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BIOCRYSTALLOGRAPHIC RESEARCH AT CEB
Silvano Geremia, Dipartimento di Scienze Chimiche, Università di Trieste
An excursus of the biocrystallographic research at the Department of Chemical Sciences of the University of Trieste and a summary of the experimental work is presented. The biocrystallography research started in 1997, thanks to a financial support from the University of Trieste, which allowed the settlement of the crystallisation laboratory for biomaterial. The first three-dimensional protein structure, the cytochrome c2 from rhodopseudomonas palustris, was determined in 1999. In 2000 we have implemented the instrumentation with an area detector and a cryo-system and it is planned for 2002 the acquisition of a rotating anode source. Three students are doing their PhD in protein crystallography. The list of crystallisation trials given below illustrates the experimental work done during these years.
| Project |
Conditions |
Collaborating University |
| Metal complexes |
Cu,Ru-sulfoxides |
Catania (Italy) |
| Supramolecular complexes |
Calixarene-Porphyrin, pH |
|
| B12 cofactors |
Several cobalamines |
|
| Peptides with uncodified a.a. |
Metals |
|
| Cytochrome c2 (R. palustris) |
Ox-red, alkaline forms |
Florence (Italy) |
| Cytochrome c (spinach) |
Ox-red |
Modena (Italy) |
| 7Fe-8S Ferrodoxin (R. palustris) |
Ox-red |
Florence (Italy) |
| Lactoperoxidase (bovine) |
Ox-red |
Turin (Italy) |
| Transcobalamin (human) |
Native, heavy atom deriv. |
Aarhus (Denmark) |
| BSA (bovin serum albumin) |
Substrates |
|
| DNA- peptide (HMG) complex |
|
|
| Four helices bundle models |
Several metals and mutants |
Naples (Italy) |
| Homotrimeric bundle |
|
Osaka (Japan) |
| Heterotrimeric bundle |
|
Pennsylvania (USA) |
| Mimochrome IV |
|
Naples (Italy) |
| Rubredoxin model METP |
Several mutants |
Naples (Italy) |
| Maltodextrin Phosphorylase |
Substrates and enzymatic cat. |
Oxford (England) |
The projects leading to full determination and refinement of the three-dimensional structure are in red and the projects leading to obtain single crystals and preliminary diffraction data but not jet the structure are in violet.
The first group of projects refers to small molecule structures from crystals obtained from hanging drop methods. The calixarene-porphyrin supramolecular complexes having a pH-tune stoichiometry are particular relevant. These structures have been recently published in Angewandte Chemie.
The following table reports the determined X-ray protein structures, which can be divided in three groups. The first group (in violet) is relative to the crystal structures of cytochrome c2 from Rhodopseudomonas palustris. The structure of this protein in both oxidation states and in the native state, and of its ammonia adduct has been determined. Dr. Garau presents in his talk some details of this research. The second group of structures (in blue) are relative to the so call "miniaturized proteins". Several derivatives of the four-helix bundle model (DF1) have been solved. Particularly important, is the structure of mimochrome IV (cytochrome b5 model) that to our knowledge is the first structure so far determined by MAD on the Co edge. Dr. Di Costanzo presents in his talk some structure details of these artificial proteins.
The third group of structures (in red) are crystal structures of maltodextrin phosphorylase obtained with several substrates and from experiments of enzymatic catalysis on crystals. The results of these experiments are presented by dr. M. Campagnolo.
X-Ray structures determined at CEB:
| |
PDB |
Res(Å) |
a(Å) |
b(Å) |
c(Å) |
b(°) |
Space group |
Resol. method |
Z |
R/R_free |
| Cyt c2 oxidised |
1fj0 |
1.7 |
50.3 |
71.5 |
66.7 |
93.5 |
P21 |
Mol. Repl |
4 |
17.8/21.8 |
| Cyt c2 reduced |
li8o |
1.9 |
50.1 |
71.7 |
67.4 |
93.7 |
P21 |
Isomorphic str. |
4 |
19.2/23.7 |
| Cyt c2-NH3 |
li8p |
1.4 |
64.8 |
64.8 |
68.6 |
: |
P3221 |
Mol. Repl. |
1 |
13.9/16.7 |
| Cyt c2 -NH3 ox. |
1hh7 |
1.1 |
64.7 |
64.7 |
68.3 |
: |
P3221 |
Isomorphic str. |
1 |
14.5/20.1 |
| Zn-DF1-Leu13 |
1ec5 |
2.5 |
36.0 |
89.0 |
79.7 |
: |
C2221 |
Mol. Repl T.M. |
3 |
24.7/30 |
| Mn-DF1-Ala13 |
1jmb |
2.1 |
37.3 |
112.7 |
80.0 |
|
C2221 |
Mol. Repl. Pack. |
3 |
25.5/30.1 |
| Mn-DF1-Ala13 |
1jm0 |
1.7 |
37.4 |
80.1 |
99.9 |
|
P212121 |
Mol. Repl |
6 |
19.6/25.6 |
| Mn-DF1-Gly13 |
|
1.9 |
38.2 |
89.3 |
146.4 |
|
P212121 |
Mol. Repl |
8 |
20.0/25.5 |
| Mn-DF1-Leu13 |
|
3.0 |
89.0 |
149.5 |
38.6 |
|
C2221 |
Packing Study |
4 |
22.9/30.5 |
| Malp G1P |
|
2.0 |
74.8 |
105.2 |
218.8 |
|
P212121 |
Isomorphic Str |
4 |
17.9/22.0 |
| Malp G1P G4 |
|
1.8 |
74.3 |
105.9 |
218.9 |
|
P212121 |
Isomorphic Str |
4 |
18.4/21.4 |
| Malp G1P G5 |
|
2.2 |
75.3 |
105.2 |
218.9 |
|
P212121 |
Isomorphic Str. |
4 |
19.5/24.0 |
| MalP G5 SO4= |
|
2.2 |
75.3 |
105.5 |
218.9 |
|
P212121 |
Isomorphic Str |
4 |
17.9/21.6 |
|
