Supporting Information of Precise and reversible protein microtubule-like structure with helicity driven by dual supramolecular interactions Guang Yang a , Xiang Zhang d , Zdravko Kochovski b,c , Yufei Zhang a , Bin Dai d , Fuji Sakai a , Lin Jiang f , Yan Lu b , Matthias Ballauff b , Xueming Li e , Cong Liu d *, Guosong Chen a *, Ming Jiang a a The State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University, Shanghai 200433, China b Soft Matter and Functional Materials, Helmholtz-Zentrum Berlin für Materialien und Energie, 14109 Berlin, Germany c TEM Group, Institute of Physics, Humboldt-Universität zu Berlin, 12489 Berlin, Germany d Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China e Ministry of Education Key Laboratory of Protein Science, Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China f Department of Neurology, Easton Center for Alzheimer’s Disease Research, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA
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Supporting Information
of
Precise and reversible protein microtubule-like structure with
helicity driven by dual supramolecular interactions
Guang Yanga, Xiang Zhang
d, Zdravko Kochovski
b,c, Yufei Zhang
a, Bin Dai
d, Fuji Sakai
a,
Lin Jiangf, Yan Lu
b, Matthias Ballauff
b, Xueming Li
e, Cong Liu
d*, Guosong Chen
a*,
Ming Jianga
aThe State Key Laboratory of Molecular Engineering of Polymers and Department of
Macromolecular Science, Fudan University, Shanghai 200433, China
bSoft Matter and Functional Materials, Helmholtz-Zentrum Berlin für Materialien und
Energie, 14109 Berlin, Germany cTEM Group, Institute of Physics, Humboldt-Universität zu Berlin, 12489 Berlin,
Germany dInterdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of
Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China eMinistry of Education Key Laboratory of Protein Science, Center for Structural Biology,
Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua
University, Beijing 100084, China fDepartment of Neurology, Easton Center for Alzheimer’s Disease Research, David
Geffen School of Medicine, University of California, Los Angeles, California 90095,
USA
S2
Sample preparation
The small molecules were synthesized and characterized as described in supporting
information (Scheme S1 and Figure S22-42). SBA protein was purchased from Sigma-
Adrich. All chemicals and proteins are used as received. The buffer solution was
prepared with HEPES {4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid} buffer
containing 20 mM HEPES, 5 mM CaCl2, 5 mM MnCl2 and 40 mM NaCl. The SBA
solution was prepared by dissolving SBA (lyophilized powder) in buffer and was stored
over 2 h at 5 °C. R3GN or RnG was also dissolved in buffer separately. The solutions
were filtered through a Millipore 0.45 µm membrane before mixing. The SBA/R3GN
protein tube was prepared by mixing SBA and R3GN together in buffer solution, fixing
the concentrition of SBA and R3GN both at 0.2 mM and then the mixture was stored at
4 °C for 24 h. The protein tube at this condition can be stable for more than one year. The
SBA/RnG (n = 2, 3, 4) protein tubes were prepared under the same condition.
Characterization
Nuclear magnetic resonance (NMR) was taken by AVANCE III HD 400 MHz of
Bruker BioSpin International. Matrix Assisted Laser Desorption Ionization-Time Of
Flight (Maldi-TOF) Mass Spectrum was taken on a AB SCIEX 5800 instrument.
Ultraviolet–vis (UV-vis) absorption spectra were recorded by a Shimadzu UV-2550
spectrophotometer with a 1 mm cuvette. Circular dicroism spectra was taken by a
JASCO-815 instrument with a 1 mm cuvette. Small Angle X-ray Scattering expeiment
was conducted on a NanoStar U SAXS System. Isothermal titration calorimetry (ITC)
experiments were conducted on a MicroCal VP-ITC system at 20.00±0.01°C. Dynamic
light scattering (DLS) was taken by Zetasizer Nano ZS90 from Malvern Instruments
(UK).
Sample preparation and data collection by electron microscopy
For the preparation of negatively stained samples, a drop of the mixture solution was
applied onto a copper grid and the excess solvent was blotted away. Samples were
subsequently stained with 1 wt% uranyl acetate. Samples for Cryo-EM were prepared by
applying 4 µL drop of mixture solution to holey carbon grids (Quantifoil R2/1) and
S3
plunge-frozen into liquid ethane with an FEI vitrobot Mark IV set at 4°C and 95%
humidity. In the case of samples for Cryo-ET, 3 µL drop of 10 nm colloidal gold solution
(Aurion) was applied to the grids and allowed to dry before plunge freezing. Vitrified
grids were either transferred directly to the microscope cryoholder or stored in liquid
nitrogen. All grids were glow-discharged before use.
Cryo-EM and negative stain micrographs were acquired at a number of
magnifications on a JEOL JEM-2100 equipped with a 4 k × 4 k CMOS digital camera
(TVIPS TemCam-F416), operated at 200 kV and on a Philips CM120 operated at 80
kV. Tomographic data were acquired on a JEOL JEM-2100 equipped with a 4 k × 4 k
CMOS digital camera (TVIPS TemCam-F416), operated at 200 kV. Tilt series were
collected to ± 60° with a 2° angular increment and a total dose of either 100 e−/Å2 or 300
e−/Å2 for vitrified or negatively stained samples respectively. In all cases tilt series were
collected at a magnification of 30,000 x, corresponding to a pixel size of 3.9 Å at the
specimen level.
Cryo-EM Data Acquisition for 3D reconstruction
Cryo-EM grids were prepared with Vitrobot Mark IV (FEI), using 8 °C and 100
percent humidity. 4 µL of sample were applied to glow-discharged Quantifoil Cu
R1.2/1.3 grids, blotted for 2.5 s, and plunged into liquid ethane cooled by liquid nitrogen.
Images were taken by an FEI Titan Krios electron microscope operating at 300 kV with a
nominal magnification of 22,500x. Images were recorded by a Gatan K2 Summit detector
(Gatan Company) with the super-resolution mode, and binned to a pixel size of 1.32 Å.
Defocus values varied from 1.1 to 2.2 µm. Each image was dose-fractionated to 32
frames with a dose rate of ~8 counts per second per physical pixel (~6 e−/sÅ2), a total
exposure time of 8 s, and 0.25 s per frame. UCSF Image41 was used for all data
collection.
Image Processing and single particle reconstruction
The images were aligned and summed using the whole image motion correction2. The
defocus value of each image was determined by ctffind33. Micrographs were selected
S4
based on the quality of the micrograph and protein microtube. Protein microtube quality
was defined by length and straightness. A total of 2303 protein microtubes were
segmented by using EMAN2′s e2helixboxer program with step size of 6.34 nm (10%
overlap), resulting in an image stack of 65035 images of 63.4 × 63.4 nm. The segement
images was binned to the pixel size of 2.64 Å for the further 3D alignment and
reconstruction.
The helical symmety was roughly measeasured based on the layer lines in the power
spectrum of a single tube. Then IHRSR4 was used for further refinement. A cylinder was
used as the initial model. After 100 cycles of IHRSR refinement, a more accurate helical
parameters was determined. The helical rotation per subunit is -36.90o and the helical rise
per subunit is 21.22 Å. The 3D reconsturction was then improved by using FREALIGN
v8.095 and finally reach the resolution of 7.9 Å. The final reconstruction was sharpened
by applying an empirical negative B-factor of -700 Å and low-pass filered to 7.9 Å with a
soft cosine edge.
Notice that although the final density map showed a left-handed twisted helical
microtube structure, we observed both left- and right-handed helical microtube from 2D
classification. However, the resolution remained relatively low when we used a right-
handed helical model, which might due to the hetergenous structures of the right-handed
SBA microtube co-exist in solution.
Model building of SBA protein microtube without ligand
The B-factor sharpened map were used for model building in UCSF Chimera. The
crystal structure of SBA from Glycine max (PDB accession code: 1SBE) was first
docked into the density map as a single rigid-body tetramer defined as the biological
assembly. Individual monomers within the tetramer were then locally fitted into the
density to maximize the correlation between the model and the map. Finally the fitted
tetramer was used as a building block to build the whole filament.
Computational modeling of designed ligands in the protein microtube
The structure of R3GN ligand was built into our 7.9 Å density map based on crystal
structure of the previously designed RhB dimer and the complex of SBA tetramer
S5
binding to the sugar ring GalNAc (PDB entries: 4P9W and 1SBE). The whole modelling
process consists of several iterative rounds of two optimization steps: 1) internal ligand
minimization: the conformation of R3GN pair is generated from the crystal structure of
RhB dimer. In order to fit the R3GN pair into the density map, we miminized the pair
while kept the rigid body orientation between two GalNAc match with the sugar binding
sites of two adjacent SBA tetramer in the density map (Figure S12). The small molecule
modelling and minimization was using the Clean feature of WebLab Viewer Pro. 2)
protein-ligand docking: The R3GN pair was then docked into two adjacent SBA tetramer
in the density map by Rosetta6. During docking, the SBA sidechain conformation at
binding site was repacked and small pertubation of rigid body degree of freedom was
refined. Several iterative optimization rounds of internal ligand minimization and
protein-ligand docking were carried until there is no steric clash between SBA tube and
R3GN ligand. Finally the R3GN ligands were fitted into the SBA tube density map to
ensure reasonable interactions at the dimer interface of R3GN pair.
Atomic force microscopy (AFM)
AFM was operated in air on a Bruker Multimode VIII SPM equipped with a J
scanner. Experiments were performed in tapping mode with NSC11 tip (spring constant
48 N·m-1, MikroMasch). Sample (5 µL) was placed on a freshly cleaved mica for AFM
test under dry conditions. Sample solution was allowed to adsorb for 5 min and then it
was washed gently with 1 mL buffer followed by air drying.
Small Angle X-ray Scattering (SAXS)
0.2 mM SBA and equimolar R3GN was mixed together in buffer solution, and the
mixture was stored at 4 °C for 48 h, then the mixture was freeze-dried to obtain red
powder for SAXS experiment. Small Angle X-ray Scattering (SAXS) results were
performed on a Nanostar U small-angle X-ray scattering system (Germany) by using Cu
Ka radiation (40 kV, 35 mA) at room temperature.
Cell experiment
RAW264.7 macrophage cell line was cultured in RPMI 1640, supplemented with