal, it is recommended to study the photograph of the whole capillary as received after the flight.

For on-ground experiment, the crystals grown in the high viscosity agarose solution will remain in the location where they nucleated and grew. However, crystals grown in space will drift away from their nucleation loci due to sedimentation during

6 J.M.Garcia-Ruiz, F. Otalora, M.L.Novella, J.A Gavira, C. Sauter, O. Vidal, A supersaturation wave in protein crystallization. Journal Crystal Growth 232 (2001) 149-155.


re-entry, landing and on-ground transport. Figure 1 shows an actual case from the Andromede Mission.

Figure 1: DHQ crystals grown in the Andromede mission. Top: crystals grown in space; Bottom: crystals grown on ground in the counterpart experiments. Diameter of the capillaries on the left column are 0.6, 0.7 and 1.0 mm. Diameter of the capillary on the right column is 1.0 mm. Note that crystals grown in space (top) have shifted from their nucleation loci due to re-entry and landing effects.

From computer simulations studies, we can assume that the precipitation pattern is basically the same in both cases. Therefore, it is important to select crystals of similar size and aspect ratio. To use this information later on for comparing X-ray data, it is recommended to photograph the crystals selected for X-ray diffraction in-situ under the microscope. Remarks:

4) Study the location of the crystal in the capillary, under the microscope and using the panoramic picture of the capillaries that will be sent after the flight.

5) Mark and label in the picture those plausible interesting crystals to be selected for crystal quality comparison. Note that they should have similar sizes.

6) Take microscopic pictures of the pre-selected labelled crystals. 7) Measure crystal size, aspect ratio, and identify crystal forms. 8) Keep all the capillaries under the same temperature at home lab and during

transportation to the synchrotron facility. You can use the 18ºC thermostated boxes that will be provided after the flight.


3. Crystal harvesting

When crystals are grown by the counter-diffusion technique inside capillaries in gelled protein solutions, it is possible to collect data at both 100 K and room temperature, directly from crystals located inside the capillary where the experiment was performed, thereby avoiding harvesting and mounting. Unfortunately, as explained above, the crystals grown in space will drift from their growth location after re-entry. Thus, the probability to find them isolated from each other (to ensure that the X-ray beam hits only the targeted crystal) is low. Therefore, to maintain the same level of manipulation it is recommended to remove the crystals from the capillaries in both, space and ground, cases7.

Remarks:

9) To keep the same level of manipulation, remove crystals from on ground and from space capillaries.

3.1. Removing crystals from the capillary

Pick up the capillary and pull it gently to extract it from the GCB. Open the upper seal by pricking with a thin needle (Figure 2). Then insert the capillary into a rubber tube

and blow very gently to push out the crystals and solution (Figure 3). Pour the crystals and protein solution on small glass vessels and cover them. Proceed very carefully to visualise continuously the crystals that you have selected for diffraction. Keep in mind that the larger crystals are those grown at locations further from the capillary end in contact with the gel. It is suggested not to extract all crystals at once, but make small drops as you blow gently. Once you have the crystals on the small glass vessels, if you find necessary to

equilibrate them, it is recommended to use the precipitating solution remaining in the GCB. If the counter-diffusion experiment is finished, the precipitating agent solution is equilibrated in the whole GCB. If the experiment is not finished, then equilibrate the crystal with the proper buffer and protein solution. Special care should be taken during soaking and/or exchange of buffers since they may be an important source of osmotic stress, dissolution or re-growth of the crystals. As a rule, it is better to work at hypertonic conditions than at hypotonic ones.

7 Ries -Kautt stresses that we then lose the benefit of having them already inside a capillary, and add the majour source of damage to the crystals. Couldn't we propose to take out those which are not expected to be analysed, and let the one of interest untouched inside the capillary?

Figure 2: Opening the upper seal of the capillary.


The above protocol also applies for on ground experiments since the agarose

inside the capillaries behaves as a viscous liquid and crystals can be extracted from the capillary and manipulated as if they were in solution. You will note that harvesting crystals grown on the ground from the agarose/protein solution is as easy as harvesting space crystals from the protein solution.

Remarks:

10) Open the upper end of the capillary. 11) Transfer the crystals from the capillary to a glass slide, making a small drop.

Each drop corresponds to consecutive volumes along the capillaries. 12) Identify the crystals that you have selected for diffraction purposes. 13) Equilibrate the crystals if necessary with the precipitating solution in the

GCB8.

4. Crystal mounting

For this study focusing on comparative purposes, we recommend to diffract most of the ground/space crystal couples at room temperature to avoid plausible problems during flash cooling. It is recommended to mount each crystal just before the diffraction experiment rather than mounting all of them at once. After mounting steps, it is recommended to double check with the microscope that the aspect of the crystal does not suffer during manipulation.

Considering that the number of crystals from six capillaries in one GCB will be in most cases large enough for room temperature data collection, it is also recommended to diffract few crystals at 100ºK to achieve higher completeness and resolution limit. Note that flash-cooling increases I/σ values but also the mosaicity. The flash-cooling conditions must be well known for the protein crystals to compare. Otherwise it is recommended to invest time on finding the best flash-cooling conditions before comparing crystals. It must be considered that any exchange of buffer during

8 It should be discussed if it is better to isolate one crystal from the capillary and mount it immediately before handling further ones.

Figure 3: Removing the crystals from the capillary.


manipulation of the crystal (i.e. soaking with the cryobuffer or mounting in the x-ray capillary) is a source of osmotic stress, dissolution or re-growth.

Remarks:

14) Double check that, after mounting the crystal, the optical quality does not suffer from manipulation.

5. Diffraction:

Data collection should be carried out on the same synchrotron beamline for all future comparative studies. While for practical and administrative reasons this might not be possible, it is obvious that the comparison of each protein crystal set must be performed on data collected in the same experimental setup and with identical values of the parameters affecting data diffraction. Among the parameters that have to be precisely defined and kept constant from experiment to experiment are:

1. Insertion device

2. Monochromator and wavelength

3. Type of detector. If possible, use CCD to speed up the collection and reduce crystal decay.

4. Parameters of the beam such as polarisation, divergence, beam size or intensity must be kept constant. Experiments must be carried at the same beamline with the same setup, same collimation and slit positions. Data must be collected on the basis of DOSE to correct intensity decays of the beam (doubling the intensity enhances the signal-to-noise ration by 21/2)9.

5. Make sure that the whole volume of the crystal is illuminated by the beam. Thus, the amount of diffracting material is known10.

6. Crystal orientation. Redundancy may be crystal orientation dependent when short data sets are collected. Crystal orientation may also limit the oscillation angle per frame.

7. Oscillation angle per frame (∆φ). In principle, the thinner the slicing the better the I/σ values (collecting 0.1º frames instead of 0.25º frames has been reported to improve the I/σ(I) for weak reflections beyond 2.0 Å by two times)11. A small ∆φ avoids spatial overlap of the spots but the minimum ∆φ to be used is limited for practical reasons during the intensity integration process: the number of partially recorded spots should not increase too much. It is important to have realistic estimates of the mosaic spread and minimum spot separation for ∆φ calculation. The oscillation angle should be greater than half the sum of the mosaic spread and beam divergence12.

9 Dauter, Z. (1999). Acta Cryst. D55: 1703-1717 10 To discuss: Wouldn't it be better to have an identical minimal volume rather than the whole crystal? 11 McRee, D.E. (1993) Practical Protein Crystallography. Academic Press, Inc. pg 79. However

consider that this could be not a rule for crystal sensitive to radiation. 12 More exactly should be greater than half of the square root of the summation of the mosaic spread to the square plus the divergente to the square.


8. Crystal to detector distance. The background decreases with the square of the crystal to detector distance13. However some compromise has to be chosen to maintain low background and recording the wanted resolution with no spot overlap.

9. Segment collected to optimise the efficiency of the data collection. For actually comparing the quality of crystals grown in different conditions it may be better to collect data from many crystals in exactly the same conditions, and not to try to get the maximum diffraction out of each crystal (see below). Short data-collection times allow for the intensity collection of many crystals. There are strategies that allow to reduce the total rotation range (to save time) and still get a maximally complete dataset, using for instance two 30 degree14 segments that give maximum completeness.

10. Dose, ∆φ, and crystal-to-detector distance should be adjusted so that all significant data for all crystals are collected under the same conditions. Adapt the crystal-to-detector distance in order to collect the high-resolution for the best diffracting crystal you have, but maintaining a minimum acceptable separation between spots. It is not recommended to use the same exposure time per image as for good data recording, it is better to use the complete dynamic range of the detector.

11. Temperature: flash-cooling increases both mosaicity and I/σ (because it improves the B-factor values), but it also minimises the radiation damage. Thus, the comparison of results from data sets collected at different temperatures is not straightforward.

Remarks:

15) Diffraction First crystal

a) If possible, align the crystal according to the best direction (i.e. the one that allows larger oscillation angles and/or maximum completeness).

b) Once the crystal is mounted on the goniometer head and both detector position and exposure dose are fixed, a few 90 degrees apart images are collected

c) These images are auto-indexed with and: a. A strategy is predicted to maximize the efficiency of the data collection

and estimate the expected redundancy. It may be worth collecting two segments apart to improve the completeness.

b. The oscillation angle is predicted in order to collect data at the largest oscillation angle that yields less than 10% overlapping between spots.

d) The data collection proceeds according to the strategy.

Subsequent crystals The data collection procedure for these crystals should be restrained to the conditions used for the first one. Global indicators of the quality of the data sets are redundancy and/or completeness dependent, and both redundancy and completeness are orientation dependent. Hence, the crystals to be compared should be aligned in a similar orientation. 1. After collecting a few images and auto-indexing them, check that:

13 Pflugrath J.W. (1999) Acta Cryst. D55: 1718-1725 14 Take this number as indicative. Remember that this number will strobgly depend on space group and crystal orientation.


a. The strategy (calculated with the same criteria as crystal 1) predicts similar completeness and redundancy for the data set (this is a clear indicator of similar orientation)

b. The oscillation angle should be very close (if possible exactly the same) as for crystal 1

6. Data reduction and analysis

Due to the dependency of many diffraction parameters and algorithms on both the software and the data reduction protocol, all the data that have to be compared should be processed with the same software and software version, using the same procedures. The processing parameters should be optimised for each dataset, in order to extract the best possible intensities for that dataset. This implies that mosaicity values should be determined and the data reprocessed using the correct mosaicity.

6.1. Main parameters to be measured

Parameters to compare crystal quality are the followings: a) Total number of unique reflections collected b) Number of observed reflections15 c) Completeness d) Rsym and Rmerge for an identical highest resolution shell e) I/σ(I) as a function of resolution for the complete dataset or a significant wedge of

data (at least 20 deg16), using always the same wedges. To this, it is important to maintain as much as possible similar crystal orientation.

f) Resolution at which I/σ(I) = 2 g) Mosaicity as refined for the entire dataset or for wedges in function of orientation17. h) Wilson plot B-value18 i) Redundancy. Redundancy biases most parameters defined to evaluate the crystal

quality19. When short data sets are collected, the orientation of the crystal in the beam becomes one of the critical parameter to take into account since it has a clear effect on the redundancy. Hence, differences in the global indicators of crystal quality should be first analysed on the basis of the redundancy and the data sets can be compared only if no relation is found.

After analysis of all the crystals, the two apparently best ones (one ground- and one

microgravity-grown) can be completely recorded in order to analyse the crystal quality

in the direct space. For high resolution diffracting crystals, three complete data sets

have to be collected: two at high resolution, then one at low resolution.

15 Criteria for observed reflections must be discussed 16 The wedge will depend on the symmetry. The larger the crystal symmetry, the smaller the wedge needed. 17 Note that the mosaicity values depend on crystal handling, temperature of data acquisition and software. 18 To be discussed. B is very sensitive to cell parameters. It is better to have B=25 with a correlation of 0.8 or B=28 with a correlation of 0.92? 19 Weiss (2001) J. Appl. Cryst. 34: 130


6.2. Comparison of structural features

After data collection has been performed according to the above protocol,

minimising bias introduced by crystal manipulation, harvesting and data collection

strategy, the next and final task is to compare the quality of the structural information

obtained from crystal grown in space and on-ground. In addition to previous paragraph

6.1, the following ones are suggested for discussion:

• R final and Rfree

• Ramachandran plots

• B Thermal factors.

• Comparison of geometries of the external chains.

• Localised solvent fraction.

• Determined hydrogen bonds.

• Geometry of the active sites.

• Metal coordination.

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