Journal: Cell / Year: 2010 Title: Insights into antiparallel microtubule crosslinking by PRC1, a conserved nonmotor microtubule binding protein. Authors: Radhika Subramanian / Elizabeth M Wilson-Kubalek / Christopher P Arthur / Matthew J Bick / Elizabeth A Campbell / Seth A Darst / Ronald A Milligan / Tarun M Kapoor / Abstract: Formation of microtubule architectures, required for cell shape maintenance in yeast, directional cell expansion in plants and cytokinesis in eukaryotes, depends on antiparallel microtubule ...Formation of microtubule architectures, required for cell shape maintenance in yeast, directional cell expansion in plants and cytokinesis in eukaryotes, depends on antiparallel microtubule crosslinking by the conserved MAP65 protein family. Here, we combine structural and single molecule fluorescence methods to examine how PRC1, the human MAP65, crosslinks antiparallel microtubules. We find that PRC1's microtubule binding is mediated by a structured domain with a spectrin-fold and an unstructured Lys/Arg-rich domain. These two domains, at each end of a homodimer, are connected by a linkage that is flexible on single microtubules, but forms well-defined crossbridges between antiparallel filaments. Further, we show that PRC1 crosslinks are compliant and do not substantially resist filament sliding by motor proteins in vitro. Together, our data show how MAP65s, by combining structural flexibility and rigidity, tune microtubule associations to establish crosslinks that selectively "mark" antiparallel overlap in dynamic cytoskeletal networks.
Mass: 18.015 Da / Num. of mol.: 216 / Source method: isolated from a natural source / Formula: H2O
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Experimental details
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Experiment
Experiment
Method: X-RAY DIFFRACTION / Number of used crystals: 1
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Sample preparation
Crystal
Density Matthews: 2.08 Å3/Da / Density % sol: 40.88 %
Crystal grow
Temperature: 277 K / pH: 9.5 Details: 1:1 v/v of protein ( 50 mg/ml in 80 mM PIPES, pH6.8, 1 mM MgCl2, 1 mM EDTA and 150 mM KCl) and reservoir buffer (100 mM CHES, pH 9.5, 30% PEG3000), VAPOR DIFFUSION, HANGING DROP, temperature 277K
Resolution: 1.75→25 Å / Cor.coef. Fo:Fc: 0.954 / Cor.coef. Fo:Fc free: 0.938 / SU B: 7.196 / SU ML: 0.097 / Cross valid method: THROUGHOUT / ESU R Free: 0.033 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
Rfactor
Num. reflection
% reflection
Selection details
Rfree
0.269
1285
5.3 %
RANDOM
Rwork
0.198
-
-
-
obs
0.202
22853
93.7 %
-
all
-
24390
-
-
Solvent computation
Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parameters
Biso mean: 28.26 Å2
Baniso -1
Baniso -2
Baniso -3
1-
6.01 Å2
0 Å2
0 Å2
2-
-
6.01 Å2
0 Å2
3-
-
-
-12.01 Å2
Refinement step
Cycle: LAST / Resolution: 1.75→25 Å
Protein
Nucleic acid
Ligand
Solvent
Total
Num. atoms
2048
0
0
216
2264
Refine LS restraints
Refine-ID
Type
Dev ideal
Dev ideal target
Number
X-RAY DIFFRACTION
r_bond_refined_d
0.008
0.022
2133
X-RAY DIFFRACTION
r_bond_other_d
0.001
0.02
1567
X-RAY DIFFRACTION
r_angle_refined_deg
1.03
1.952
2857
X-RAY DIFFRACTION
r_angle_other_deg
0.801
3
3794
X-RAY DIFFRACTION
r_dihedral_angle_1_deg
4.71
5
251
X-RAY DIFFRACTION
r_dihedral_angle_2_deg
34.716
24.444
117
X-RAY DIFFRACTION
r_dihedral_angle_3_deg
15.632
15
444
X-RAY DIFFRACTION
r_dihedral_angle_4_deg
18.374
15
16
X-RAY DIFFRACTION
r_chiral_restr
0.065
0.2
287
X-RAY DIFFRACTION
r_gen_planes_refined
0.004
0.02
2321
X-RAY DIFFRACTION
r_gen_planes_other
0.001
0.02
443
X-RAY DIFFRACTION
r_nbd_refined
X-RAY DIFFRACTION
r_nbd_other
X-RAY DIFFRACTION
r_nbtor_refined
X-RAY DIFFRACTION
r_nbtor_other
X-RAY DIFFRACTION
r_xyhbond_nbd_refined
X-RAY DIFFRACTION
r_xyhbond_nbd_other
X-RAY DIFFRACTION
r_metal_ion_refined
X-RAY DIFFRACTION
r_metal_ion_other
X-RAY DIFFRACTION
r_symmetry_vdw_refined
X-RAY DIFFRACTION
r_symmetry_vdw_other
X-RAY DIFFRACTION
r_symmetry_hbond_refined
X-RAY DIFFRACTION
r_symmetry_hbond_other
X-RAY DIFFRACTION
r_symmetry_metal_ion_refined
X-RAY DIFFRACTION
r_symmetry_metal_ion_other
X-RAY DIFFRACTION
r_mcbond_it
0.391
1.5
1221
X-RAY DIFFRACTION
r_mcbond_other
0.103
1.5
486
X-RAY DIFFRACTION
r_mcangle_it
0.724
2
1943
X-RAY DIFFRACTION
r_scbond_it
1.35
3
912
X-RAY DIFFRACTION
r_scangle_it
2.169
4.5
907
X-RAY DIFFRACTION
r_rigid_bond_restr
X-RAY DIFFRACTION
r_sphericity_free
X-RAY DIFFRACTION
r_sphericity_bonded
LS refinement shell
Resolution: 1.75→1.79 Å / Total num. of bins used: 20
Rfactor
Num. reflection
% reflection
Rfree
0.292
82
-
Rwork
0.19
1633
-
obs
-
-
91.17 %
Refinement TLS params.
Method: refined / Refine-ID: X-RAY DIFFRACTION
ID
L11 (°2)
L12 (°2)
L13 (°2)
L22 (°2)
L23 (°2)
L33 (°2)
S11 (Å °)
S12 (Å °)
S13 (Å °)
S21 (Å °)
S22 (Å °)
S23 (Å °)
S31 (Å °)
S32 (Å °)
S33 (Å °)
T11 (Å2)
T12 (Å2)
T13 (Å2)
T22 (Å2)
T23 (Å2)
T33 (Å2)
Origin x (Å)
Origin y (Å)
Origin z (Å)
1
0.5985
-0.046
-0.1318
0.5565
0.4544
2.0958
0.0281
-0.0673
-0.0116
0.0768
0.0232
0.0036
0.0609
0.1027
-0.0513
0.0584
-0.0291
0.0114
0.0606
-0.0192
0.0193
15.3699
-13.7169
11.5583
2
0.6311
0.0843
0.2748
0.6436
-0.1495
2.9563
0.0319
-0.0454
0.011
0.0526
0.0249
0.0111
-0.0129
-0.1287
-0.0568
0.0371
-0.0278
-0.0076
0.0573
0.0287
0.0086
9.1887
-29.2801
10.3303
Refinement TLS group
ID
Refine-ID
Refine TLS-ID
Auth asym-ID
Auth seq-ID
1
X-RAY DIFFRACTION
1
A
3 - 42
2
X-RAY DIFFRACTION
1
A
48 - 130
3
X-RAY DIFFRACTION
2
B
3 - 40
4
X-RAY DIFFRACTION
2
B
49 - 128
+
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