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Joint Sino–U.K. Protein Symposium: a Meeting to Celebrate the Centenary of the Biochemical Society
Non-homologous end-joining partners in a helical
dance: structural studies of XLF–XRCC4 interactions
Qian Wu*1 , Takashi Ochi*, Dijana Matak-Vinkovic†, Carol V. Robinson‡, Dimitri Y. Chirgadze* and Tom L. Blundell*
*Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, U.K., †University Chemical Laboratory, Department of
Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K., and ‡Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road,
Oxford OX1 3TA, U.K.
Abstract
XRCC4 (X-ray cross-complementation group 4) and XLF (XRCC4-like factor) are two essential interacting
proteins in the human NHEJ (non-homologous end-joining) pathway that repairs DNA DSBs (doublestrand breaks). The individual crystal structures show that the dimeric proteins are homologues with
protomers containing head domains and helical coiled-coil tails related by approximate two-fold symmetry.
Biochemical, mutagenesis, biophysical and structural studies have identified the regions of interaction
between the two proteins and suggested models for the XLF–XRCC4 complex. An 8.5 Å (1 Å = 0.1 nm)
resolution crystal structure of XLF–XRCC4 solved by molecular replacement, together with gel filtration
and nano-ESI (nano-electrospray ionization)–MS results, demonstrates that XLF and XRCC4 dimers interact
through their head domains and form an alternating left-handed helical structure with polypeptide coiled
coils and pseudo-dyads of individual XLF and XRCC4 dimers at right angles to the helical axis.
XLF and XRCC4 play roles in recruiting and
stabilizing DNA ligase IV at DSBs in NHEJ
DNA DSBs (double-strand breaks) can be caused by ionizing
radiation or toxic chemical exposure, but are also present
as intermediates in V(D)J recombination and class switch
recombination for antigen receptor diversity formation.
Unrepaired DSBs lead to chromosome fragmentation and
rearrangement and are lethal to cells, changing cell gene
regulation and expression, and often leading to cancer cell
formation. The two major DSB repair pathways are HR
(homologous recombination) and NHEJ (non-homologous
end-joining).
Our current understanding of the NHEJ repair pathway
(Figure 1) is that it comprises three major steps: first,
the Ku heterodimer and DNA-PKcs (DNA-dependent
protein kinase catalytic subunit) recognize DSBs and
generate a protein-binding platform for XRCC4 (X-ray
cross-complementation group 4), XLF (XRCC4-like factor)
and other proteins [1,2]; secondly, Artemis containing
endonuclease activity and other end-processing proteins,
such as PNKP (polynucleotide kinase/phosphatase) and
PolX family DNA polymerases, process the DSBs ends
before ligation [3,4]; and thirdly, the XRCC4–LigIV (DNA
ligase IV) complex ligates the two ends of the DNA promoted
by XLF [5]. Understanding how these transient NHEJ
Key words: double-strand break (DSB), non-homologous end-joining (NHEJ), X-ray crosscomplementation group 4 (XRCC4), XRCC4-like factor (XLF).
Abbreviations used: ATM, ataxia telangiectasia mutated; BRCT, BRCA1 C-terminal; DNAPKcs, DNA-dependent protein kinase catalytic subunit; DSB, double-strand break; EM, electron
microscopy; LigIV, DNA ligase IV; nano-ESI, nano-electrospray ionization; NHEJ, non-homologous
end-joining; PNKP, polynucleotide kinase/phosphatase; SAXS, small-angle X-ray scattering;
XRCC4, X-ray cross-complementation group 4; XLF, XRCC4-like factor.
1
To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2011) 39, 1387–1392; doi:10.1042/BST0391387
complexes assemble structurally in both space and time is
a challenging, but timely, research focus. In the past, we have
defined the crystal structures of XRCC4 with LigIV peptide
[6], XLF [7] and more recently DNA-PKcs [8]. The next
step in exploring the NHEJ protein assembly is to study the
complexes of these key protein components.
Although XLF itself cannot directly ligate DSBs, it
performs an essential NHEJ function by interacting with
XRCC4 and stabilizing XRCC4–LigIV broken DNA ends,
thereby enhancing the LigIV end-joining process [9]. The
mechanism of XLF mediating ligation enhancement is
through enhancement of LigIV recharging following ligation
in the presence of ATP [10]. How XLF is structurally
involved in the NHEJ pathway is not clear. In the present
paper, we focus on current biophysical studies of XLF and
XRCC4 and XLF–XRCC4 interactions and recent results
from the crystal structure of the XLF–XRCC4 complex,
which shed light on this question.
XLF and XRCC4 are dimeric coiled-coil
proteins with a common ancestor
Despite the low sequence identity (13.6%), the crystal
structures of XLF and XRCC4 demonstrate that the two
proteins are homologous homodimers comprising globular
head domains and C-terminal helices that form coiled-coil
tail structures [6,7,12,13]. However, the structural differences
between the two are large. The head domains form sevenstranded antiparallel β-sheets sandwiching a helix–turn–helix
motif between β4 and β5, but XLF contains an extra helix in
the N-terminal region. Whereas the tail structure of XRCC4
comprises an elongated coiled-coil, the equivalent extended
helix α4 of XLF is followed by further helices, α5 and α6,
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Figure 1 An overall view of the NHEJ system
A schematic representation of NHEJ and crystallographic structures of the core components Ku70/80 [11], DNA-PKcs [8],
XRCC4-LigIV [6] and XLF [7].
which fold back around the coiled-coil formed by α4 so
that the C-termini come close to the α1 helices of the head
domains. The sequence and structural differences between
XLF and XRCC4 tails explain why LigIV does not bind
to XLF in the same way as XRCC4. A further significant
structural difference is the angle formed between head domain
and helical tail structures for XLF and XRCC4. There is
an approximately 45◦ difference between XLF and XRCC4
coiled-coil tail structures when the head domains from both
proteins were aligned. This is presumably because the helix
α6 of XLF folds back and contacts the head domain, pushing
it further away from the coiled-coil helices.
The highly flexible and disordered C-termini of both XLF
(residues 234–299) and XRCC4 (residues 214–336) were
removed for the crystal structure analyses [6,7,13]. The Cterminal sequence of XLF is important for DNA binding,
and DNA-PKcs targets both protein C-terminal structures
for phosphorylation [14,15]. DNA-PKcs phosphorylates
XRCC4 to regulate its binding with DNA [14]. The
phosphorylation of XLF residues in the unstructured Cterminal affects neither XLF DNA-binding ability nor DNArepair efficiency [16]. The approximate location of the XLF
C-terminal region is predicted to be near the N-terminal
head domain according to the direction of helix α6. EM
(electron microscopy) studies have revealed that the mouse
XRCC4 C-terminal structure is a dimeric globular domain
[17]. SAXS (small-angle X-ray scattering) studies indicated
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observed in XLF [18]. Characterization of the structures of
these regions is needed in future in order to complete our
understanding of the function of XLF and XRCC4 in NHEJ.
XLF and XRCC4 interact through their head
domains
Interactions between XLF and XRCC4 identified through
a yeast two-hybrid study led to the discovery of XLF [5],
even though interactions are dynamic, salt-sensitive and
not dependent on DNA [5,10,18,19]. Another yeast twohybrid study demonstrated that XLF (residues 1–128) and
XRCC4 (residues 1–119) are the minimal regions required for
their interaction, implying that XLF and XRCC4 contacts
are through their head domains [19]. Indeed, when XLF
is immobilized to glutathione-conjugated Sepharose beads
through its C-terminal GST (glutathione transferase) tag,
it is still able to pull down XRCC4–LigIV, implying that
the C-terminal of XLF is not important for interaction
with XRCC4–LigIV [15]. Although both proteins are
present in solution as stable homodimers, a heterodimeric
interaction model between XLF and XRCC4 is unlikely [19].
Furthermore, domain swapping between XLF and XRCC4
indicated that the head domains and coiled-coil regions of
XLF and XRCC4 are not interchangeable, but rather each
has a specific role [20].
Joint Sino–U.K. Protein Symposium: a Meeting to Celebrate the Centenary of the Biochemical Society
The first mutagenesis studies of XLF and XRCC4 revealed
that the structurally exposed Leu115 located in the XLF β6–β7
loop is important for XLF–XRCC4 interaction. Lys63 , Lys65
and Lys99 of XRCC4 essential for XLF–XRCC4 interaction
are located in the beginning of α2 (just after the loop in the
helix–turn–helix structure) and the end of β6 (near the β6–β7
loop). These studies led to the first proposal of a linear sideby-side XLF–XRCC4 interaction model, in which XLF head
domains slide into the space created by XRCC4 head domains
and the N-terminal part of the coiled-coil tail structure [13].
Further extensive mutagenesis studies indicated that
two more XLF head domain residues, Arg64 and Leu65 ,
both located in the loop between helix–turn–helix α2–
α3, are important for interaction with XRCC4 [21].
Leu115 , Arg64 and Leu65 are located in XLF conserved
regions (residues 57–65 and 108–123) [13]. Isothermal
titration calorimetry of the interaction between XLF and
XRCC4 in solution indicated weak enthalpic but significant
entropic contributions, implying a hydrophobic interface
[21]. Together with protein–protein docking analysis, a new
XLF–XRCC4 interaction model was proposed in which
XLF does not slide into the space created by the XRCC4
head domain and the N-terminal part of the coiled coil for
interaction. Instead, interaction between XLF and XRCC4
is mediated through relatively small regions located at the
sides of the head domains and contain the helix–turn–helix
structures and the β6–β7 loop [21].
SAXS structural studies of XLF-(1–248)–XRCC4-(1–
140), XLF-(1–248)–XRCC4 and XLF-(1–248)–XRCC4–
LigIV BRCT (BRCA1 C-terminal) domains suggested a
similar XLF–XRCC4 linear, rather than sliding, binding
model. In addition, SAXS also revealed there is an
approximately 45◦ rotation between XRCC4 and XLF
coiled-coil tails [18].
XLF–XRCC4 partners form an alternating
helical fibre
In order to study the XLF–XRCC4 complex formation,
XLF-(1–233) and XRCC4-(1–164) have been expressed,
purified individually and then run together on an analytical
gel filtration column (Q. Wu, T. Ochi, D. Chirgadze and
T.L. Blundell, unpublished work). An elution peak, indicated
by SDS/PAGE (Figure 2A, left-hand panel) to be an XLF–
XRCC4 complex, runs further to the left and separately
from individual proteins. Increasing the concentration of the
complex shifts the elution peak further to the left, indicating
formation of larger complexes at higher concentrations (Figure 2A, right-hand panel). This XLF–XRCC4 concentrationdependent higher-order complex formation is confirmed
by nano-ESI (nano-electrospray ionization)–MS (Figure 2B)
(see the Supplementary Online Data at http://www.
biochemsoctrans.org/bst/039/bst0391387add.htm for experimental details) [22]. As the concentration of XLF-(1–
233)–XRCC4-(1–164) sample was decreased from 20 μM
to 10 μM (calculated using the molecular mass of 1XLF–
1XRCC4), the size of the largest complex was reduced from a
4XLF–4XRCC4 octamer to a 4XLF–2XRCC4 hexamer. The
observation that large amounts of XLF and XRCC4 dimers
are still present is consistent with previous observations that
the interaction between XLF and XRCC4 is very dynamic
[18].
The heterogeneous XLF-(1–233)–XRCC4-(1–164) complex samples can be crystallized using the hanging drop
method in 0.1 mM Tris/HCl (pH 7.5) and 2 M sodium
formate (Figure 2C, left-hand panel) and SDS/PAGE of
the washed protein crystals confirms the presence of both
proteins (Figure 2C, right-hand panel). The crystal of the
complex diffracts to a resolution of 8.5 Å (1 Å = 0.1 nm)
at the Diamond beamline I04, and the structure of XLF-(1–
233)–XRCC4-(1–164) complex structure was solved at this
resolution by molecular replacement (see the Supplementary
Online Data).
The interaction between XLF and XRCC4 is mediated
through the helix–turn–helix and β6–β7 loop structures
from the head domains of each protein. The binding of
the two proteins generates a tilt angle between the pseudodyads relating head domains and coiled-coil tail structures
(Figure 3A). The XLF-(1–233)–XRCC4-(1–164) proteins
form a left-handed helical filament structure (Figure 3B).
In the crystals, six such helical filaments together create a
tubular structure with a 120 Å diameter central cylindrical
cavity (Figure 3B). The crystal lattice is stabilized through
contacts between the coiled-coil domains of XLF and
XRCC4 (Figure 3A). The interactions appear to be mediated
by hydrophobic contacts between XLF and XRCC4. The
packing arrangement of the tubes, viewed along the c-axis,
appears to be a series of engaged gear cogs (Figure 3A).
The biological role of helical
XLF-(1–233)–XRCC4-(1–164) assemblies
The helical alternating XLF–XRCC4 complex structure does
not contain the region of XRCC4 that binds LigIV. The
crystal structure of XRCC4–LigIV BRCT domains shows
that the BRCT2 domain of LigIV interacts with the coiledcoil region of XRCC4 and is positioned close to one XRCC4
protomer head domain [23] where it can be accommodated
without interfering with the observed helical structure of
the XLF–XRCC4 complex. An EM study concluded that
the catalytic domains of LigIV are located near the XRCC4
head domain and is connected to BRCT1 through a flexible
linker [17]. As XLF interacts with the XRCC4 head domain,
the location and flexibility of catalytic domains of LigIV
when bound to XRCC4 requires further analysis in order
to establish whether the presence of LigIV catalytic domains
affects the interaction of XLF and XRCC4 in the helical fibre.
In classical chromosomal NHEJ, the function of XLF
overlaps with that of ATM (ataxia telangiectasia mutated),
which detects DSBs and activates DSB responses by
phosphorylating histone H2AX and other substrates [24].
XLF, which is also targeted for phosphorylation by ATM in
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Figure 2 XLF-(1–233)–XRCC4-(1–164) complex formation, identification and crystallization
(A) Gel filtration results show that XLF-(1–233) and XRCC4-(1–164) form a complex under 150 mM NaCl conditions. mAu,
milli-absorbance units. Complex formation is concentration-dependent. Insets show SDS/PAGE of the samples, with molecular
masses indicated in kDa. (B) Nano-ESI–MS shows that the major complex is a XLF-(1–233)–XRCC4-(1–164) heterotetramer
with a measured molecular mass of 95661 ± 58 Da, composed of one XLF and one XRCC4 homodimer with peaks between
4000 and 5000 m/z. Both homodimers are present individually in solution (2500–4000 m/z region) with measured molecular
masses of 42 190 ± 23 Da for XRCC4 and 53 455 ± 49 Da for XLF homodimers. Higher oligomers, hexamers and octamers
appear above 5000 m/z and their intensities are concentration-dependent. The mass spectrum on the left-hand side was
obtained from the sample of higher concentration and contains both hexamers and octamers of XLF–XRCC4 complexes,
whereas the spectrum from the sample at half the concentration shows only hexamers. (C) The XLF-(1–233)–XRCC4-(1–164)
protein sample was crystallized (left-hand panel), and the components of the complex was confirmed using SDS/PAGE
(right-hand panel). Molecular masses are indicated in kDa.
its C-terminal region, may have a role in this process [24]. The
crystal structure of the core nucleosome (PDB code 1KX5)
with a diameter of approximately 100 Å can fit within the
XLF–XRCC4 helical filament (diameter of approximately
120 Å), opening up the possibility that the XLF–XRCC4
fibre might wrap around chromatin interacting with DNA
and histones. This would explain the earlier observation that
the C-terminus of XLF, which would be located at the inner
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DNA interaction [10]. It is also possible to accommodate
the Ku70/80 heterodimer (PDB code 1JEY) and DNAPKcs (PDB code 3KGV) within the helical fibre. The Cterminal structures of XLF and XRCC4 are both targeted for
phosphorylation by DNA-PKcs [15,16] and XLF can bind to
the Ku heterodimeric core structure through its C-terminal
structure [25]. Having both DNA-PKcs and Ku70/80 located
Joint Sino–U.K. Protein Symposium: a Meeting to Celebrate the Centenary of the Biochemical Society
Figure 3 Structure of the XLF–XRCC4 complex at 8.5 Å resolution
(A) The XLF–XRCC4 model involves head-to-head interactions and tilt angles between coiled-coil tails of each protein in the
helical structure. (B) One helical turn of the XLF–XRCC4 filament shown from two different viewpoints. XLF and XRCC4 are
represented in red and blue respectively. (C) Tubular structure of XLF–XRCC4.
within the helical fibre would assist these functions. Indeed,
the XLF–XRCC4 helical filament may act as a ‘reaction shell’,
which stabilizes chromatin near-IR foci, and gathers Ku70/80
and DNA-PKcs together for efficient NHEJ function.
The recently defined crystal structures of the N-terminal
regions of the centriole protein SAS-6 in Caenorhabditis
elegans, Chlamydomonas reinhardtii and Danio rerio have
revealed similar protein folds to those of XLF and
XRCC4 [26,27]. The homodimeric SAS-6 proteins form
nine-fold symmetrical ring structures with head domains
interacting together. The coiled-coil tails of the SAS-6 dimers
extend outwards towards the assemblies of microtubules.
A mutagenesis study has shown that the head-to-head
interaction of SAS-6 proteins during oligomerization is
mediated by the β6–β7 loop inserting into the hydrophobic
pocket created by the helix–turn–helix structure and β7 from
the neighbouring homodimer head domain. This is very
similar to the binding model described here between XLF and
XRCC4. The interaction region between XLF-(1–233)
and XRCC4-(1–164) is relatively small, which makes the
helical complex structure rather flexible and could also allow
the formation of a closed ring structure as in SAS-6.
In addition to the proteins bound within the XLF–
XRCC4 helical structure, there may be other NHEJ proteins
assembled around it interacting with the coiled-coil Cterminal regions as in SAS-6. One of these proteins is likely
to be LigIV, which binds to the XRCC4 coiled-coil tail.
This would be required at the DSBs and therefore might
not be bound at every XRCC4, but rather could destabilize
or rearrange the helical structure. Further proteins may
interact with the extension at the C-terminus of XRCC4;
for example, PNKP interacts with XRCC4, both through
a site phosphorylated by protein kinase CK2, as well as
with the unphosphorylated protein [28,29]. XLF does not
bind to LigIV, but the folded-back loop sequence between
XLF α4 and α5 is evolutionarily conserved [13]. Site-directed
mutagenesis studies of XLF at Leu174 , Arg178 and Leu179 ,
which are all located in this evolutionarily conserved hinge
region, reduces the stimulation of the DNA end ligation
activity without affecting the association with XRCC4 or
DNA [13]. This XLF conserved region of unknown function
may bind to other as yet unidentified NHEJ proteins. XLF
is also required for alignment-based gap filling by DNA
polymerases λ and μ [30]. Thus XLF–XRCC4 may provide
a similar safety-belt function elsewhere by securing key
proteins close to the DSB site, therefore assisting DNA repair,
which is crucial for cells to survive.
Indeed, the XRCC4–XLF assembly now described may
be the first dance steps of the protein partners XLF and
XRCC4. The next steps may involve further NHEJ proteins
in a synchronized formal dance that will reveal more of the
complex process of DNA DSB repair.
Acknowledgements
We thank Dr Victor Bolanos Garcia and Dr Lynn Sibanda for helpful
discussions.
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Funding
T.L.B. and D.C. thank the Wellcome Trust for funding through a
programme grant.
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29 Mani, R.S., Yu, Y., Fang, S., Lu, M., Fanta, M., Zolner, A.E., Tahbaz, N.,
Ramsden, D.A., Litchfield, D.W., Lees-Miller, S.P. and Weinfeld, M. (2010)
Dual modes of interaction between XRCC4 and polynucleotide
kinase/phosphatase. J. Biol. Chem. 285, 37619–37629
30 Akopiants, K., Zhou, R.-Z., Mohapatra, S., Valerie, K., Lees-Miller, S.P., Lee,
K.-J., Chen, D.J., Revy, P., de Villartay, J.-P. and Povirk, L.F. (2009)
Requirement for XLF/Cernunnos in alignment-based gap filling by DNA
polymerases λ and μ for nonhomologous end joining in human
whole-cell extracts. Nucleic Acids Res. 37, 4055–4062
Received 22 May 2011
doi:10.1042/BST0391387
Joint Sino–U.K. Protein Symposium: a Meeting to Celebrate the Centenary of the Biochemical Society
SUPPLEMENTARY ONLINE DATA
Non-homologous end-joining partners in a helical
dance: structural studies of XLF–XRCC4 interactions
Qian Wu*1 , Takashi Ochi*, Dijana Matak-Vinkovic†, Carol V. Robinson‡, Dimitri Y. Chirgadze* and Tom L. Blundell*
*Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, U.K., †University Chemical Laboratory, Department of
Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K., and ‡Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road,
Oxford OX1 3TA, U.K.
Nano-ESI MS
For MS of the intact complexes, 20 ml of the XLF–XRCC4
complex was buffer-exchanged into 200–250 mM ammonium
acetate Biospin columns (Bio-Rad Laboratories). Nano-ESI
mass spectra were acquired on a modified QSTAR XL
(MDS Sciex, Applied Biosystems) mass spectrometer using
a protocol described previously [1].
Typical instrument parameters used, in positive-ion mode,
on the QSTAR XL were: capillary voltage 1.4 kV; declustering
potential 100 V, focusing potential 150 V; quadrupole voltage
(Q0) 20–90 V, collision gas (CAD) 3–8. Data were acquired
using Analyst QS software (Applied Biosystems) and
MassLynx v4.1 (Waters).
Molecular replacement
Phaser 2.3 of Phenix software suite [2] first identified two
possible solutions for the position of the XLF using the XLF(1–233) dimer (PDB code 2QM4) as a molecular replacement
search probe resulting in the Translation Function Z-scores
of 7.3 and 6.6. The subsequent molecular replacement search
for the position of the XRCC4 using XRCC4-(1–164) dimer
(PDB code 1FU1) was performed in two different ways. For
each, the position of the XLF search probe found in the
previous step was fixed. This led to identification of two
possible positions of XRCC4 located in close proximity to
the two positions of XLF, resulting in Translation Function
Z-scores of 6.6 and 6.1. These two solutions for the XLF–
XRCC4 heterotetramers are crystallographically identical;
they are related by the half-translation along the c-axis (results
not shown). The rigid-body refinement of the molecular
replacement model obtained was carried out using the
phenix.refine protocol in the Phenix software suite [2] where
each dimer was taken as a rigid body. However, the R-factor
and Rfree of the model did not improve after the refinement and remained over 50 % for the both in R-factor
and Rfree . Therefore the position of the XRCC4 dimer was
manually adjusted using Coot [3] in such a way as to make
the interactions of XRCC4 with XLF similar for both chains
of the XRCC4 dimer. The rigid-body refinement of the
1
To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2011) 39, 1387–1392; doi:10.1042/BST0391387
manually adjusted model reduced the R-factor and Rfree to
40.3 % and 41.5 % respectively. The crystals have one XLF(1–233)–XRCC4-(1–164) heterotetramer in the asymmetric
unit, resulting in a high solvent content of 87 %, which
probably explains the low resolution of the diffraction from
the crystals.
Table S1 Crystallographic data collection and refinement
statistics
Values in parentheses show the corresponding statistics for the highest
resolution shell. Rsym = h |I h − <I > |/ h I h , where Ih is the intensity
of reflection h, and <I> is the mean intensity of all symmetry-related
reflections. R cryst = ||F obs | − |F calc ||/ |F obs |, where F obs and F calc
are observed and calculated structure factor amplitudes. Rfree as for Rcryst
using a random subset of the data (approximately 10 %) excluded from
the refinement.
Parameter
Statistic
X-ray diffraction data
Space group
P65
Resolution range (Å)
a = b = 236.76 Å, c = 103.23 Å,
α = 90◦ , β = 90◦ , γ = 120◦
50.0–8.50 (8.80–8.50)
Rsym (%)
Completeness (%)
Number of unique reflections
0.05 (44.5)
99.9 (100)
3019
Unit cell
Average redundancy
Average intensity, <I/σ (I)>
Refinement
6.4 (6.2)
12.0
Resolution range (Å)
Number of reflections: work/test
Rcryst (%)
49.8–8.49
2717/278
40.3
Rfree (%)
41.5
The presence of the two non-crystallographic two-fold
axes perpendicular with the c-axis (Figure S1B, Calculated)
was confirmed by calculating the self-rotation function using
the X-ray diffraction data (Figure S1B, Observed). The
tubular XLF–XRCC4 structure has solvent gaps between the
C The
C 2011 Biochemical Society
Authors Journal compilation Biochemical Society Transactions (2011) Volume 39, part 5
Figure S1 Crystallographic packing of the XLF–XRCC4 complex
(A) Crystallographic packing of the XLF–XRCC4 complex viewed along the c-axis. Black arrows indicate pseudo-two-fold
axes. The blue line is a unit cell. XLF and XRCC4 are represented in red and blue respectively. (B) Observed and calculated
self-rotation maps of the XLF–XRCC4 complex. The images show χ = 180◦ of the self-rotation maps that were calculated
using Molrep [4]. The structural factors of the model were calculated using SFALL.
filaments (Figure S1A). The gaps can be filled with another set
of the six filaments related by pseudo-translation along the
c-axis to the original ones. However, since strong peaks were
absent from the native Patterson map except for the origin
(results not shown), 12-filament tubes are unlikely to exist in
our crystal.
References
1 Hernández, H. and Robinson, C.V. (2007) Determining the stoichiometry
and interactions of macromolecular assemblies from mass spectrometry.
Nat. Protoc. 2, 715–726
2 Adams, P.D., Afonine, P.V., Bunkóczi, G., Chen, V.B., Davis, I.W., Echols, N.,
Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W. et al. (2010)
PHENIX: a comprehensive Python-based system for macromolecular
structure solution. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 213–221
3 Emsley, P., Lohkamp, B., Scott, W. and Cowtan, K. (2010) Features and
development of Coot. Acta Crystallogr. Sect. D Biol. Crystallogr. 66,
486–501
4 Vagin, A. and Teplyakov, A. (1997) MOLREP: an automated program for
molecular replacement. J. Appl. Crystallogr. 30, 1022–1025
Received 22 May 2011
doi:10.1042/BST0391387
C The
C 2011 Biochemical Society
Authors Journal compilation