Supplementary Experimental Methods
Protein expression and purification
Recombinant proteins were overexpressed in E. coli strain BL21(DE3) from pET
vectors (Novagen) and purified from bacterial lysate by either conventional chromatography
(untagged or MBP-tagged ) or by affinity chromatography on Ni-NTA resin (His6-tagged).
Wild type Bni1p FH2 domain (residues 1327-1769) and homodimeric point mutants were
expressed untagged and His6-tagged, respectively. MBP-tagged tethered FH2 construct was
generated by genetically fusing two FH2 domains through an Ala-Met-Gly linker. MBP was
removed by thrombin digestion followed by anion-exchange chromatography. Actin was
prepared from rabbit muscle acetone powder1, and labeled with pyrene or
Tetramethylrhodamine (Molecular Probes) as reported2,3.
Structure determination of the FH2-actin complex
Diffraction quality
The diffraction quality of both our native and selenomethionine-derivatized (SeMet)
crystals was limited when using synchrotron radiation in several ways. First, the SeMet
crystals were prone to radiation damage. Second, their average diffraction intensity was
relatively strong to a Bragg spacing (dmin) of about 4 Å, but suffered a dramatic decrease to
the high-resolution limit (2.95 Å—but see below, only data to 3.50 Å were ultimately used in
phase determination). Third, the SeMet crystals diffracted isotropically to a dmin of about 4.5
Å, but anisotropically to a dmin of 2.95 Å. Anisotropy was less pronounced but still
significant for our highest-quality native dataset for resolution higher than 4 Å. Finally,
calculation of Wilson B-values (although limited in its information content, due to the low
resolution) indicated a significant degree of disorder (113 Å2 for our native dataset). These
features are not atypical for diffraction data of proteins that contain large -helical bundles4-6.
Phasing
Data were indexed, integrated and scaled using HKL20007. Intensities were converted
to structure factor amplitudes and placed on an approximate absolute scale using
TRUNCATE from the CCP4 package8,9. We found that a single anomalous dispersion
(SAD) experiment was not sufficient to locate selenium sites in our SeMet crystals.
However, these sites could be identified successfully using a three-wavelength MAD dataset.
Data near the high-resolution limit (2.95 Å) showed limited completeness (30-40 %), due to
anisotropy and radiation damage. Because of these shortcomings, only data to 3.5 Å, which
coincides roughly with the resolution limit for which anomalous signal was observed, were
used for locating and refining selenium sites as well as in density modification. Six selenium
sites were identified from the peak and inflection point energy data sets using the Patterson
search routine in CNS 1.110. Phases were refined between 35.0 and 3.5 Å using all three
SeMet data sets in the program MLPHARE11. The figure-of-merit was 0.36 after phase
refinement. Phases were further improved by density modification with histogram matching
in the program DM12, resulting in a final overall figure of merit of 0.56.
The resulting electron density map was sufficiently clear to place rigid body models
of TMR-actin (PDB code 1NWK), including the bound ATP, and the FH2 domain of Bni1p
(PDB code 1UX5). To confirm the validity of the SeMet-derived phases, we carried out
molecular replacement using TMR-actin (PDB ID code 1nwk, stripped of the Ca2+-ATP) as a
search model against the native data set for the actin/FH2 complex in the program AMoRe.
The resulting solution coincides with the actin density observed in the SeMet-phased map. In
addition, strong density for the missing Ca2+-ATP moiety and the Bni1p FH2 domain were
clearly observed in this map.
Model refinement
The structure was refined in the CNS 1.1 program suite with a random 4.7% subset of all
data set aside for Rfree calculation. We corrected the model and refined it against our native
dataset using all data between a dmin of 30.0 and 3.05 Å. Iterative cycles of simulated
annealing, standard positional and grouped atomic displacement parameter (“B-value”)
refinement via a maximum likelihood target were carried out in the program CNS. B-value
refinement, although limited in its information content at this resolution, helped to stabilize
the overall refinement procedure. The resulting electron density map was reasonable for this
type of crystal, but its quality was heterogeneous: some regions had the appearance of a
“typical 3 Å structure”, whereas others were significantly worse. Many side chains did not
have electron density associated with them. Either pruning these side chains to alanine (for
refinement purposes) or setting their occupancy to zero did not lead to improvements in the
refinement behavior nor in the resulting electron density. The best agreement between model
and data was obtained when all side chains were present, probably because the resulting
molecular mask was more accurate and thus the bulk solvent correction more powerful. The
B-values for individual subdomains of actin and FH2 in our model range from 91 Å2 to 139
Å2 with an average of 103 Å2, which is typical for this type of crystal (for a recent example of
a similar refinement behavior, but at even higher resolution, see Bowman, G.D., O’Donnell,
M., & Kuriyan, J., Nature, 2004, 429, 724). TLS-refinement using the program RefMac (G.
N. Murshudov, Acta Crystallogr D Biol Crystallogr 53, 240 (1997)) was used to extract TLS
parameters for actin and FH2, but did not converge when the model was subdivided into
smaller portions. TLS-refinement did not improve the quality of the electron density map,
and even led to an increase in the R-factors. A detailed list of average B-values (without
TLS-refinement) for the individual actin and FH2 subdomains is presented in the Methods
section of the main text to indicate their relative degrees of order.
The final model was validated using a composite simulated-annealing omit map
calculated in CNS. The omit map was virtually identical wi
-weighted 2Fo-Fc
electron density map obtained with a maximum-likelihood refinement target and showed
contiguous density for all but three isolated residues in our model, residues 230 and 252 in
actin, and residue 1488 in the FH2 domain.
NMR Analyses of the mDia1 FH2 domain
For NMR, the FH2 domain of mDia1 (745-1209) was expressed in 99% D2O M9
media supplemented with 40 mg/l 2-keto-3-methyl -13C, 3,4,4,4-D4-butyric acid initially and
68 mg/l 2-keto-3,3-D2-butyric -4-13C- acid at induction to achieve U-[15N, 2H] Ile 1-[13CH3]
and Leu, Val-[13CH3/12CD3] labeling13. NMR experiments were carried out at 25 °C on a
Varian Inova 800 MHz spectrometer. Samples were in D2O buffer containing 10 mM Hepes
pH 7.0, 150 mM KCl, 0.1 mM CaCl2, 1 mM DTT.
1
H-13C TROSY HMQC data were
acquired on free U-[15N,2H], Ile 1-[13CH3], Leu, Val -[13CH3/12CD3]-labeled mDia1 FH2
domain (100 M), and in the presence of 25 M TMR-actin. Data processing and analysis
was carried out using NMRPipe and NMRview, respectively14,15.
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