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Structure, Volume 23
Supplemental Information
Near-Atomic Resolution for One State of F-Actin
Vitold E. Galkin, Albina Orlova, Matthijn R. Vos, Gunnar F. Schröder, and Edward H. Egelman
Supplementary Figure 7
0 2 4 6 8 10 12 14 16 18Refinement cycle #
0.525
0.53
0.535
0.54R
free
(4.4
-4.5
Å)
Supplementary Figure 1, related to Figure 1. Preparation of grids for cryo-EM involves large
forces on actin filaments. A) An image of a field of filaments showing a common orientation,
almost certainly due to fluid flow in one direction during the blotting that occurs prior to freezing.
B) Breaks (black arrows) can occasionally be observed in actin filaments due to the mechanical
forces that are placed on these filaments from the fluid flow. The sharp bend (white arrow) is
inconsistent with the known rigidity of the actin filament and thermal flexing, and must also arise
from mechanical forces.
Supplementary Figure 2, related to Figure 1. Out-of-plane tilt of segments correlates with the
perceived thickness of the ice. Typical images of actin in what were perceived as thin ice (A) and
thick ice (C), both using lacey carbon grids imaged with a Titan Krios. In the thin ice, actin
filaments (A, arrows) can be frequently found that are anomalously straight (Galkin et al., 2012)
due to the large mechanical forces that are present. Histograms for out-of-plane tilt from thin ice
(B) and thick ice (D) have been fitted with Gaussians, and the distributions show that the amount
of out-of-plane tilt is increased when the ice is thicker, consistent with the fact that filaments
have a greater freedom to deviate away from the perpendicular to the beam when the film
containing the filaments is thicker prior to freezing.
Supplementary Figure 3, related to Figure1. Comparison of twist distributions of F-actin in
thinner ice (A) and thicker ice (B) were calculated using cross-correlation of images with
projections from a set of models having a range of twist from 162° to 170° with a step of 0.5°.
The reliability of the sorting was established by computing averaged power spectra from
segments having smaller (red bins) or larger (blue bins) angles. A) Twist distribution based on a
sorting of 55,052 segments in thinner ice, with the two classes used to calculate power spectra
marked in red (n=8,727) and blue (n=11,900). Comparison of the two power spectra is shown in
insert where positions of the n=4 layer line are marked with white arrows. B) Twist distribution
based on a sorting of 89,160 segments in thicker ice, with the two classes used to calculate power
spectra in red (n=6,770) and blue (n=8,995). Comparison of the two power spectra is shown in
insert where positions of the n=4 layer line are marked with white arrows.
Supplementary Figure 4, related to Figure 2. (A) The Fourier Shell Correlation (FSC)
between reconstructions from half data sets, with each half set containing from 6,250 to 25,000
segments. Completely independent data sets were generated, so that there was no overlap of
boxes (as opposed to the extensive overlap that would exist if one took odd/even images to create
the two sets). (B) The resolution (using the FSC=0.143) found in (A) is plotted versus the
number of segments, using a log10 scale. This can be fit nicely by a straight line (dotted red line)
as observed by others (Stagg et al., 2014). The estimate for the resolution of 50,000 segments
(approximately the number of segments in the full data set) is 0.223 (vertical black line) or 1/(4.5
Å).
Supplementary Figure 5, related to Figure 2. The FSC between the atomic model for the
canonical state of F-actin and the actual reconstruction. The FSC is 0.5 at 4.66 Å.
Supplementary Figure 6, related to Figure 4. The FSC for the T2 reconstruction. The data
were split into two halves with almost no overlap of boxes. Each subset was iterated
independently for five cycles, and then aligned to generate the FSC curve.
Supplementary Figure 7, related to Experimental Procedures. Rfree value during the final
refinement and model building cycles. Simulated annealing refinements were performed with
CNS using DEN restraints. Structure factors between 4.4-4.5 Å were chosen for the calculation
of Rfree.
Oda et al., Nature (2009) Fujii et al., Nature (2010)
Current manuscript
Longitudinal SD3-up→SD4-low
200-208→283-294 241-247→283-294
205→286 241→324 244→290 245→322
241→324 244→325 245→322
Longitudinal SD3-up→SD2-low
61-65→283-294 40→169 61→167 62→288 64→166
38→169 44→168 61→167 62→288 64→166
Longitudinal SD1-up→SD2-low
38-49→139,140,143 38-49→346,351,374 43→346 44→375
45→143
44→143 47→352
Lateral HP plug→SD2-across the strand
265-271→39-42 265→40 268→40 270→39
N/A
Lateral HP plug→SD3-across the strand
265-271→170-174 267→173
267→173
Lateral HP plug→SD4-across the strand
265-271→201-203 265-271→285-286
none 271→201/202
Lateral SD4→SD1
191-199→110-115 191→110 194/195→110 195→113
194→111 195→113
Lateral SD4→SD3
none 194→177
none
Supplementary Table I, related to Figure 3 The residues predicted to be involved in the subunit-subunit interface are shown for the fiber diffraction model of Oda et al. (Oda et al., 2009), the cryo-EM model of Fujii et al. (Fujii et al., 2010) and this paper. Differences between our model and that of Fujii et al. are shown in red.
All-Atom Contacts
Clashscore, all atoms: 3.24 97th percentile* (N=1784, all resolutions) Clashscore is the number of serious steric overlaps (> 0.4 Å) per 1000 atoms.
Protein Geometry
Poor rotamers 65 20.44% Goal: <1% Ramachandran outliers 23 6.17% Goal: <0.05% Ramachandran favored 314 84.18% Goal: >98% MolProbity score^ 2.78 32nd percentile* (N=27675, 0Å - 99Å) Cβ deviations >0.25Å 23 6.63% Goal: 0
Supplementary Table II, related to Supplementary Experimental Procedures
Supplemental Experimental Procedures
Model building of F-actin
The starting structure for the model building was based on the X-ray structure 2BTF.PDB
(Schutt et al., 1993), which we refined against an earlier EM reconstruction of lower resolution
using the program DireX (Schröder et al., 2007). A short six monomer filament was then
extracted from this model which we used to start a 88 ns MD simulation at a temperature of 300
K in explicit water using Gromacs (Hess et al., 2008) with the Amber-SB99-ILDN force field.
From the resulting trajectory 230 equally spaced frames were extracted. One of the two central
protomers in the short 6-mer filament was extracted from each frame yielding 230 protomer
structures. These protomer structures were then refined against a masked protomer density from
our 4.7 Å EM reconstruction using DireX with cross-validation (free interval 4.0–4.5 Å). The
best 100 structures with the lowest Cfree values were averaged. The geometry of this averaged
structure was then optimized by energy minimization with CNS (Brunger et al., 1998) and again
refined against the EM density with DireX.
A minimal filament model was built by surrounding one protomer with four others using
the helical symmetry of the filament such that this single protomer makes all possible
interactions within the filament. Several rounds of manual model building and correction with
Coot (Emsley et al., 2010), followed by simulated annealing structure refinement with CNS with
DEN restraints, were performed. For the structure refinement in CNS, the EM density for the
five protomers was masked with a soft mask and then converted to an HKL structure factor file
using the CNS task em_map_to_hkl.inp.
NCS restraints were used during the refinement to keep the protomers similar to each
other. The MLHL target function was used while the standard X-ray scattering factors were
replaced by electron scattering factors in CNS. The final refinement yielded an R-value of 36.1%
(Supplementary Fig. 7) and the grouped and restrained B-factor refinement yielded an average
atomic B-factor of 111 Å2. The Molprobity analysis is shown in Supp. Table II.
Supplemental References Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W.,
Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. (1998). Crystallography & NMR
system: A new software suite for macromolecular structure determination. Acta
CrystallogrDBiolCrystallogr 54, 905-921.
Emsley, P., Lohkamp, B., Scott, W.G., and Cowtan, K. (2010). Features and development of
Coot. Acta crystallographica Section D, Biological crystallography 66, 486-501.
Fujii, T., Iwane, A.H., Yanagida, T., and Namba, K. (2010). Direct visualization of secondary
structures of F-actin by electron cryomicroscopy. Nature 467, 724-728.
Galkin, V.E., Orlova, A., and Egelman, E.H. (2012). Actin filaments as tension sensors. Current
Biology 22, R96-R101.
Hess, B., Kutzner, C., van der Spoel, D., and Lindahl, E. (2008). GROMACS 4: Algorithms for
Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. Journal of Chemical
Theory and Computation 4, 435-447.
Oda, T., Iwasa, M., Aihara, T., Maeda, Y., and Narita, A. (2009). The nature of the globular- to
fibrous-actin transition. Nature 457, 441-445.
Schröder, G.F., Brunger, A.T., and Levitt, M. (2007). Combining efficient conformational
sampling with a deformable elastic network model facilitates structure refinement at low
resolution. Structure 15, 1630-1641.
Schutt, C.E., Myslik, J.C., Rozycki, M.D., Goonesekere, N.C.W., and Lindberg, U. (1993). The
structure of crystalline profilin:·-actin. Nature 365, 810-816.