|
BIGS protein YecD (1J2R) : an E. coli protein
belonging to the isochorismatase family
|
|
The Escherichia coli gene yecD encodes a 20 kD protein of unknown
function annotated in Swiss-Prot as belonging to the
isochorismatase family (Swiss-Prot: P37437). The crystals
belong to the orthorhombique space group P21212 with 4
molecules in the asymmetric unit. The YecD protein shares
19.5% and 24.6% identity with the 1IM5 and the 1NBA
structures. Out of the 60 models generated and screened
through the CaspR procedure, 30 correspond to truncated models
(positions 1 to 18, 35 to 39 and 68 to 103). In the sorted
list of the best ranking solutions in AMoRe, the 1NBA template
is 13th and 1IM5 is 16th. The ten best solutions all
correspond to truncated models and all converged through the
pre-refinement process while 1NBA and 1IM5 did not (best
solution: Rfree: 50.6, Rwork: 42.0; 1NBA: Rfree: 58.0 Rwork:
56.5; 1IM5: Rfree: 59.6 Rwork: 57.1). This case is actually
the first structure that has been solved entirely using the
method now automated in CaspR.
|
| To look at the general result page click
here
|
| To look at the T-COFFEE alignment click
here
|
| To look at the AMoRe result page click
here
|
| To look at the CNS result page click
here
|
|
|
|
1AJX: the HIV1 protease
|
|
The HIV1 protease structure belongs to the acid proteases fold. It is
a homodimer of a 99 residue long protein and can adopt two
conformations upon ligand binding (RMSD between 1.0 and 1.2 Å over 99
residues based on C. superimposition of the two structural
conformations). Each of these structures has been solved independently
and correspond to 1AJX (1) and 1HHP (2) PDB accession numbers. We used
the available 1HHP structure to solve the 1AJX structure with
CaspR. In this example, the crystals belong to the orthorhombic space
group P21212 and there are 2 molecules in the asymmetric unit. While
the 1HHP structure clearly identifies the first position in the AMoRe
step of the CaspR process, it fails to identify the second molecule
using the standard procedure. On the other hand, using the CaspR
automated protocol, 10 out of the 30 models produce a two molecule
solution in AMoRe and converge during the refinement process (best
model Rfree: 38.4, Rwork: 31.4; 1HHP Rfree: 54.8, Rwork: 50.6).
|
| To look at the general result page click
here
|
| To look at the T-COFFEE alignment click
here
|
| To look at the AMoRe result page click
here
|
| To look at the CNS result page click
here
|
|
|
|
1K6D : the E. coli . subunit of the acetate
CoA-transferase
|
|
The 1K6D structure (3) belongs to the CoA transferase fold. It
crystallizes in the hexagonal space group P62 with two molecules in
the asymmetric unit. This 220 residues long protein has a closely
related structural homologue exhibiting 35% sequence identity over its
entire length (1M3E PDB accession number) as well as a more distant
homologue with less than 25% sequence identity (1POI PDB accession
number). Both potential templates were tested using the CaspR
web-server. In the case of 1M3E, 30 models were produced by MODELLER
and residues that were poorly aligned to the target were automatically
excised based on the T-COFFEE core index (residues 1 to 7, 124 to 134
and 216 to 220). As a result, the 14 best AMoRe solution were obtained
using the truncated models. 28 out of the 30 truncated and 21 out of
the 30 non truncated ones produced an AMoRe solution for both
molecules in the a.u.. All of them present a higher correlation
coefficient and a lower R-factor in AMoRe than the unperturbed 1M3E
structure. The 10 best solutions all correspond to the truncated
models and converge during the pre-refinement procedure using CNS
(best solution Rfree: 43.7, Rwork: 36.3; 1M3E: Rfree: 56.9 Rwork:
53.2)
|
|
In the second case (1POI), 60 models were generated, 30 of them
corresponding to the truncated forms based on the T-COFFEE core index
(excision of residues 58 to 60 and 218 to 220). In this case, the best
AMoRe solution is given by the 1POI structure itself, while none of
the solutions converged during the pre-refinement process. This is a
case where CaspR was not able to improve the solution with a homology
model. Neither the models nor the related structure could produce a
result in this standard procedure. A way to retrieve a solution may be
to change the core index threshold value to be used in the design of
truncated models.
|
|
|
|
BIGS protein YhbO (1OI4): The E. coli potential
cysteine peptidase protein
|
|
YhbO corresponds to a 193 residues long protein annotated as a
potential cysteine peptidase (Swiss-Prot: P45470). It
belongs to the Flavodoxin-like fold and shares 41%
identity with its structural homologue 1G2I. The crystals
belongs to the orthorhombic space group P212121 with 2
molecules in the asymmetric unit. CaspR produced 60
models corresponding to 30 truncated forms (excision of
residues 1 to 24; 56 to 63 and 168 to 171) which went
through the molecular replacement procedure using
AMoRe. The best scoring AMoRe solution is given by the
1G2I structure itself. However all 60 models also produce
a correct solution. After refinement procedure with CNS,
the 1G2I solution eventually only ranks third below two
truncated models with a better convergence, which again
can be explained by the replacement of the 1G2I sequence
by the proper YhbO one (best solution Rfree: 44.9, Rwork:
37.5; 1G2I: Rfree: 45.7 Rwork: 38.4). This again is a
case were CaspR is helpful to decrease the time spent in
refinement procedure.
|
| To look at the general result page click
here
|
| To look at the T-COFFEE alignment click
here
|
| To look at the AMoRe result page click
here
|
| To look at the CNS result page click
here
|
|
|
|
BIGS protein YahK (1UUF): The E. coli Zinc-type
alcohol dehydrogenase-like protein
|
|
YahK is a 349 residue long protein annotated as a Zinc-type alcohol
dehydrogenase-like protein (Swiss-Prot: P75691). Like
1MP0, it is a two domain structure belonging to the
GroES-like fold for the first domain and to the
NAD(P)-binding Rossmann-fold for the second domain. It
shares 33% identity with its closest homologue 1LLU and
27.6% and 26.5% identity with the two related structures
1H2B and 1JVB. The crystals belongs to the monoclinic
space group C2 and there is one molecule in the
asymmetric unit. Out of the 60 models produced using the
3 template structures through the CaspR process, 30
correspond to truncated forms based on the T-COFFEE core
index (10 excised segments corresponding to positions 1
to 48, 72 to 80, 110 to 115, 129 to 146, 156 to 168, 191
to 193, 248 to 260, 290 to 304, 327 to 336 and 364 to
369). Both 1LLU and 1JVB produces higher scoring solution
in the AMoRe search, however, the best solution in the
pre-refinement procedure is produced by one of the 30 non
truncated models (best solution Rfree: 49.9, Rwork: 40.7;
1LLU: Rfree: 51.1 Rwork: 42.0). Due to their ranking,
none of the truncated models went through the refinement
process.
|
| To look at the general result page click
here
|
| To look at the T-COFFEE alignment click
here
|
| To look at the AMoRe result page click
here
|
| To look at the CNS result page click
here
|
|
|
|
BIGS protein YggV (also available as 1K7K) : The
E. coli HAM1 NTPase protein
|
|
YggV is a 197 residue long protein belonging to the HAM1 family of
pyrophosphatase (Swiss-Prot: P52061). According to the
SCOP database it belongs to the Anticodon-binding
domain-like and shares 33.5% identity with the related
2MJP structure. The crystals belong to the tetragonal
space group P43212 and there is one molecule in the
asymmetric unit. 30 full length models having been
produced through the automated procedure and generated 30
truncated models based on T-COFFEE core index (excision
of segments 22 to 39, 89 to 111, 119 to 133 and 191 to
197). In AMoRe, the 2MJP template structure is ranked in
position 27th in the list, while the best ranking
solutions corresponds to the full length models and after
the pre-refinement process, 2 full length models comes
first and are immediately followed by the 2MJP template
structure (best solution Rfree: 54.3, Rwork: 44.7; 2MJP:
Rfree: 55.3 Rwork: 45.2). The amplitude of the R-factors
and difference between that of the free and the working
set prompt for a visual inspection of the corresponding
electron density maps, based on for highest scoring model
and on 2MJP itself, in order to validate the CaspR
solution(s). It turned out that sole the solution based
on the homology model provided a usable map for further
refinement (most likely due to the presence of the
correct side chains in the model).
|
| To look at the general result page click
here
|
| To look at the T-COFFEE alignment click
here
|
| To look at the AMoRe result page click
here
|
| To look at the CNS result page click
here
|
|
|
|
| REFERENCES |
| 1.Backbro, K., Lowgren, S., Osterlund, K., Atepo, J., Unge, T.,
Hulten, J., Bonham, N. M., Schaal, W., Karlen, A. & Hallberg,
A. (1997) Unexpected binding mode of a cyclic sulfamide HIV-1 protease
inhibitor. J. Med. Chem., 40, 898-902. |
| 2.Spinelli, S., Liu, Q. Z., Alzari, P. M., Hirel, P. H. & Poljak,
R. J. (1991) The three-dimensional structure of the aspartyl protease
from the HIV-1 isolate BRU. Biochimie, 73, 1391-1396. |
| 3.Korolev, S., Koroleva, O., Petterson, K., Gu, M., Collart, F.,
Dementieva, I. & Joachimiak, A. (2002) Autotracing of E. Coli Acetate
Coa Transferase A-Subunit Structure Using 3.4 A MAD and 1.9 A Native
Data. Acta Cryst., D58, 2116-2121. |
| 4.Sanghani, P. C., Robinson, H., Bennett-Lovsey, R., Hurley, T. D. &
Bosron, W. F. (2003) Structure-Function Relationships in Human Class
III Alcohol Dehydrogenase (Formaldehyde
Dehydrogenase). Chem. Biol. Interact., 143, 195-200. |