User:Eric Martz/Cavities tests: Difference between revisions

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<applet load='Dnac_from_2ggz_a.pdb' size='500' frame='true' align='right'
<StructureSection load='' size='350' side='right' caption='' scene=''>
scene='User:Eric_Martz/Sandbox_4/Dnac_model_from_2ggz_a/8' />
==Homology Model of DnaC==
The following sequence was provided for DnaC from E. coli:


<tt>
*1 <scene name='31/319439/Surface_test/1'>Surface test 1</scene>: simple surface on chain A of 1d66. Colored translucent green. This surface was generated with the '''representations''' tab of the SAT.
MKNVGDLMQR LQKMMPAHIK PAFKTGEELL AWQKEQGAIR SAALERENRA
<br>
MKMQ<b>RTFNRS GIRPLHQNCS FENYRVECEG QMNALSKARQ YVEEFDGNIA
<br>
SFIFSGKPGT GKNHLAAAIC NELLLRGKSV LIITVADIMS AMKDTFRNSG
<br>
TSEEQLLNDL SNVDLLVIDE IGVQTESKYE KVIINQIVDR RSSSKRPTGM
<br>
LTNSNMEEMT KLLGERVMDR MRLGNSLWVI FNWDSYR</b>SRV TGKEY
</tt>


This sequence was submitted to Swiss Model, which [http://tinyurl.com/4nek2q generated the homology model] shown here (<scene name='User:Eric_Martz/Sandbox_4/Dnac_model_from_2ggz_a/8'>restore initial scene</scene>) using [[2qgz]] chain A as a template, which has 18.6% sequence identity. Apparently Swiss Model used predicted secondary structure to help in the sequence alignment, but details are not clear to me. The homology model represents residues 55-237, shown in boldface in the above sequence. ''Because of the low sequence identity, this model may well contain major errors, or even be wholly incorrect.''


Swiss Model has apparently used the [[temperature value]] field in the PDB file to indicate regions that are highly unreliable, namely the regions that are <font color="red"><b>red</b></font> when the model is <scene name='User:Eric_Martz/Sandbox_4/Dnac_model_from_2ggz_a/4'>colored by temperature</scene>. These regions are shown as '''translucent white''' in the initial scene (using the Jmol command <i>select temperature >50</i>). The uncertainty in three of these regions is explained by gaps in the template model (see below). Although the details of these regions are even more uncertain than other regions, it seems likely that these loops are on the surface, if the homology model turns out to be substantially correct.
*2 <scene name='31/319439/6zgi_translucen_backbone/1'>6zgi as translucent backbone, chain colors</scene> (no isosurfaces). After clicking this standard SAT-generated green link, then
<jmol>
<jmolButton>
<script>
isosurface delete; isosurface "http://proteopedia.org/wiki/images/6/67/6zgi-cavities.jvxl";
</script>
<text>Load isosurface cavities</text>
</jmolButton>
</jmol>
(From uploaded file http://proteopedia.org/wiki/images/6/67/6zgi-cavities.jvxl -- see [[Image:6zgi-cavities.jvxl]])


===Evolutionary Conservation in the Homology Model===


The [[Conservation, Evolutionary|evolutionary conservation]] pattern, revealed by ConSurf, is quite interesting, showing <scene name='User:Eric_Martz/Sandbox_4/Dnac_model_from_2ggz_a/9'>two conserved patches</scene>.<ref>ConSurf found only 10 sequences in SwissProt, with an Average Pairwise Distance of 1.6. The [http://consurf.tau.ac.il/results/1222995227/output.html run shown here] used 100 sequences from Uniprot, with an APD of 1.4.</ref>
*3 <jmol>
<jmolButton>
<script>
load "http://proteopedia.org/wiki/images/c/c6/6zgi-cavities.pngj";
</script>
<text>Load .PNGJ file</text>
</jmolButton>
</jmol>
(From uploaded file http://proteopedia.org/wiki/images/c/c6/6zgi-cavities.pngj -- see [http://proteopedia.org/wiki/index.php/Image:6zgi-cavities.pngj Image:6zgi-cavities.png])


===Homology Model in FirstGlance===


In order to find specific residues, or see charge distribution or other aspects of this homology model, please use:
*4 <jmol>
<jmolLink>
<script>
load "http://proteopedia.org/wiki/images/c/c6/6zgi-cavities.pngj";
</script>
<text>Green link loading uploaded PNGJ file</text>
</jmolLink>
</jmol>. SAT not involved. PNGJ file generated externally in the [[Jmol/Application|Jmol Java application]], then uploaded to Proteopedia as for the previous item above.


[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=http%3A//proteopedia.org/wiki/images/3/3e/Dnac_from_2ggz_a.pdb View DnaC Homology Model in FirstGlance in Jmol]


===Structural Alignment with Closest Hit at PDB===
*5 <scene name='31/319439/6zgi_cavities_from_pngj/1'>Test dropping pngj with cavities into SAT</scene>, then saving scene. (No molecule loaded with the SAT "load molecule" tab.)
<applet load='2chg9-63_aligned_with_dnac_model.pdb' size='300' frame='true' align='right' caption='Residues 9-63 structurally aligned with the homology model of dnaC.' />




When the [[PDB]] is searched with the dnaC sequence, the closest hit is 39% identity with residues 9-63 of chain A of replication factor C (2CHG), which align with 72-124 of dnaC. When the above homology model of dnaC (made with template 2QGZ) is structurally aligned with residues 9-63 of 2CHG<ref>Structural alignment done with DeepView 3.6b3 using Magic Fit of carbon alphas.</ref>, 43 alpha carbons (out of 54) aligned with RMS deviation 2.3 &Aring;. Residues 21-63 of 2CHG aligned with residues 80-124 of the dnaC homology model. This result adds some confidence to the homology model, since the structural alignment of 2CHG:A21-63 occurred in the same range as the sequence alignment (which was 72-124 in dnaC).
Tested in Firefox (current version) on macOS Mojave, mid-2014 MacBook Pro, 2.2 GHz Intel Core i7.


==Crystal Structure of DnaC==
*6 <scene name='31/319439/6zgi_cavities/1'>Cavities in coronavirus spike protein 6zgi</scene>. It took 8.5 min to generate the cavity isosurfaces after I entered the command "isosurface minset 100 interior cavity 3.0 10.0" into the command slot in the SAT. (This command takes ~45 sec in the Jmol Java app which is therefore 11-fold faster for this operation.) After clicking this green link, I waited 11 min and the scene still did not appear. So I think the isosurfaces are being generated again when the green link is clicked.


A sequence-based search at the international [http://targetdb.pdb.org/ Structural Genomics TargetDB] reveals that the closest completed structure is 2QGZ, the one chosen by SwissModel as a template. A number of crystal and NMR structures have sequence identities up to 37% but over shorter stretches, and with higher E values.


Diffraction data have been obtained (but the solved structure not yet deposited) for a ''Listeria monocytogenes'' sequence of 307 residues, pI 5.2, with an E value of 1.6e-05, though only 21% sequence identity. Diffraction-quality crystals (but not yet diffraction data) have not been obtained for any sequence with such a low E value.


''E. coli'' dnaC (245 residues, pI 9.4) has been crystallized by RIKEN Structural Genomics Initiative (Japan), but the crystals may not be of diffraction quality. It has been cloned, expressed as a soluble protein, and purified (but not yet crystallized) by 3 Structural Genomics Groups (RIKEN Structural Genomics Initiative (Japan), Montreal-Kingston Bacterial Structural Genomics Initiative, Midwest Center for Structural Genomics), as have several proteins with >40% sequence identity. So there is reason for optimism that either a crystal structure, or a more suitable template for homology modeling, will be forthcoming soon. One might consider contacting the groups who have reported purification of dnaC to inquire about progress, and possibly request priority for dnaC.
</StructureSection>
 
==Gaps in the Template Model==
<applet load='Dnac_from_2ggz_a.pdb' size='500' frame='true' align='right'
scene='User:Eric_Martz/Sandbox_4/2qgz/3' />
The template was 2QGZ (<scene name='User:Eric_Martz/Sandbox_4/2qgz/3'>initial scene</scene>). The portion of the template used was Glu107-Arg300. Only the amino-terminal 6 residues were not used as template (translucent). Note that there are <scene name='User:Eric_Martz/Sandbox_4/2qgz/5'>three loops</scene> in this segment of the template that lack coordinates due to [[disorder]] in the crystal (marked with spacefilled alpha-carbon atoms).
 
The missing loops are 202-205 (NGSV), 226-231 (EQATSW), and 268-275 (TIKGSDET). These gaps, which occur between the residues marked /\ below, were apparently ignored in making the model, which has a continuous main chain.
 
{{Clear}}
Below is the alignment produced by Swiss Model, used in making the 3D model. Vertical bars for identity were inserted by hand (I may have missed some).
<pre>
                                                |    | |  |    ||
TARGET    55            R TFNRSGIRPL HQNCSFENYR VECEGQMNAL SKARQYVEEF
2qgzA    100  qkqaais--e riqlvslpks yrhihlsdid vnnasrmeaf saildfveqy
                                                                     
TARGET                    sssss    h h            hhhhhhh hhhhhhhhh
2qgzA              hhh  h  sss    h h            hhhhhhh hhhhhhhhh
 
                            |        | ||  ||    | |              |
TARGET    96    DGN-IASFIF SGKPGTGKNH LAAAICNELL L-RGKSVLII TVADIMSAMK
2qgzA    148  psaeqkglyl ygdmgigksy llaamahels ekkgvsttll hfpsfaidvk
                                                                     
TARGET                ssss ss    hhh hhhhhhhhhh h h  ssss sshhhhhhh
2qgzA                ssss ss    hhh hhhhhhhhhh hh    ssss sshhhhhhh
 
                                  ||  |  | ||                |
TARGET    144  DTFRNSGTSE EQLLNDLSNV DLLVIDEIGV QTESKYEKVI INQIVDRRSS
2qgzA    198  naiske---- --eidavknv pvlilddiga vrde-----v lqvilqyrml
                  /\                          / \
TARGET                        hhh    ssssss              hhhhhhhhhh
2qgzA                        hh  h    ssssss              hhhhhhhhhh
 
                  |    |                ||| |  |              |
TARGET    194  SKRPTGMLTN SNMEEMTKLL ---GERVMDR MRLGNSLWVI FNWDSYR 
2qgzA    247  eelptfftsn ysfadlerkw awqakrvmer vr-ylarefh leganrr- 
                                      /\
TARGET          h  ssssss    hhhhh          hhhh hh  ssssss s       
2qgzA          h  ssssss    hhhh          hhhh hh hh ssss s
</pre>
 
Below is the sequence with ATOM records (coordinates) from 2QGZ, numbered 100-300, showing the gaps as "...". This sequence listing was used to locate the positions marked /\ above.
<pre>
    1 .......... .......... .......... .......... ..........
  51 .......... .......... .......... .......... .........Q
  101 KQAAISERIQ LVSLPKSYRH IHLSDIDVNN ASRMEAFSAI LDFVEQYPSA
  151 EQKGLYLYGD MGIGKSYLLA AMAHELSEKK GVSTTLLHFP SFAIDVKNAI
  201 S....KEEID AVKNVPVLIL DDIGA..... .VRDEVLQVI LQYRMLEELP
 
  251 TFFTSNYSFA DLERKWA... .....WQAKR VMERVRYLAR EFHLEGANRR
</pre>
(Copied from Protein Explorer's sequence display.)
 
Below is the alignment of full-length dnaC with 2QGZ according to TargetDB (see above). Note that the 2QGZ structure begins at residue 100, and so the homology model begins with residue 55 of dnaC, indicated with &gt; below.
<pre>
ID:  DR58  Center: NESGC
E-value: 0.00028  Identity: 19.737%
 
                                    10        20        30       
Query                        MKNVGDLMQRLQKMMPAHIKPAFKTGEELLAWQKEQGA
                                    Q+ Q  P++I  +++    +    + +
Subjct EVASFISQHHLSQEQINLSLSKFNQFLVERQKYQLKDPSYIAKGYQPILAMNEGYADVSY
              40        50        60        70        80        90
 
      40        50    >  60        70        80        90       
Query  IRSAALERENRAMKMQRTFNRSGIRPLHQNCSFENYRVECEGQMNALSKARQYVEEF-DG
      +++  L + ++  +++ ++  ++  +++  + +  V+  ++M+A+S  ++VE++ ++
Subjct LETKELVEAQKQAAISERIQLVSLPKSYRHIHLSDIDVNNASRMEAFSAILDFVEQYPSA
              100      110      120      130      140      150
 
      100      110      120        130      140      150     
Query  NIASFIFSGKPGTGKNHLAAAICNELLLR-GKSVLIITVADIMSAMKDTFRNSGTSEEQL
      +  ++ + G  G GK++L AA+ +EL  + G S+ ++  ++  +K+++ N++++EE 
Subjct EQKGLYLYGDMGIGKSYLLAAMAHELSEKKGVSTTLLHFPSFAIDVKNAISNGSVKEE--
              160      170      180      190      200         
 
        160      170        180      190      200      210   
Query  LNDLSNVDLLVIDEIGV-QTESKYEKVIINQIVDRRSSSKRPTGMLTNSNMEEMTK----
      ++ ++NV +L++D+IG+ Q+ S  +  +++ I++ R  + PT + +N ++ ++ +   
Subjct IDAVKNVPVLILDDIGAEQATSWVRDEVLQVILQYRMLEELPTFFTSNYSFADLERKWAT
      210      220      230      240      250      260       
 
                    220      230      240   
Query  LLG-------ERVMDRMRLGNSLWVIFNWDSYRSRVTGKEY
      + G      +RVM+R+R                     
Subjct IKGSDETWQAKRVMERVRYLAREFHLEGANRR       
      270      280      290      300       
</pre>
 
==Notes==
<references />

Latest revision as of 18:59, 13 August 2020


  • 1 : simple surface on chain A of 1d66. Colored translucent green. This surface was generated with the representations tab of the SAT.


  • 2 (no isosurfaces). After clicking this standard SAT-generated green link, then

(From uploaded file http://proteopedia.org/wiki/images/6/67/6zgi-cavities.jvxl -- see File:6zgi-cavities.jvxl)


  • 3

(From uploaded file http://proteopedia.org/wiki/images/c/c6/6zgi-cavities.pngj -- see Image:6zgi-cavities.png)


  • 4 . SAT not involved. PNGJ file generated externally in the Jmol Java application, then uploaded to Proteopedia as for the previous item above.


  • 5 , then saving scene. (No molecule loaded with the SAT "load molecule" tab.)


Tested in Firefox (current version) on macOS Mojave, mid-2014 MacBook Pro, 2.2 GHz Intel Core i7.

  • 6 . It took 8.5 min to generate the cavity isosurfaces after I entered the command "isosurface minset 100 interior cavity 3.0 10.0" into the command slot in the SAT. (This command takes ~45 sec in the Jmol Java app which is therefore 11-fold faster for this operation.) After clicking this green link, I waited 11 min and the scene still did not appear. So I think the isosurfaces are being generated again when the green link is clicked.



Drag the structure with the mouse to rotate
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