Assembly of the Inner Rod Determines Needle Length in the Type III Secretion
Injectisome
Thomas C. Marlovits, Tomoko Kubori, María Lara-Tejero, Dennis Thomas, Vinzenz M.
Unger, and Jorge E. Galán
Supplementary Methods
Needle complex sample preparation and biochemical analysis
Needle complex sample preparation for electron microscopy or biochemical analysis was
carried out as previously described1 with some modifications. Briefly, overnight cultures
of the S. typhimurium grown in LB broth supplemented with 0.3 M NaCl were harvested
when they reached an OD600 of 1.0. The needle complex and base substructures were
then separated by CsCl density-gradient centrifugation. Samples were prepared
essentially as described previously (1) except that the pH was kept at 8.0 at all times.
Briefly, harvested cells were resuspended in 150mM Tris-HCl (pH 8), 0.5M sucrose,
0.5mg/ml hen egg lysozyme, 5mM EDTA, and kept first on ice for 45min and then at
37°C for 15min. Cells were lysed with 0.3% lauryldimethylamine oxide (LDAO) before
adding 10mM MgCl2 and 500mM NaCl to the lysate. Cell debris was removed by lowspeed centrifugation and bases and needle complexes were pelleted by high-speed
centrifugation (Beckman, 45Ti rotor, 35krpm, 4 hs, 12°C). The pellet was resuspended in
0.5% LDAO in 10mM Tris-HCl (pH 8), 0.5M NaCl, 5mM EDTA and adjusted to a final
concentration of 30% w/v of CsCl. Two-milliliter samples were centrifuged for 12 hours
at 50k rpm in a Beckman TLS-55 rotor. Half-milliliter aliquots were combined with
2.5ml CsCl-free buffer and samples were pelleted in a TLA-110 rotor at 100krpm for
30min. The complexes were resuspended in 0.1ml 0.2% LDAO, 10mM Tris-HCl (pH 8),
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0.5M NaCl and stored at 4°C. Western-blot analysis of needle complex preparations was
carried out as previously described2.
Electron microscopy, image processing, and determination of rotational symmetry
Electron microscopy, image processing and the determination of the rotational symmetry
were carried out as previously described1. Briefly, samples were placed onto glowdischarged carbon-coated Cu/Rh-grids and vitrified by flash-freezing in liquid ethane.
During data collection, the specimens were held at –175°C, and low-dose micrographs
were recorded on Kodak SO163 film using a Tecnai F20 field emission gun electron
microscope operated at 200kV, 50,000-fold magnification, and underfocus values of 1.2 2.9 µm. Micrographs were digitized on a Zeiss Scanner using a 7 µm stepsize, which
corresponds to 1.4Å sampling at the level of the specimen. Individual particle projections
were extracted, combined into a dataset, and subjected to 2x2 pixel averaging in
IMAGIC-53 (final box size of 200x200 pixel). The contrast reversals imposed by the
contrast transfer function (CTF) of the objective lens were corrected for each particle
projection using the mean underfocus value of the respective micrographs as determined
by the program CTFFIND34. Subsequent image processing was done in SPIDER5 as well
as IMAGIC-5. Volumes were rendered using CHIMERA6. The rotational symmetry was
determined using a multi-reference method to both classify the symmetry and determine
the alignment from side views, as previously described (D. R. Thomas, N. R. Francis, and
D. J. DeRosier, unpublished results). Briefly, a single subunit from a three dimensional
map of the M-ring of the flagellar basal body, which is analogous to IR1, was used to
create initial three dimensional models for hypothetical 18 through 28-mers. An
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appropriate number of subunits were placed into the IR1 of cylindrically averaged maps
whose diameters were adjusted to fit the different number of subunits. To model IR2, an
appropriate number of spheres were placed into IR2 in register with the subunits in IR1.
A multi-reference dataset was created by projecting all the different symmetry models
using a limited number of angles about the long axis of the complex, including out of
plane tilt. This dataset was then utilized to sort the individual particles into several
subpopulations with different and well-defined symmetries. Particles that migrated
between different rotational symmetries were discarded and only those particle images
whose symmetries remained stable during iterations of the alignment were used for the
final reconstructions. To generate 3D volumes, individual particles were back-projected
using the Euler angles assigned during projection matching. To calculate density
difference maps between fully assembled needle complex, and the wild type and ∆invJ
base structures, the 3D maps of these structures were normalized (10 and filtered to a
resolution of 20 Å. The difference map was then generated by subtraction of the
normalized ∆invJ or wild type base maps from the wild-type base or fully assembled
needle complex, as appropriate, utilizing the IMAGIC-5 software package.
References for Supplementary Methods
1.
Marlovits, T. C. et al. Structural insights into the assembly of the type III
secretion needle complex. Science 306, 1040-2 (2004).
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2.
Sukhan, A., Kubori, T., Wilson, J. & Galán, J. E. Genetic analysis of assembly of
the Salmonella enterica serovar Typhimurium type III secretion-associated needle
complex. J Bacteriol 183, 1159-67 (2001).
3.
van Heel, M., Harauz, G., Orlova, E., Schmidt, R. & Schatz, M. A new generation
of the IMAGIC image processing system. J. Struct. Biol. 116, 17-24 (1996).
4.
Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and
specimen tilt in electron microscopy. J. Struct Biol. 142, 334-47 (2003).
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Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D
electron microscopy and related fields. J Struct Biol. 116, 190-9. (1996).
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Pettersen, E. F. et al. UCSF Chimera--a visualization system for exploratory
research and analysis. J. Comput. Chem. 25, 1605-12 (2004).
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