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ECCA and HICA nickel affinity data files

Chromatographs

How to upload records

Exported excel files of nickel column runs containing the following data should be exported: mL, time, A280 trace, A260 (if available), % buffer B. Name the files according to the following format: proteinmutation-date-initialsofgroupmembers

R160HHICA-1Jan2013-KHJW.xls

Uploading files to the galleries

  1. Open a window to the File Galleries in a separate browser window by shift-clicking (Firefox) on File Galleries in the Menu. You will see a listing of available File Galleries. Choose the CHEM 443L Data folder, then the subfolder Endogenous Nickel Affinity
  2. To upload a file, click on the Upload file link in the Menu under Image Galleries. Give the file a name, or use the existing filename. It is highly recommended that you use filenames without spaces.
  3. Check that the drop-down menu has selected the Endogenous Nickel Affinity gallery.
  4. Click the upload button

  1. Still in the file gallery, click browse images. Right-click on the file you would like to insert into a wiki page and (Firefox) select Copy Link Location.
  2. To insert this link into your wiki page paste this code into your wiki page at the appropriate location using CRTL-V or by right-clicking and selecting Paste. Surround your link with square brackets and add descriptive text after a vertical bar, e.g. [http://pasted-link-to-file|R160HHICA-1Jan2013-KHJW.xls].
  3. Alternatively, use the File ID number in this format: e.g. [tiki-download_file.php?fileId=74|R160HHICA-1Jan2013-KHJW.xls].
  4. To show the image of the file, use the code e.g. {img fileId="376" width="250"}.

images and figures



Figure 1. R160H purification chromatograph. R160H HICA eluted as a mixture of peaks at ~55% buffer B (275 mM imidazole). Possibly a sizing column run, or a higher imidazole concentration in the wash step would remove some of these contaminants.

Figure 2. R160H and ECCA purification gel 1. Possibly this is reversed?

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Figure 1. R160H purification gel attempt 2. 4-20% SDS PAGE gel run at 180V for 50 minutes. Lanes are from left to right: 1. Whole cell 2. cell free extract 3. Nickle column pure protein (post-desalting column) 4. sizing column fraction 12 mL 5. sizing column fraction 13 mL 6. sizing column fraction 14 mL 7. sizing column fraction 15 mL 8 BLUEstain 3 protein marker. Sizing column fraction 14, when compared to the GEC column standards spreadsheet, is consistent with the HICA tetramer at ~100 kDA.


Alysha Labrum, Dave Lee, Ingmar Bolinger

Excel data files:

[[Konis&Blount 1/28/13]]

[[Labrum, Lee, & Bolinger, 3/4/13]]

[[Labrum, Lee, & Bolinger, 5/4/13]]

Summary Results


Table 1
Elution Statistics

proteinelution volume (mL)elution %Byield protein (mg)total cell pellet (g)group members
ECCA 45 mL 45% 2.1 mg 32.5 g K.Hoffmann & J.Watson
HICA 42.3 mL 0% 0 mg 9.697 g Mary Konis & Adam Blount
HICA R160H X mL Batch gradient was used X mg X g Mary Konis & Adam Blount
HICA R160H 52.70 mL 55.60% 6.7 mg 13.1 g A. Labrum, D. Lee & I. Bolinger
ECCA 34.93 mL 59.00% 2.84 mg 14.6 g A. Labrum, D. Lee & I. Bolinger
protein X mL X% X mg X g name & name
protein X mL X% X mg X g name & name
protein X mL X% X mg X g name & name
protein X mL X% X mg X g name & name
protein X mL X% X mg X g name & name

Note

Elution volumn should be normalized to the beginning of the gradient, if possible, to account for differences in load volumes earlier in the run.

Crystallography Hits

Conditions

R160H HICA protein set up in 1x1uL drops of stock 8.4 mg/mL concentration, gave hits in 4 days in the following conditions:
2.0 M (NH4)2SO4; 0.1 HEPES-Na pH 7.5,
0.8 M HNa2PO4, 0.8 M HK2PO4*H2O; and
0.05 H3Na-citrate*H2O, 10% 2-Propanol and 10% polyethylene glycol (PEG) 4,000.
Another hit was observed at 3 weeks, but may have appeared earlier:
10% PEG 8,000 with 0.1 M TRIS-HCl, pH 8.5.

Screens were set up to methodically step down the precipitant in the well solution, but no crystals were grown in time observed.
[[Labrum, final paper Sp13]]

Images of drops

Take a focused photograph of a crystal hit and upload the image as for excel files, above. When linking in this page, use the file ID number and this code: {TAG(tag=center)}{img fileId=1 width=300}__Figure 1.__ Figure caption text.{TAG}.
For each hit, note in the figure legend: protein mutation and concentration, drop size, well solution, and time to crystal appearance (approximately).

Solving Structure in CCP4 and COOT

Solved ECCA structure MTZ and PDB files found in File Galleries under CHEM 443 Data
-GP 4/30/13

The X-ray data for nickel-bound ECCA indicates preliminarily that Ni is bound by surface His residues 72 and 160 at the tetramerization interface (Figure 4).


Figure 4: The solved crystal structure of Ni-ECCA (pink) is symmetry mirrored (grey) to produce the tetramer and overlaid by an electron potential map. Both His160 and His72 are in proximity to areas of positive potential (green) that could indicate presence of a Ni ligand at the tetramerization interface. Figure was taken at 2.10σ/background overall and 4.0σ/background for unresolved potentials, made in COOT.