User:Karsten Theis/Takustr6
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Arylsulfatase A (1AUK) | Arylsulfatase A (1AUK) | ||
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| + | ::"This was a side project for me just after I graduated from the lab working on the biochemistry of the DNA bending protein FIS. I was hanging around in the lab looking for a postdoctoral position, and a new graduate student, Gordian Lukatela, visited bringing crystals of the human lysosomal protein arylsulfatase A. We made good progress, but the project got delayed twice. First, the antibody-affinity column used in purification failed, and we lost our supply of fresh protein. Second, a nasty bug in data processing compromised our structure factors, leading to abnormally high R-factors for a structure that was essentially correct. | ||
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| + | ::The resulting structure is fascinating because of its unusual modification in the active site with an amino acid side chain carrying an aldehyde functional group. In the crystal structure, the density looked like the aldehyde was hydrated (geminal diol), but we weren't bold enough to commit to that (at least not for the pdb - there is a figure with the density and interpretation in the paper). However, we came up with a mechanism that utilized this hydrated aldehyde. A higher resolution structure of a bacterial homolog (1hdh, solved three years later in 2001) showed the hydrated aldehyde as the resting state of the active site, supporting our mechanistic hypothesis." (Karsten) | ||
=== 1996 - 2001<ref>Karsten: Looking back, I'm amazed that only five years passed between the 4 Angstrom model and the complete atomic model of photosystem I. To me, folks working on that project were in a different space-time continuum. Everything took longer, the unit cell dimensions were crazy, the solvent content unbelievable, and then the did these things like advancing the spindle every three images because they were burning through the crystal, or using a cylindrical image plate in Japan in hopes of spreading out the overlapping diffraction spots. And then there was airport security: "No, you can't X-ray that, sorry. It's in the thermos to keep it cool. No, you can't look inside, it is light sensitive. Yes, trust me." I am still impressed by the graduate students who dared to join this project.</ref> === | === 1996 - 2001<ref>Karsten: Looking back, I'm amazed that only five years passed between the 4 Angstrom model and the complete atomic model of photosystem I. To me, folks working on that project were in a different space-time continuum. Everything took longer, the unit cell dimensions were crazy, the solvent content unbelievable, and then the did these things like advancing the spindle every three images because they were burning through the crystal, or using a cylindrical image plate in Japan in hopes of spreading out the overlapping diffraction spots. And then there was airport security: "No, you can't X-ray that, sorry. It's in the thermos to keep it cool. No, you can't look inside, it is light sensitive. Yes, trust me." I am still impressed by the graduate students who dared to join this project.</ref> === | ||
Revision as of 18:12, 27 July 2018
Introduction
The Institute of Crystallography, part of the Chemistry department of the Free University Berlin, was located at Takustrasse 6. The macromolecular crystallography was on the third floor. It is still there, but the institutional framework has changed, and a new group has moved in. This page reminisces about research done from when the group moved to Berlin to the PIs retirement.
Results
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Methods
The third floor was one big corridor, outlining the steps in structure solution. Cold rooms on one end, crystallization and X-ray labs in the middle (along with the two grown-up offices and the seminar room) and data crunching on the other end.
Purification
Crystallization
In-house data collection
Synchrotron data collection
Data processing
Structure solution
Structure refinement
1992: For every new structure, there would be two papers. The first describing the fold (in Science or Nature), the second "full paper" describing the details after refining the structure. Then, if you wanted, you would deposit your coordinates in the PDB. The diffraction data would be moved to magnetic tape as soon as a new project took up to much of your precious disk space quota.
Making figures
1992: FTP (yes, that's a verb) your coordinates to the Evans-and-Sutherland vector graphic machine (sign up first). Find the perfect orientation of your model (in stereo, if necessary), apply rub-off lettering directly on the screen for labels, and set up a tripod for the camera to take pictures. If you were in a hurry, run down to the photography lab in the basement to develop your film and make copies of the negatives (unless you were making slides for a talk). Then, patiently take off all the lettering and start over because someone wanted a different view, maybe 3 degrees rotation around the y-axis.
Publishing
1992: We had computers, even PCs, but they were slow. You would find out if you had your entire PhD thesis in a single document and tried to go from chapter 1 to chapter 4 after making changes. Word had to re-paginate the entire document, which would take longer than making a pot of coffee and drinking it. Others used LaTeX on the mainframe, entering text on a VT100 terminal.
Model building
1990s: Every time a new structure was solved, we would build a physical model. Well, we would have a crystallography course, and participants would build stretches of ten amino acids from a list of main-chain and side-chain torsion angles. Then, we would assemble those on a wooden board with aluminum stakes, guided by the projection of the C-alpha trace taped to the wooden board. At the end, the technician from downstairs would build a plexiglass housing to avoid tampering with the structure (see Discussion, occupational hazards). It was reminiscent of a hunter putting skulls and antlers of the kill on the wall.
Conferences
Discussion
Weekends
Weekends were the time you really could make progress on the graphics system or one of the purification machines. Sometimes, though, the PI would walk in and have suggestions (like "did you try to add calcium") and your plans would change.
Smoking
Famously, Joerg Labahn and Hui-Woog Choe swore on their own brands of cigarettes to generate smoke to induce nucleation in crystallization. They would smoke in the lab no matter whether they were setting up crystallization trays or not.
Secretaries
Students and guest scientists came and went, but the secretary provided institutional memory.
IT dept
Leibniz keks
1988: 
Surprises
The day the FAST detector got flooded... The day in seminar the PI shouted "that alpha helix is left-handed" and the speaker, after some consternation, flipped the overhead slide... The day a grad student wanted to announce to the world that crystallization of novel proteins in space works, and the post-doc recognized the units cell dimensions as those of hen-egg white lysozyme, the positive control.
Occupational hazards
Sometimes, you would get yelled at. Like the new students asking what the red button on the X-ray generator does while reaching for it. Or like the PI when he picked up the physical model of FIS (still in 10 amino acid segments) and started fidgeting with it, changing side chain torsion angles. Sometimes, you would get embarrassed. Like the diploma student who first learned how to crystallize RNAse T1 with TTP, and then tried to crystallize RNAse A with TTP ("what do you think the A stands for?", asked the old-hand grad student laughing out loud after the entire tray had already been set up, with the PI grinning in the background"). And folks wore special undergarments when they knew they had to spend hours in the cold room.
, respectively.
Magic word you had to use at least twice or four times in posters and papers, respectively.
Culture clashes
We had a variety of people, some who grew up in Berlin and had never left it (and didn't speak much English), and visitors from all over the world. Interactions weren't always smooth. The 16-year old lab tech started calling a visitor from Japan Kamikaze because he was so polite that he would quickly move to the wall of the main hallway when someone else approached him. Everyone learned how to say "yes" in Russian because one of the co-workers kept uttering "da, da, da, da" on the phone.
Myths
The one about the pipette tips floating in the bottled buffer solutions ("they increase the shelf-life of the buffer").
Food supply
Olfert Landt started two businesses during his time at Takustr 6. The Biobar was a drawer below one of the fridges stocked with candy bars. The checklist, which was evaluated weekly, was taped to the fridge. You could see who had deadlines or no other place to be by looking at the list. There was another list for coffee. Tobacco was not provided. Olfert's other business is still going strong.
Sleeping arrangements
Most folks had homes most of the time. On synchrotron trips, though, sleeping arrangements were sketchy. For one trip to DESY in Hamburg, the PI determined the number of reserved beds by dividing the number of travelers by two. After all, the synchrotron runs 24/7, and half of the crew should always be at the beam.
Visitors
Footnotes
- ↑ Karsten: back then, you could ask for a specific PDB ID, and researchers tried to get one that fit their project. nRNT for RNase T1 is pretty good, but at one point the lab ran out of digits
- ↑ Karsten: Looking back, I'm amazed that only five years passed between the 4 Angstrom model and the complete atomic model of photosystem I. To me, folks working on that project were in a different space-time continuum. Everything took longer, the unit cell dimensions were crazy, the solvent content unbelievable, and then the did these things like advancing the spindle every three images because they were burning through the crystal, or using a cylindrical image plate in Japan in hopes of spreading out the overlapping diffraction spots. And then there was airport security: "No, you can't X-ray that, sorry. It's in the thermos to keep it cool. No, you can't look inside, it is light sensitive. Yes, trust me." I am still impressed by the graduate students who dared to join this project.
