!
43!
necessary and fresh crystals from the same drop were flash-cooled in liquid nitrogen
and sent to a synchrotron X-ray facility for data collection.
HLH24-82-L and the seleno-methionine version HLH24-82-L-Se-Met were sent to
the Argonne National Laboratory synchrotron for data collection.
The native crystal resulted in a 3.1Å resolution dataset which indexed in the
space group P2
1
2
1
2
1
with unit cell parameters a=68.052, b=86.803, c=93.638,
α=β=γ= 90.00°. Based on the volume of the unit cell, and a molecular weight of 8.3
kDa, the Matthews coefficient was 2.77 Å
3
/Da at a solvent content of 55% assuming
6 molecules (3 dimers) per asymmetric unit. A self rotation function in Molrep (CCP4
suite) (Lebedev, et al., 2008) clearly showed 2-fold and 3-fold symmetry, indicating 3
dimers per asymmetric unit.
A MAD dataset was collected for HLH24-82-L-Se-Met at 3 energies: Peak
(12,658.3 eV), Inflection (12,656.5 eV), and Remote (13,058.3 eV) at a resolution
range of 50-2.5Å. Only the peak dataset was used to identify selenium peaks and
was indexed and scaled in space group P3
2
21 with unit cell parameters a=51.5Å,
b=51.5Å, c=111.72Å and α=β=90° γ=120°. Matthews coefficient was 2.52 Å
3
/Da
assuming 2 molecules (1 dimer) in the asymmetric unit at 51% solvent content.

240
275
Rotation/image (°)
1 and 2
1
Number of images
180 and 90 (merged)
180
Crystal Data Space Group*
P 31 2 1
P 32 2 1
Unit Cell Dimensions (Å) a
51.62
51.5
b
51.62
51.5
c
111.47
111.72


10.3 (10.5)
†
Rmerge = ∑
hkl
∑
i
|I
i
(hkl) – [I (hkl)]|/ ∑
hkl
∑
i
I
i
(hkl), where I
i
(hkl)and [I (hkl)] are the intensity of
measurement i and the mean intensity for the reflection with indices hkl, respectively.
* See Section 4.1 for differing space group explanation

!
!
45!
Figure 12: HKL view of reflections in the kl plane in reciprocal space for N-HLH82-L crystal at
2.1Å resolution.

!
46!
CHAPTER 4: RESULTS and DISCUSSION
(Structure Solution and Insights)

Values in parantheses are for the highest resolution shell.
Phasing Statistics
HLH24-82-L-Se-Met
Ranom
†
(%)
7.7
Rpim (%)
6.3
Selenium sites
4
Anomalous multiplicity
5.5 (5.4)
Anomalous completeness
100 (100)
DelAnom correlation between half-sets
0.495
Mid-Slope of Anom Normal Probability
1.139
†
Ranom = Sum |Mn (I+) - Mn (I-)| / Sum (Mn (I+) + Mn (I-))

MR on the native HLH24-82-L dataset using the same strategy as before still
provided no viable solution and was abandoned in favour of the higher resolution
dataset of the longer form of ID2, N-HLH82-L. The strategy for running MR was to
use 1-2 ensembles with the Se-Met dimer and monomer respectively while
stipulating that the scaled input file, originally indexed in P3
1
21, apply the alternative
P3

Protein
870
Water
24
Potassium
2
Average isotropic (or equivalent) B factors

Macromolecule
54.4
Solvent
55.3
R.M.S deviations from ideal

Bond angles (°)
1.07
Bond lengths (Å)
0.007
Ramachandran analysis (%)

Favoured
97.09
Allowed
2.91
Outliers
0
†
Rwork = Σhkl[""Fobs" - k"Fcalc""] / Σhkl["Fobs"]; Rfree = Σhkl⊂ T[""Fobs" - k"Fcalc""] / Σ
hkl⊂ T["Fobs"]; hkl⊂T – test set.


RAMPAGE by Paul de Bakker and Simon Lovell available at
Please cite: S.C. Lovell, I.W. Davis, W.B. Arendall III, P.I.W. de Bakker, J.M. Word, M.G. Prisant, J.S. Richardson & D.C. Richardson (2002)
Structure validation by C geometry: ! and C deviation. Proteins: Structure, Function & Genetics. 50: 437-450
!
50!
4.2 Overall Structure
The structure of the ID2 homodimer was solved to 2.1Å resolution. The
asymmetric unit of the crystal contained two monomers of the HLH domain (A and B
chains) (Figure 14A). Even though the protein contained residues 1-82 that included
the N-terminus up to the end of the predicted HLH domain, the first 31 residues had
no interpretable density. The final model of ID2 unambiguously showed the
boundaries of the HLH domain to center around residues 32 to 82 in chain A and
residues 39 to 81 in chain B with the loop region for both chains hinging between
residues 51 to 59. Overall, chain A contained 59 residues corresponding to residues
30 to 82 of ID2 and 6 residues (83 to 88) belonging to the polypeptide stabilizer.
Chain B contained 47 residues corresponding to residues 35 to 82 of ID2 with no
sign of the stabilizing polypeptide.