Question 1: Name, PDB codes
• Eric Martz
• emartz@microbio.umass.edu
• Keiichi Namba
• Macromolecular visualization
I chose:
• 3onz
• HUMAN TETRAMERIC
HEMOGLOBIN: PROXIMAL
NITRITE LIGAND AT BETA
• Function: Oxygen transport
• Resolution: 2.09 Å
• R = 0.218
• Rfree = 0.280 “WORSE
THAN AVERAGE at this
resolution”
Minamino-san gave to
me:
• 1d66
• DNA-binding domain of
Gal4.
• Transcriptional
regulation.
• Resolution: 2.70 Å
• R = 0.230
• Rfree not published
Question 2: Number of chains
Protein: 2 chains (2 distinct)
DNA: 0 chains
RNA: 0 chains
Chain A:
HEMOGLOBIN SUBUNIT ALPHA
141 residues (1 missing) of Protein.
Source: Homo sapiens (Human).
Other_details: Blood.
Chain B:
HEMOGLOBIN SUBUNIT BETA
146 residues (12 missing) of Protein.
Source: Homo sapiens (Human).
Other_details: Blood.
Question 3: Ligands and non-standard residues
3orn:
2 HEM: Protoporphyrin ix containing Fe
2 NO2: Nitrite ion
4 MBN: Toluene
1d66:
4 CD : Cadmium ion
Question 4: Full length vs. crystallized sequence
Chain A:
Crystal: 146 amino acids
Full length: 147 amino acids
Chain B:
Crystal: 141 amino acids
Full-length: 142 amino acids
Question 5: Secondary structure
This structure is all alpha
helices (red) and “coil”
(white). It has no beta
strands.
80.7% alpha helices
0% beta strands
19.3% neither
Question 6: Charge distribution
I found no patches of all
positive or all negative
charges.
For 3onz chain A:
pI = 8.69.
Charges at pH
• 4.0: +20.7
• 7.0: +4.3
• 10.0: -7.0
Question 7: Local uncertainty
Glu43 in chain B
has a high
temperature.
Question 8 - Hydrophobic cores
Yes. Each of the
two chains in
3onz has a
hydrophobic
core (circled in
red).
Question 9 - Water solubility
3onz appears to be soluble
because there are polar
residues everywhere on
the surface.
3onz is hemoglobin which
is known to be a soluble
protein.
Question 10 - Disulfide bonds
Question 11 - Missing residues
13 Missing Residues including
2-, 3+ charged amino acids!
All sidechains are complete.
Question 12 - Non-covalent interactions
• Top: hydrophobic van der
Waals interaction
between two carbon
atoms.
• Bottom: histidine
nitrogen interacting with
negatively charged
oxygen (carboxyl). Partial
salt bridge since His will
have partial positive
charge at pH 7, or
hydrogen bond since His
N epsilon has a hydrogen
to donate..
Question 13 - Biological unit
Asymmetric unit: 2 chains
Biological unit: 4 chains
Question 14 - Evolutionary Conservation
http://consurf.tau.ac.il/results/1368001022/output.php
Top: Lys 61
unexpected.
Bottom: Lys 66
expected because it
forms salt bridges
with the carboxyls
on the heme ligand.
Question 15 - Proteopedia Scene I
Sandbox Reserved 20 (1d66)
Question 16 - Proteopedia Scene II
Sandbox Reserved 20 (1d66)
Question 17a - Cation-pi interaction
There are no significant cation-pi interactions in 3onz.
1d66 contains 3 significant cation-pi interactions. One is Lys25 with
Tyr40 in chain B. See next slide for snapshot.
Here is the report from CaPTURE:
Question 17b - Cation-pi interaction
(see previous slide for explanation)
Question 18 - Polyview-3D static image
This image is for 1d66.
Deoxyguanosine 26 in chain E is red.
Question 19 - Polyview-3D animation for Powerpoint
This animation is for 1d66.
Deoxyguanosine 26 in chain E is red.
Question 20a - Intrinsically disordered regions
3onz chain B is 141 amino acids. The full length sequence has 142. Below
are results for the full length sequence.
FoldIndex predicts no
intrinsically disordered regions,
despite the presence of 3
segments of missing residues in
the crystal model. Therefore I
analyzed 2gry which is more
interesting (next slide).
Question 20b - Intrinsically disordered regions
2GRY is an X-ray crystallographic structure with resolution 2.35 Å. The full
length sequence is 679 amino acids. The crystal includes residues 126-526
(plus an N-terminal His tag). Thus, 1-125 and 527-679 (length 153) were
removed before crystallization. FoldIndex predicts that the removed portions
are intrinsically disordered.
Continued on next slide …
Question 20c - Intrinsically disordered regions
FoldIndex predicts that 5 segments of the crystallized sequence will
be disordered.
Segments predicted to
be disordered:
1. All but 7 of these 69 N
terminal residues are
missing in 2GRY.
2. The middle 4 of these
6 residues are missing in
2GRY.
3, 4, 5: None of these
predictions overlap with
the four additional
segments of missing
residues in 2GRY.
Continued on next slide …
Question 20d - Intrinsically disordered regions
The two segments of amino acids missing in 2GRY are at the N terminus
and near the N terminus. The flanking residues (marked with yellow halos)
have mostly low temperatures, while other missing segments (not
predicted by FoldIndex) have higher temperatures.
2GRY colored by
temperature
showing “empty
baskets”, regions
with missing
residues. Yellow
halos mark the
alpha carbons
flanking missing
residues predicted
to be disordered
by FoldIndex.