seminar05pptdv2.ppt - High Throu

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					  High Throughput Technique in
    Structural Bioinformatics-
Application to Catalase, an enzyme
   of 57 kDa molecular weight

              By

        Prof. D. VELMURUGAN
   DEPARTMENT OF CRYSTALLOGRAPHY &
                           BIOPHYSICS
         UNIVERSITY OF MADRAS
            GUINDY CAMPUS
            CHENNAI – 600 025
       One of the main interests in the molecular
biosciences is in understanding structure function
relations and X-ray crystallography plays a major role in
this.

        ab initio solutions of the crystal structures of small
molecules are possible by using atomic-resolution
diffraction data, usually at ~0.8 Å. Most of these small
molecular crystal structures are usually solved using
direct-methods programs.

       Macromolecules have mainly been solved at
resolutions less than atomic and this has necessitated
determination of initial phases either from experimental
techniques such as Molecular replacement techniques,
MIR or MAD .
        During the last decade, admirable advances have
taken place in the data-collection facilities and
techniques       available   to    the     macromolecular
crystallographer. To get better X-ray intensity data for this
purpose, new techniques like cryo temperature data
collection, halide soaking and passing of Ar, Ne, Hg gas
have been developed.

        With the above advances, more data sets appear
to be coming from atomic-resolution data. The above
possibility of gaining atomic resolution data even for
macromolecules       prompted   the    direct methods
practitioners to make attempts to extend the direct
methods using other macromolecular techniques to
enable them to tackle the structure solution of
macromolecules.
         X-ray Crystallography has become a central tool in modern
drug and target discovery, providing important insights into
molecular interactions and biological function. The past few years
have seen many advances in the methods underlying
macromolecular crystallography such as protein production,
crystallization, cryo-crystallography and synchrotron technology.
Together these advances mean that X-ray data can be collected
extremely quickly for many different crystals and ligand-bound
complexes. The challenge is to ensure rapid and accurate
interpretation of the data to provide valuable structural information.
         The High Throughput Crystallography (HTC) Consortium
offers scientists a valuable new dimension to the drug discovery
process. The HTC Consortium aims to accelerate crystallographic
structure determination by developing new science as well as
utilizing current technology to go from initial phasing through to
structure refinement and analysis while minimizing the amount of
human intervention that is required. The ability to examine in
atomistic detail the interactions between many different proteins
and ligands provides scientists unprecedented insight into the
mechanics of drug binding.
        Rapid and revolutionary developments in genome
sciences, combinatorial chemistry, informatics and robotics are
having major impacts on drug discovery. Genome sequencing
projects in man and micro-organisms have provided an
unprecedented number of potential drug targets. These have
given impetus to the study of protein expression (proteomics)
and structure (structural genomics), and have allowed a clearer
description of drug targets as molecular components of disease
processes. At the same time, there is rapidly expanding range of
screening technologies, as well as consolidations in medicinal
chemistry arising from the combinatorial approaches that were
pioneered in the 1990s. These developments have created an
environment for the emergence of new strategies for drug
discovery.
        High-Throughput Crystallography is essential for
structure-based lead discovery – a strategy that combines
features of random screening and rational structure-based
design.
       More than 29,000 protein structures are deposited in
the Protein Data Bank (PDB) and more than 1,50,000
sequence (SWISS-PROT) entries exist for which the three
dimensional structures are not available.

       In Structural Genomics, one is interested in determining
the structure in the fastest way to understand new folds and
this has opened up the “High Throughput Crystallography”. An
understanding of the three-dimensional structure (fold)
correlates the function of the molecule.

       High Throughput Crystallography using Automated
procedures promotes a quicker elimination of the structure
having the same fold among the deposited ones when
analyzing thousands of macromolecular structures for which
functional assignments are yet to be known.
        ACORN is a comprehensive and efficient phasing
procedure involving direct methods for the determination of
protein structures when atomic-resolution data are
available (better than 1.2 Å) (Foadi et al., 2000; Mcauley et
al., 2001; Yao, 2002; Foadi, 2003; Dodson & Yao, 2003).

      The fragment can be as a small-idealized piece of
secondary structure (Rajakannan et al., 2004a, b;
Selvanayagam et al., 2004) or an experimental
substructure, such as a metal or a set of S, Se or similar
atoms which can be located from anomalous scattering
measurements.

       ACORN then uses a combination of approaches,
most importantly dynamic density modification, to develop
a refined set of phases. Key to the procedure is the use of
a correlation factor for the weak amplitudes as a criterion of
phase quality.
Dynamic Density Modification (DDM) is designed to modify
the densities in three steps:

    ‟ = 0                               if <0
    ‟ = tanh{0.2[/()]3/2}           if >0
    ‟ = kn()                          if ‟>kn(),

   It sets all negative densities to zero.
   It modifies the positive densities according the ratio
    /().
   It truncates the modified densities to a value of kn(),
    where k is a constant given by the user (default value
    is 3); n is the cycle number of DDM.
   The reflections are divided into three groups (strong,
medium and weak) according to their normalized structure-
factor (E) values.

   The strong reflections (E > 1.2) are used in the phase
refinement by the Dynamic density modification (DDM)
and Patterson superposition (SUPP) procedures.

  Both strong and weak reflections (E < 0.1) are used in
Sayre-equation refinement (SER).

  The medium reflections (0.1 < E < 1.2) are used to
calculate a correlation coefficient (CC) for each potential
solution of DDM.
An important component of ACORN is a CC that describes
the extent to which the magnitudes of the calculated
normalized structure factors (Ec) resemble the observed
normalized structure-factor amplitudes (Eo). A fragment in
a particular position and orientation in the unit cell will have
an associated set of structure factors and the CC will be
expressed by
                        Ec Eo    Ec           Eo
                 CC                                ,
                            E c   Ε ο 




 where
                   = <E2> - <E2>½
   Ec and CC values are calculated from the starting
fragment for all reflections to find the correct orientation
and position in molecular replacement (MR) or random MR
or for single random-atom searching.

   In phase refinement Ec and CC values are calculated
from the modified map for medium reflections, which are
not used for computing the map, to indicate solutions of
DDM.
   The ACORN procedure, as implemented in CCP4, is
divided into two parts, ACORN-MR and ACORN-PHASE,
as illustrated in the flow diagram.

  Large       AMoRe           PDBSET      Positioned       Known                           Patterson
  Motif       in CCP4         in CCP4     fragment         phases                        superposition

                                                                             Yes

                                                                                   No                No
                                                                          SUPP?             SER?


                                                                                               Yes
 Standard       Molecular replacement      Structure-           Initial
                of random molecule re-       factor             phase
  helices
              placement by search on CC    calculation           sets                   Sayre-equation
                                                                                          refinement




                                                                                        Dynamic density
                                                                                         modification
   Small               Single random-        Known heavy-atom
   motif             atom search on CC           positions

                                                                                          Best set of
                                                                                        refined phases



                        ACORN-MR                                                   ACORN-PHASE
     ACORN-MR, deals with finding the position of a fragment
of the structure, even a single atom, that provides an initial set
of estimated phases. This set is passed into ACORN-PHASE,
where phase refinement by a number of real-space processes
is performed.

     For locating a single atom, this approach randomly
generates thousands of positions in the asymmetric unit. For
each random position, the calculated normalized structure
factor values and corresponding CCs are calculated for all
reflections. 1000 sets with highest CCs are saved as starting
points for further calculations. In most cases, the solution is
normally found in the top 100 sets. This approach can be used
to determine a native protein structure from AR data, if the
structure contains at least one heavy atom (sulphur or
heavier).
       Foadi (2003) has given a detailed explanation of the
reasons for the failure of ACORN when the resolution is below
1.2 Å. At atomic resolution, two neighbouring atomic peaks will
be two separate entities and DDM will enhance both of them.
At lower resolutions, these two peaks will merge into a single
peak and DDM will just enhance it and no positive phase
refinement can be expected in this situation.

       The present work overcomes the above problem at low
resolution using the fragments for seed phasing information.
   The use of ACORN in solving a 57 kDa macromolecule
with atomic resolution (0.88 A) / truncated synchrotron data
(1.5Å resolution)
   Micrococcus lysodeikticus catalase (Murshudov et
al., 2002)
                                       12         17

                                                                          19
                                                                               20
                                                             18
                              7    6                    3
                          8                      14
                      2                     5          8 7
                                                                  6   5
                                                              4
                                            9
                      1           4
                                        3               10

                                            11

                                  15,16
                                            13




                                                        1




                                    PDB i.d.:1gwe
                                  Total residues: 503
Details of the crystallographic data, helices, sheets and sets
Ab initio phasing using ACORN
        ACORN was run with 5000 random single atom
  trials and the 40 positions with highest CCs‟ were
  selected.
        ACORN refined the phases from the random atom
  trials using DDM and led to the solution with good
  agreement of CC.
        In this run, 78 cycles of DDM increased the CC for
  medium reflections with E values from 0.0285 to 0.5246
  in 14.2 hours of CPU time.
        In this ab initio case 8 chains could be automatically
  built with the ARP/wARP (Perrakis et al., 1999) followed
  by REFMAC (Murshudov et al., 1999) (482 residues).
  Manual model building was carried out for the missing
  residues and the final Rw and Rf- values are 14.0 and
  16.2% respectively. The superposition using PROFIT of
  the backbone atoms of this structure with the backbone
  atoms of the same structure solved using conventional
  technique gives the r.m.s deviation of 0.143 Å.
Details of ACORN, ARP/wARP and REFMAC results for
                   ab initio case
Applications of truncated data at 1.5 Å resolution
       For set 23(minimum input), all sheets and one helix
  (helix4) containing 76 residues were given as input to
  ACORN. Here, the ACORN-PHASE option was selected for
  the structure solution. The R-factor and correlation coefficient
  for the medium E value reflections of the initial model are
  54.2% and 0.0469, respectively. Within 56 cycles of DDM the
  R-factor and correlation coefficient attained 53.9% and
  0.0771 indicating a good solution.
       The phases were then fed to ARP/wARP (Perrakis et al.,
  1999) followed by REFMAC (Murshudov et al., 1999). After
  the initial model building by ARP/wARP, the Rw and Rf values
  were 44.8 and 44.4% respectively. This initial model was
  refined for ten cycles of auto building along with five cycles of
  REFMAC in each auto-building cycle. Finally, ARP/wARP was
  able to build 212 residues. At this stage Rw and Rf values
  were 28.9 and 36.3% respectively. An iterative cycle carried
  out with these output phases revealed 481 residues out of
  503 residues with a connectivity index of 0.97.
     Manual model building was carried out in the missing
regions as densities were clear. After the manual model
building, 20 cycles of maximum-likelihood refinement were
performed using REFMAC and solvent atoms were updated
after the refinement using ARP/wARP „build solvent atoms‟
script. The final Rw and Rf values were 13.6 and 15.6%
respectively.
     The backbone of this final model was superimposed with
the structure conventionally solved by the molecular
replacement method. The root-mean square deviation was
0.176 Å and the details are shown in Table 2. The results for
sets 1-16 and 23 are also shown in Table 2.
     Figs 3a to 3q describe the final models obtained after all
the sets were used for „seed-phasing‟ information to ACORN.
     Table 2 lists the ACORN statistics and the ARP/wARP
details for all these cases. The final results obtained in each
case are also mentioned in this table.
Table 2.
Details of ACORN phasing, ARP/wARP model building and
REFMAC refinement
                 PROGRAM                                                            SET 9             SET 10                                     SET 11                                SET 12
                  ACORN                         STARTING             R-factor (%)           CC        R-factor (%)          CC       R-factor (%)           CC          R-factor (%)            CC
                                                Large E     (L)      0.399                  0.2110    0.402                 0.2070   0.411                  0.1871      0.417                   0.1708
                                                Medium E (M)         0.521                  0.1212    0.523                 0.1153   0.526                  0.1044      0.529                   0.0935
                                                                                                      H 1,4-5,7,10-11,14 &17-20      H 1,5,7,10-11,14&17-20 (135
                    Input                                 H 1,4-5,7,10-11,13-14 &17-20 (151a.a)                                                                         H 1,5,10-11,14&17-20 (125 a.a)
                                                                                                      (145a.a)                       a.a.)
                                                          After 37 cycles of DDM                     After 39 cycles of DDM          After 37 cycles of DDM             After 41 cycles of DDM
                                                FINAL         L      0.269                  0.6329    0.269                 0.6330   0.270                  0.6331      0.271                   0.6308
                                                              M      0.525                  0.1199    0.526                 0.1166   0.526                  0.1156      0.528                   0.1108
                 ARP/wARP                                            R-factor (%)           Rfree     R-factor (%)          Rfree    R-factor (%)           Rfree       R-factor (%)            Rfree

AUTOBUILDING : 10 Cycles                        INITIAL              43.5                   42.8      43.5                  42.8     43.6                   43.7        43.8                    43.2
REFMAC : 5 Cycles for each auto building;
Side dock after 6 cycles of auto building
                                                FINAL                14.4                   18.1      13.6                  17.1     15.3                   19.3        14.6                    18.2

                                                                                                      7 chains, 482 residues,
                                                                                                                                     8 chains, 481 residues, missing    8 chains, 481 residues, missing
                                                                                                      missing residues 1 -7, 59,
                                                8 chains, 475 residues, missing residues 1 -7, 59,                                   residues 1 -7, 59, 60, 113, 114,   residues 1 -7, 59, 60, 1 42, 143,
                                                                                                      60, 113, 114, 142, 143,
                                                60, 113, 114, 142, 143, 175, 176, 186, 187,195 -                                     142, 143, 174,175, 186, 187,       186, 187, 201, 202, 331, 332,
        Details of ARP/wARP result                                                                    174-176, 388, 389, 401,
                                                202,388, 389,503,dummy atoms 1288,connectivity                                       201, 202, 388, 389, 503, dummy     388, 389, 401, 402,503, dummy
                                                                                                      402, 503, d ummy atoms
                                                index 0.96                                                                           atoms 1235, connectivity index     atoms1203, connectivity index
                                                                                                      1273, connectivity index
                                                                                                                                     0.97                               0.97
                                                                                                      0.97

                                                                     R-factor (%)           Rfree     R-factor (%)          Rfree    R-factor (%)           Rfree       R-factor (%)            Rfree

Without dummy atoms made by ARP/wARP                                         28.4             29.4           27.3             28.2           27.6             28.9              27.2                 28.3


 After manual model building for missing
                                                                             14.2             15.8           13.6             15.4           13.9             15.7              13.8                 15.6
       residues and solvent building

 r.m.s. deviations of the model with backbone
                                                                       0.145                                        0.191                           0.161                              0.169
     atoms superposed with that of 1gwe
                PROGRAM                                                       SET 13                        SET 14                             SET 15                        SET 16                       SET 23
                                                                                                                                                                                                 R-factor
                 ACORN                           STARTING          R-factor (%)         CC      R-factor (%)           CC          R-factor (%)          CC      R-factor (%)          CC                          CC
                                                                                                                                                                                                   (%)
                                                Large E (L)            0.423           0.1513       0.424            0.1418            0.432            0.1225       0.437            0.1058      0.437          0.1143
                                                 Medium E (M)          0.533           0.0821       0.534            0.0760            0.539            0.0614       0.542            0.0544      0.542          0.0469
                                                                                                                                                                                                  Sheets (1-8) & H4 (76
                   Input                             H1,10-11,14&17-20 (114 a.a)                H 1,10-11,14&18-20 (103 a.a)       H 10-11,14&18-20 (91 a.a)       H 11,14&18-20 (79 a.a)
                                                                                                                                                                                                            a.a)
                                                        After 42 cycles of DDM                     After 46 cycles of DDM            After 55 cycles of DDM         After 55 cycles of DDM        After 56 cycles of DDM
                                               FINAL        L         0.272            0.6286      0.274              0.6283          0.273           0.6289         0.272           0.6275         0.273          0.6211
                                                            M         0.529            0.1085      0.531              0.1022          0.534           0.0961         0.536           0.0869         0.539          0.0771
                                                                    R-factor                                                                                                                       R-factor
                ARP/wARP                                                               Rfree    R-factor (%)          Rfree        R-factor (%)         Rfree    R-factor (%)         Rfree                        Rfree
                                                                       (%)                                                                                                                            (%)
       AUTOBUILDING : 10 Cycles
                                                  INITIAL              43.8             43.8         44.2             44.2             44.5              44.5         45.0             44.8          44.8           44.4
 REFMAC : 5 Cycles for each auto building;
  Side dock after 6 cycles of auto building        FINAL               14.9             19.0         15.2              19.1             14.5            18.1          29.1             35.7          28.9           36.3
                                                                                                8 chains, 474 residues, missing    7 chains, 482 residues,
                                              6 chains, 485 residues, missing residues 1 -      residues 1 -9, 59 60, 1 42, 143,   missing residues 1 -8, 59,
                                              7, 59, 60, 113, 114, 142, 143, 174, 175, 388,     174-180, 186, 187, 201, 202,       60, 113, 114, 142, 143,       20 chains, 3 10 residues,       22 chains, 212 residues,
      DETAILS OF ARP/wARP result
                                              389, 503, dummy atoms 1200, connectivity          258, 259, 388, 389, 503,dummy      186,187, 201, 202, 503,       connectivity index 0.88         connectivity index 0.79
                                              index 0.98                                        atoms 1250, connectivity index     dummy atoms 1224,
                                                                                                0.97                               connectivity index 0.97
                                                                                                                                                                                                  R-factor
                ARP/wARP                                                                                                                                         R-factor (%)         Rfree                         Rfree
                                                                                                                                                                                                    (%)
       AUTOBUILDING : 10 Cycles
                                                                                                                                                                      29.1             35.8          28.9           36.3
 REFMAC : 5 Cycles for each auto building;
  Side dock after 6 cycles of auto building                                                                                                                           13.0             16.6          14.0          17.6
                                                                                                                                                                                                 7 chains, 481 residues,
                                                                                                                                                                 8 chains, 481 residues,         missing residues 1-9, 59,
                                                                                                                                                                 missing residues 1-7, 59, 60,   60, 142, 143, 174, 175,
       Details of ARP/wARP result                                                                                                                                142, 143, 186, 187, 258, 259,   186, 187, 388, 389, 401,
                                                                                                                                                                 388, 389, 401, 402, 503,        402, 5 03, dummy
                                                                                                                                                                 dummy atoms 1337,               atoms 1287, connectivity
                                                                                                                                                                 connectivity index 0.97         index 0.97
                                                                         R-factor                                                                                                                  R-factor
                                                                                       Rfree    R-factor (%)          Rfree        R-factor (%)         Rfree    R-factor (%)         Rfree                        Rfree
                                                                           (%)                                                                                                                        (%)
Without dummy atoms made by ARP/wARP                                       27.0         28.2         28.0             29.3             27.3              28.3         27.2             28.1          27.1           28.2

 After manual model building for missing
                                                                           13.3         14.9         15.0             16.9             14.3              15.8         14.6             16.2          13.6           15.6
       residues and solvent building
    r.m.s. deviations of the model with
 backbone atoms superposed with that of                          0.146                                       0.182                             0.146                         0.204                          0.176
                   1gwe
            Fig. 1                   Fig. 2                     Fig. 3a
      PDB-id: 1GWE                 Ab initio                     SET 1
      Total:503residues     Auto Built: 482 residues      Input: 187 residues
                                                        Auto Built: 476 residues




        Fig. 3b                     Fig. 3c
         SET 2                       SET 3                      Fig. 3d
                                                                 SET 4
  Input: 184 residues         Input: 181 residues
                                                          Input: 177 residues
Auto Built: 470 residues    Auto Built: 474 residues
                                                        Auto Built: 482 residues




         Fig. 3e                     Fig. 3f                    Fig. 3g
          SET 5                      SET 6                       SET 7
   Input: 172 residues        Input: 167 residues         Input: 162 residues
 Auto Built: 477 residues   Auto Built: 472 residues    Auto Built: 479 residues




         Fig. 3h                      Fig. 3i                    Fig. 3j
          SET 8                      SET 9                      SET 10
   Input: 157 residues         Input: 151 residues        Input: 145 residues
 Auto Built: 484 residues    Auto Built: 475 residues   Auto Built: 482 residues
        Fig. 3k                      Fig. 3l                       Fig. 3m
        SET 11                      SET 12                         SET 13
  Input: 135 residues         Input: 125 residues            Input: 114 residues
Auto Built: 481 residues    Auto Built: 481 residues       Auto Built: 485 residues




         Fig. 3n                      Fig.3o                       Fig. 3p
         SET 14                      SET 15                        SET 16
   Input: 103 residues          Input: 91 residues            Input: 79 residues
 Auto Built: 474 residues    Auto Built: 482 residues      Auto Built: 481 residues




                                        Fig. 3q
                                        SET 23
                                   Input: 76 residues
                                Auto Built: 481 residues
Seed phasing using Cα atoms

     Only the 503 Cα atoms from the known structure
 were used for seed phasing to ACORN with the
 truncated data extending to 1.3 Å resolution. Successful
 model could be built with 474 amino acids (a.a), the
 backbone atoms of which had an r.m.s deviation 0.132 Å
 with the actual structure (1gwe).

     To mimic the above „seed feeding‟ in real situations,
 mean positional errors (MPE, hereafter) of 0.1, 0.2 Å
 were introduced for the above Cα atoms using
 MOLEMAN (Kleywegt, 1992-2004). Successful model
 could be built with 483, 481 a.a corresponding to input
 fragments with MPE of 0.1 and 0.2 Å respectively. The
 backbone atoms of these had an r.m.s deviation of
 0.169, 0.163 Å respectively with the actual structure
 (1gwe).
        Results of ACORN and ARP/wARP using only Cα
                        atoms (1gwe)
                      Resolution 20-1.3 Å
                   PROGRAM                                                        Cα atoms alone            Mean Positional Error (MPE) of Cα atoms
                                                                                                                     0.1 Å                           0.2 Å
                     ACORN                            STARTING             R-factor (%)    CC              R-factor (%)           CC       R-factor (%)    CC
                                                      Large E (L)          0.446           0.0607          0.445                  0.0640   0.446                  0.0543
                                                      Medium E (M)         0.551           0.0368          0.551                  0.0348   0.552                  0.0303
  Input (1 atom /a.a - ~13% of the total structure)                 503 atoms                                        503 atoms                       503 atoms
                                                              After 123 cycles of DDM                     After 121 cycles of DDM          After 165 cycles of DDM
                                                      FINAL          L     0.254           0.6542          0.255                  0.6520   0.257                  0.6452
                                                                  M        0.503           0.1944          0.505                  0.1904   0.508                  0.1826
                   ARP/wARP                                                R-factor (%)    Rfree           R-factor (%)           Rfree    R-factor (%)           Rfree
AUTOBUILDING : 10 Cycles                              INITIAL              39.9            40.6            40.5                   40.6     40.7                   40.4
REFMAC : 5 Cycles for each auto building;
Side dock after 6 cycles of auto building             FINAL                13.3            16.8            14.7                   18.4     14.5                   18.2
                                                                                                                                           8 chains, 481 residues,
                                                                                                           7 chains, 483 residues,
                                                                                                                                           missing residues 1-7, 59,
                                                      9 chains, 474 residues, missing residues 1-7, 34-    missing residues 1-7, 59, 60,
                                                                                                                                           60, 113, 114, 142, 143, 175
                                                      40,59,60,113,114,142,143,174,175,186,187,388,38      113, 114, 142, 143, 186,
          Details of ARP/wARP result                                                                                                       ,176, 201, 202, 331, 332,
                                                      9,401, 402,503, dummy atoms 1389, connectivity       187, 331, 332, 388 ,389,
                                                                                                                                           388 ,389, 503, dummy
                                                      index 0.96                                           503, dummy atoms 1225,
                                                                                                                                           atoms 1247, connectivity
                                                                                                           connectivity index 0.97
                                                                                                                                           index 0.97
                                                                           R-factor (%)    Rfree           R-factor (%)           Rfree    R-factor (%)           Rfree

 Without dummy atoms made by ARP/wARP                                             28.2          28.9               26.9             28.1           27.5             28.5

    After manual model building for missing
                                                                                  15.1          17.1               14.2             16.0           14.4             16.1
          residues and solvent building

r.m.s. deviations of the model with backbone atoms
           superposed with that of 1gwe
                                                                           0.132                                          0.169                           0.163
              PDB i.d. : 1gwe               Input: Calpha atoms (503)
            Total residues:503               Auto built: 474 residues




Input: 0.1Angstrom error at calpha atoms   Input: 0.2Angstrom error at calpha atoms
         Auto built: 483 residues                   Auto built: 481 residues
Seed phasing using 120 a.a as polyala

      The first 120 a.a from the actual structure were
 treated as polyala model and the above procedures were
 carried out to obtain the final model. Results are detailed
 in Table.

      With the 120 residues as polyala model, ARP/wARP
 was able to build 111 residues in 15 chains when the
 above procedures were followed. An iterative cycle
 carried out with this output as input revealed 480
 residues out of 503 residues with a connectivity index of
 0.98. In the case of first 120 residues of polyala model
 with 0.1 Å MPE, ARP/wARP initially built only 6948
 dummy atoms. Two iterative cycles carried out with this
 as input finally built 481 residues. These two models
 have an r.m.s deviation of 0.176, 0.173 Å respectively
 with the backbone atoms of the actual structure (1gwe).
     Results of ACORN and ARP/wARP using polyala model
                    (5atoms/a.a) (1gwe)
                    Resolution 20 – 1.5 Å                                                          First 120 residues of polyala with a mean positional
               PROGRAM                                          First 120 residues of polyala
                                                                                                                   error (MPE) of 0.1 Å
                 ACORN                          STARTING        R-factor (%)            CC               R-factor (%)                      CC
                                                Large E (L)         0.439             0.0915                 0.442                       0.0853
                                               Medium E (M)         0.541             0.0525                 0.541                       0.0499

                                                                   After 56 cycles of DDM                          After 55 cycles of DDM
                                              FINAL    L            0.276              0.6196                0.274                        0.6195
                                                      M             0.537              0.0807                0.540                        0.0775
               ARP/wARP                                         R-factor (%)           Rfree             R-factor (%)                     Rfree
      AUTOBUILDING : 10 Cycles
                                                 INITIAL             45.1               44.4                 45.1                           44.9
REFMAC : 5 Cycles for each auto building;
 Side dock after 6 cycles of auto building        FINAL              32.3               42.7                 24.5                           45.1

                                                              15    chains,    111    residues,
                                                                                                   0 chains, 0 residues, connectivity index 0.00, dummy
               DETAILS OF ARP/wARP result                     connectivity index 0.88, dummy
                                                                                                   atoms 6948
                                                              atoms 4437

                ARP/wARP                                        R-factor (%)           Rfree             R-factor (%)                      Rfree
       AUTOBUILDING : 10 Cycles
                                                 INITIAL             32.3               42.8                 24.5                           45.2
REFMAC : 10 Cycles for each auto building;
  Side dock after 6 cycles of auto building       FINAL              13.1               16.6                 26.8                           35.8

                                                              6 chains, 480 residues, missing
                                                              residues 1-9, 59, 60, 110-114,       20 chains, 282 residues, dummy atoms 3271,
                 Details of ARP/wARP result
                                                              142, 143, 174, 175, 388, 389, 503,   connectivity index 0.85
                                                              dummy atoms 1354, connectivity
                                                              index 0.98
               ARP/wARP                                                                                  R-factor (%)                      Rfree
       AUTOBUILDING : 10 Cycles                  INITIAL                                                     26.9                           35.8
REFMAC : 10 Cycles for each auto building;
  Side dock after 6 cycles of auto building       FINAL                                                      13.6                           17.5
                                                                                                   8 chains, 481 residues, missing residues 1-7, 39, 40, 59,
                 Details of ARP/wARP result                                                         60, 142, 143, 174, 175, 186, 187, 388, 389, 401, 402,
                                                                                                      503, dummy atoms 1201, connectivity index 0.97
                                                                R-factor (%)           Rfree             R-factor (%)                      Rfree

         Without dummy atoms made by ARP/wARP                        27.3               28.0                 27.1                           28.0

   After manual model building for missing residues and
                                                                     14.0               15.7                 14.0                           15.5
                      solvent building
    r.m.s. deviations of the model with backbone atoms
                                                                            0.176                                           0.173
                superposed with that of 1gwe
                                              PDB i.d. : 1gwe
                                             Total residues:503




Input: First 120 a.a as polyala model   Input: First 120 a.a as polyala model after
       Auto built: 480 residues              introducing the MPE of 0.1Angstrom
                                                  Auto built: 481 residues
 STEREO VIEW OF THE ELECTRON DENSITY (2FO-FC|) MAP SUPERPOSED
WITH FINAL MODEL (Input: Polyala model for the first 120a.a with a MPE of 0.1 Å)
STEREO VIEW OF THE FINAL ELECTRON DENSITY (2FO-FC|)
MAP STARTING WITH THE POLYALA MODEL OF FIRST 120A.A
                 WITH MPE OF 0.1 Å
FINAL ELECTRON DENSITY (2FO-FC|) MAP FOR POLY ALA MODEL
ELECTRON DENSITY (2FO-FC|) MAP FOR HEME GROUP IN
                POLYALA MODEL
Seed phasing using Ncap, Ccap and Middle
        portions of helices/sheets

    Instead of feeding the entire helices or
sheets [Selvanayagam et al., 2004 (a minimum
of 76 residues were found to be sufficient for
seed phasing with 1.5 Å truncated data to solve
the three dimensional structure of catalase)]
either the N cap/C cap regions or the mid portion
in the helices or sheets could also be fed as
input for seed phasing. Successful model can be
built in these cases also. The results obtained
are listed in Table.
Results of ACORN and ARP/wARP using Ncap, Ccap and Middle portions
                      of helices/sheets (1gwe)
                         Resolution 20-1.5 Å

                                                                     Ncap region of helices/sheets          Ccap region of helices/sheets              Middle region of
              PROGRAM
                                                                                                                                                        helices/sheets
                ACORN                       STARTING              R-factor (%)            CC              R-factor (%)            CC           R-factor (%)            CC
                                            Large E    (L)        0.435                   0.1118          0.437                   0.1076       0.434                   0.1319
                                            Medium E (M)          0.539                   0.0569          0.542                   0.0553       0.538                   0.0623
                  Input                                               76 a.a                                             76 a.a                               76 a.a
                                                      After 51 cycles of DDM                             After 53 cycles of DDM                After 52 cycles of DDM
                                            FINAL            L    0.275                   0.6270          0.271                   0.6307       0.272                   0.6248
                                                          M       0.534                   0.0901          0.533                   0.0946       0.533                   0.0926
               ARP/wARP                                           R-factor (%)            Rfree           R-factor (%)            Rfree        R-factor (%)            Rfree
AUTOBUILDING : 10 Cycles
REFMAC : 5 Cycles for each auto             INITIAL               44.5                    44.8            44.4                    44.9         44.7                    44.5
building;
Side dock after 6 cycles of auto building   FINAL                 14.9                    18.6            14.5                    18.2         15.2                    19.1

                                                                                                          9 chains, 474 residues, missing
                                                                                                                                               8 chains, 479 residues, missing
                                            10 chains, 470 residues, missing residues 1-7, 39, 40, 59,    residues 1-7, 39-40, 59, 60, 142,
                                                                                                                                               residues 1-7, 60, 110-114, 142,
                                            60, 113, 114, 142, 143, 174, 175, 176, 195-202, 331, 332,     143, 174, 175, 186, 187, 201, 202,
      Details of ARP/wARP result                                                                                                               143, 175, 176, 186, 187, 388,
                                            388, 389, 401, 402, 503, dummy atoms 1308, connectivity       331, 332, 388, 389, 503, dummy
                                                                                                                                               389, 503, dummy atoms 1230,
                                            index 0.95                                                    atoms 1277, connectivity index
                                                                                                                                               connectivity index 0.97
                                                                                                          0.96

                                                                  R-factor (%)            Rfree           R-factor (%)            Rfree        R-factor (%)            Rfree
   Without dummy atoms made by
                                                                           28.9                29.3               28.3                 29.3            27.8                 28.7
            ARP/wARP

   After manual model building for
                                                                           13.3                16.2               14.6                 17.4            13.0                 15.8
 missing residues and solvent building
    r.m.s. deviations of the model with
 backbone atoms superposed with that of                               0.218                                              0.183                                0.151
                   1gwe
Input: Ncap region of helices/sheets(76 a.a)   Input: Ccap region of helices/sheets(76 a.a)
         Auto Built: 470 residues                       Auto Built: 474 residues




                                     Input: Middle region of helices/sheets(76 a.a)
                                               Auto Built: 479 residues




                           Black shaded regions correspond
                            to the input residues from 1gwe
Conclusion
• Based on the published work and the work being carried
  out by our group (Rajakannan et al., 2004a; 2004b), it
  has now become very clear that very little information
  (15%) is needed to determine the structure of a protein
  using ACORN.
• Ours is the first case of ACORN applications using seed-
  phasing information to solve even larger molecular
  weight protein (57 kDA) when the resolution extends to
  1.5 Å.
• Among the multiple solutions, the correct solutions can
  be obtained in all trials with high reliability by the working
  of correlation coefficient and hence high resolution and
  fairly complete diffraction data enable one to solve a
  protein ab initio, in a relatively short amount of time.
• ACORN has the great potential to establish itself
  as program for high-throughput structure
  determination.

• Currently, in order to extend the applicability of
  ACORN to lower resolutions, the seed phasing
  has been obtained from the native structure itself
  (as the structure had already been solved by
  traditional   macromolecular     crystallographic
  methods). Data mining approach to feed
  fragments using the PDB entries is in progress.
References
Banumathi, S., Rajakannan, V., Velmurugan, D., Dauter, Z., Dauter, M., Tsai,
   M. D. & Sekar, K. (2002). Japanese Crystallographic Society Meeting,
   Poster, P3-II-27, 123.
Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50, 760-
   763.
Dodson, E. J. & Yao, J. -X. (2003). Crystallogr. Rev. 9, 67-72.
Foadi, J. (2003). Crystallogr. Rev. 9, 43-65.
Foadi, J., Woolfson, M. M., Dodson, E. J., Wilson, K. S., Yao, J. -X. & Chao-de,
   Z. (2000). Acta Cryst. D56, 1137-1147.
Kleywegt, G. J. (1992-2004). Uppsala University, Uppsala, Sweden.
   Unpublished program.
McAuley, K. E., Yao, J. –X., Dodson, E. J., Lehmbeck, J., Ostergaard, P. R. &
   Wilson, K. S. (2001). Acta Cryst. D57, 1571-1578.
Murshudov, G. N., Lebedev, A., Vagin, A. A., Wilson, K. S. & Dodson, E. J.
   (1999). Acta Cryst. D55, 247-255.
Murshudov, G. N., Grebenko, A. I., Brannigan, J. A., Antson, A. A., Barynin, V.
   V., Dodson, G. G., Dauter, Z., Wilson, K. S. & Melik-Adamyan, W. R.
   (2002). Acta Cryst. D58, 1972-1982.
Perrakis, A., Morris, R. M. & Lamzin, V. S. (1999). Nature Struct. Biol. 6, 458-
   463.
Rajakannan, V., Velmurugan, D., Yamane, T., Dauter, Z., Dauter, M., Tsai, M.
   D. & Sekar, K. (2002). Japanese Crystallographic Society Meeting, Poster,
   P3-I-22, 84.
Rajakannan,V., Yamane, T., Shirai, T., Kobayshi, T., Ito, S. &
   Velmurugan, D. (2003). International Symposium on Diffraction
   Structural Biology, Tsukuba, Japan, 28-31 May 2003, Poster P-085.
Rajakannan, V., Yamane, T., Shirai, T., Kobayshi, T. Ito, S. &
   Velmurugan, D. (2004a). J. Synchrotron Rad. 11, 64-67.
Rajakannan, V., Selvanayagam, S., Yamane, T., Shirai, T., Kobayshi,
   T., Ito, S. & Velmurugan, D. (2004b). J. Synchrotron Rad. 11, 358-
   362.
Selvanayagam, S., Velmurugan, D., Yamane, T. (2004). Asian
   Crystallographic Association Meeting (AsCA‟04) Poster(P0165).
Velmurugan, D., Rajakannan, V., Yamane, T., Dauter, Z. & Sekar, K.
   (2002). Japanese Crystallographic Society Meeting, Poster, P3-II-
   26, 122.
Yao, J. -X. (2002). Acta Cryst. D58, 1941-1947.

				
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