Structural basis of tubulin recruitment and assembly by microtubule polymerases with tumor overexpressed gene (TOG) domain arrays

8 figures, 1 video, 6 tables and 1 additional file

Figures

Figure 1 with 3 supplements
TOG1 and TOG2 domains bind αβ-tubulins and exchange them at different rates, within Alp14 TOG arrays.

(A) Top, domain organization of Alp14-monomer. Bottom, SEC-MALS for wt-Alp14-monomer at 100 mM KCl with and without DRP, revealing two αβ-tubulins bound in a non-polymerized state. (B) Top, domain …

https://doi.org/10.7554/eLife.38922.002
Figure 1—figure supplement 1
The Alp14 TOG array:αβ-tubulin binding capacities reveal non-equivalent behavior of TOG1 and TOG2 in different conditions.

We determined the molar ratio for four Alp14 constructs (described in Figure 1) in binding to soluble αβ-tubulins at 100 and 200 mM KCl conditions using quantitative-size exclusion chromatography …

https://doi.org/10.7554/eLife.38922.003
Figure 1—figure supplement 2
Molar ratios of Alp14 constructs binding to αβ-tubulin binding in the presence of Darpin-D1 (DRP) show no change in stoichiometry, and control SEC-MALS traces.

For each condition, 1 μmol of each Alp14 construct (described in Figure 1) was mixed with defined molar amount of soluble αβ-tubulin (1–2 μmol moles per Alp14 subunit) and Darpin-D1 (DRP). (A, D) …

https://doi.org/10.7554/eLife.38922.004
Figure 1—figure supplement 3
Isothermal titration calorimetry (ITC) reveals Alp14-TOG1 and TOG2 αβ-tubulin-binding affinities.

(A) Top and bottom panels: scheme for recombinant TOG1 and TOG2 domains studied and SDS-PAGE of purified 10 and 1 μM TOG1 and TOG2 domains, respectively. (B) Summary of measured αβ-tubulin binding …

https://doi.org/10.7554/eLife.38922.005
Figure 2 with 2 supplements
X-ray structures reveal αβ-tubulins bound in a wheel-like organization around a pseudo-dimeric TOG square complex.

(A–B) 3.6 Å X-ray crystal structure of the S. kluyveri 1:2:2 sk-Alp14:αβ-tubulin:DRP reveals pseudo-dimeric head-to-tail subunits (red and orange) in a TOG square assembly consisting of four TOG …

https://doi.org/10.7554/eLife.38922.007
Figure 2—figure supplement 1
X-ray crystallography and structure determination of 2:4:4 Alp14-monomer:αβ-tubulin:DRP complexes.

(A) Images of square crystals of 1:2:2 sk-Alp14-monomer:αβ-tubulin:DRP assemblies. (B) Molecular replacement search results for αβ-tubulin placement in the asymmetric unit. Note the distinct …

https://doi.org/10.7554/eLife.38922.008
Figure 2—figure supplement 2
Sequence conservation in TOG square interfaces across each TOG1 and TOG2 domain.

(A) Top scheme for Alp14, TOG1, TOG2, and linker sequence. Bottom, structure of TOG square with two TOG1 (blue) and two TOG2 (cyan) domains forming interfaces via inter-HEAT repeat elements with the …

https://doi.org/10.7554/eLife.38922.009
Figure 3 with 1 supplement
Cysteine mutagenesis/crosslinking and mass spectrometry-based peptide-mapping reveal that dimeric sk-Alp14 forms TOG square conformations in solution.

(A) TOG square structure showing two cysteine pairs (green space fill) mutated in interfaces 1 (black-dashed lines) and 2 (red-dashed lines). (B) Close-up views of interfaces 1 (left) and 2 (right) …

https://doi.org/10.7554/eLife.38922.011
Figure 3—figure supplement 1
Yeast dimeric TOG arrays form a TOG square assembly in solution as measured by cysteine crosslinking and mass spectrometry.

(A) Mass-spectrometry-based strategy for disulfide peptide mapping for the sk-Alp14 S180C L304C mutant reveals that sequences of peptides in interface 1 form crosslinked disulfides, confirming that …

https://doi.org/10.7554/eLife.38922.012
Figure 4 with 2 supplements
Inactivation of interfaces 1 and 2 destabilizes TOG square organization without affecting αβ-tubulin binding.

(A–D) Generation of structure-based TOG square assembly-defective mutants using wt-Alp14-dimer (A) through inactivation of interface 1 in the INT1 mutant (B: INT1, pink; eight mutant residues), …

https://doi.org/10.7554/eLife.38922.014
Figure 4—figure supplement 1
Inactivating interfaces 1 and 2 destabilizes the TOG square organization but does not influence αβ-tubulin-binding activity.

(A) Purification of INT1 mutant. Top, SEC-elution profile and trace for INT1 showing that it behaves homogenously similarly to wt-Alp14-dimer. Bottom panel show SDS-PAGE for SEC purified fractions. …

https://doi.org/10.7554/eLife.38922.015
Figure 4—figure supplement 2
Negative stain EM micrographs and class averages for the TOG square and its inactivated assemblies.

(A), (D), (G), and (J) Raw negative stain EM micrographs of WT-Alp14-dimer:αβ-tubulin, INT1, INT2, and INT1 +2, respectively. Note that the highlighted rectangle regions are shown in Figure 1. Boxes …

https://doi.org/10.7554/eLife.38922.016
Figure 5 with 2 supplements
X-ray structure of 1:2:1 TOG-array:αβ-tubulin:DRPΔN reveals unfurled TOG1-TOG2 array bound to two polymerized αβ-tubulins.

(A, B) Top, schemes of DRP and DRPΔN binding to αβ-tubulin. DRP shifts the equilibrium toward dissociation from αβ-tubulin. Bottom, isothermal titration calorimetery studies reveal a three-fold …

https://doi.org/10.7554/eLife.38922.018
Figure 5—figure supplement 1
Strategy to promote αβ-tubulin polymerization using DRPΔN and the structural comparison of DRP and DRPΔN interfaces with αβ-tubulin.

(A) SEC trace for the purified DRPΔN mutant. (B) SEC trace of 1:2:2 TOG1-TOG2:αβ-tubulin:DRPΔN complex (blue) in comparison to DRPΔN (red) and αβ-tubulin (black). (C) and (D) SDS-PAGE fractions …

https://doi.org/10.7554/eLife.38922.019
Figure 5—figure supplement 2
X-ray crystallographic structure determination of 1:2:1 Sk-Alp14-monomer:αβ-tubulin:DRPΔN complex.

(A) View of rectangular 1:2:1 TOG1-TOG2:αβ-tubulin:DRPΔN crystals formed in the same conditions as crystals in Figure Figure 2—figure supplement 1. (B) Top, molecular replacement solution to …

https://doi.org/10.7554/eLife.38922.020
Unfurling the TOG array: TOG2 rotation around TOG1 promotes the bound αβ-tubulins to polymerize.

(A and B) Conformational change of TOG2 (blue) around TOG1 (red) while each is bound to αβ-tubulin (green and cyan) from a corner subunit in the wheel assembly (left) and in the extended …

https://doi.org/10.7554/eLife.38922.022
Figure 7 with 1 supplement
Docking of atomic structures onto protofilament ends reveals the molecular details of unfurling.

(A) Right, atomic model for four αβ-tubulin-bound TOG square X-ray structures (Figure 2) docked using αβ-tubulin bound to TOG1 at the terminal αβ-tubulin in a curved protofilament (PDB ID: 3RYH). …

https://doi.org/10.7554/eLife.38922.023
Figure 7—figure supplement 1
All-atom docking models for structures superimposed onto curved protofilament plus-ends.

(A) An all-atom model for a TOG square bound to four αβ-tubulins docked onto the protofilament plus-end by overlaying the terminal αβ-tubulin with the αβ-tubulin bound onto TOG1. Note some minor …

https://doi.org/10.7554/eLife.38922.024
A polarized unfurling model for TOG arrays as MT polymerases.

An animation for this model is shown in Video 1. (A) Assembly of yeast MT polymerase dimeric TOG1-TOG2 subunits with four αβ-tubulins into an αβ-tubulin:TOG square. TOG squares diffuse along MT …

https://doi.org/10.7554/eLife.38922.025

Videos

Video 1
Animation of the mechanism of polarized unfurling for multiple TOG domains in a yeast MT polymerase.

This animation describes the ‘polarized unfurling’ mechanism for multiple TOG domains in promoting MT polymerization. Briefly, Yeast MT polymerases are dimers with each subunit including TOG1 and …

https://doi.org/10.7554/eLife.38922.026

Tables

Table 1
The stoichiometry for MT polymerases TOG1-TOG2 binding αβ-tubulin and DARPin (DRP)
https://doi.org/10.7554/eLife.38922.006
Protein complexExpected Mass (kDa)SEC-MALS Measured
Mass
(kDa)
SEC
elution
volume
(mL)
Apparent
Mass (kDa)
αβ-Tubulin100 kDa98 ± 0.323*12.9104
Alp14-dimer150 kDa143 ± 1.7010.0675
1 Alp14-dimer: 1 Tubulin 80 mM KCl350 kDa387 ± 2.749.27463
1 Alp14-dimer: 2 Tubulin 80 mM KCl550 kDa578 ± 1.418.98784
1 Alp14-dimer: 1Tubulin 200 mM KCl350 kDa304 ± 11.89.22693
1 Alp14-dimer: 2 Tubulin 200 mM KCl350 kDa392 ± 9.669.67549
1 Alp14-dimer-TOG1M: 1 Tubulin 80 mM KCl350 kDa400 ± 7.310.13433
1 Alp14-dimer-TOG2M: 2 Tubulin 80 mM KCl350 kDa382 ± 149.76524
Alp14-monomer62 kDa77.8 ± 1.2112.9899
1 Alp14-monomer: 2 Tubulin 80 mM KCl262 kDa264 ± 1.3111.17253
1 Alp14-monomer: 2 Tubulin: 2 DRP 80mM KCl298 kDa312 ± 2.3210.94285
1 Alp14-dimer-INT1: 2 Tubulin 80mM KCl550 kDa533 ± 3.118.95775
1 Alp14-dimer-INT2: 2 Tubulin 80mM KCl550 kDa580 ± 3.118.90770
1 Alp14-dimer-INT1+2: 2 Tubulin 80mM KCl550 kDa540 ± 3.228.80790
  1. *Standard error is defined based on fitting data across peaks using Astra-software.

Table 2
Capacities of MT polymerase TOG1-TOG2 to bind αβ-tubulin, influenced by ionic strength
https://doi.org/10.7554/eLife.38922.010
Protein Complex
(Alp14-monomer concentration 4.43 uM)
µM tubulin* boundµM tubulin
free
TOG1-TOG2
:αβ-tubulin
wt-Alp14-monomer (2 µM)
1 Alp14-monomer: 1 Tub 100 mM KCl4.57 ± 0.080.43 ± 0.081.04
1 Alp14-monomer: 2 Tub 100 mM KCl8.82 ± 0.161.18 ± 0.162
1 Alp14-monomer: 1 Tub 200 mM KCl3.37 ± 0.271.63 ± 0.270.76
1 Alp14-monomer: 2 Tub 200 mM KCl5.97 ± 0.124.03 ± 0.121.35
1 Alp14-monomer:1 Tub:1 DRP 100 mM KCl4.33 ± 0.130.67 ± 0.130.98
1 Alp14-monomer:2 Tub:2 DRP 100 mM KCl7.23 ± 0.012.77 ± 0.011.64
1 Alp14-monomer:1 Tub:1 DRP 200 mM KCl4.13 ± 0.090.87 ± 0.090.94
1 Alp14-monomer: 2 Tub 2 DRP 200 mM KCl5.67 ± 0.034.34 ± 0.031.28
wt-Alp14-dimer (1 µM)
1 Alp14-dimer: 1 Tub 100 mM KCl4.31 ± 0.070.69 ± 0.070.98
1 Alp14-dimer: 2 Tub 100 mM KCl7.11 ± 0.142.89 ± 0.141.61
1 Alp14-dimer: 1 Tub 200 mM KCl3.44 ± 0.371.56 ± 0.370.78
1 Alp14-dimer: 2 Tub 200 mM KCl5.46 ± 0.344.55 ± 0.341.24
1 Alp14-dimer: 1 Tub: 2 DRP 100 mM KCl4.23 ± 0.020.78 ± 0.020.96
1 Alp14-dimer: 1 Tub: 2 DRP 100 mM KCl8.33 ± 0.091.67 ± 0.091.89
1 Alp14-dimer: 1 Tub: 1 DRP 200 mM KCl4.17 ± 0.290.83 ± 0.290.95
1 Alp14-dimer: 2 Tub: 2 DRP 200 mM KCl6.02 ± 0.173.99 ± 0.171.36
TOG square Interface mutants (1 µM)
1 INT1: 2 Tub 100mM KCl8.34 ± 0.071.66 ± 0.071.74
1 INT1: 2 Tub 200mM KCl5.67 ± 0.044.33 ± 0.041.18
1 INT2: 2 Tub 100mM KCl9.52 ± 0.30.48 ± 0.22.02
1 INT2: 2 Tub 200mM KCl6.82 ± 0.23.1 ±0.11.45
1 INT1+2: 2 Tub 100mM KCl8.44 ± 0.051.56 ± 0.051.86
1 INT1+2: 2 Tub 200mM KCl6.85 ± 0.043.15 ± 0.041.46
Inactivated TOG mutants (1 µM)
1 TOG1M: 1 Tub 100 mM KCl4.04 ± 0.070.97 ± 0.070.92
1 TOG1M: 2 Tub 100 mM KCl4.94 ± 0.065.06 ± 0.061.12
1 TOG1M: 1 Tub 200 mM KCl1.66 ± 0.253.34 ± 0.250.38
1 TOG1M: 2 Tub 200 mM KCl2.23 ± 0.17.77 ± 0.10.51
1 TOG2M: 1 Tub 100 mM KCl3.53 ± 0.041.48 ± 0.040.8
1 TOG2M: 2 Tub 100 mM KCl6.24 ± 0.103.76 ± 0.11.41
1 TOG2M: 1 Tub 200 mM KCl3.15 ± 0.241.85 ± 0.240.72
1 TOG2M: 2 Tub 200 mM KCl4.28 ± 0.185.72 ± 0.180.97
  1. *Standard error is defined based on combined data from duplicated SEC runs.

Table 3
X-ray Crystallographic and Refinement statistics of MT polymerase:αβ-tubulin:DRP.
https://doi.org/10.7554/eLife.38922.013
Data collection1:2:2 sk-Alp14-monomer:
αβ-Tubulin:DRP
1:2:2 sk-Alp14-monomer-SL:
αβ-Tubulin:DRP
1:2:2 sk-Alp14-monomer:
αβ-Tubulin:DRPΔN
1:2:2 sk-Alp14-dimer:
αβ-Tubulin:DRPΔN
Resolution range (Å)96.59 - 4.40 (4.64- 4.40)*59.45 – 3.60 (3.79 – 3.60)*57.56 – 3.20 (3.37 – 3.20)*99.83 – 3.5 (3.69 – 3.50)*
Space groupP 21P 21P 21P 21
Wavelength (Å)0.97920.97920.97920.9792
Unit cell (Å): a, b, c
(°): β
218.80, 107.65, 282.74
90.38
218.48, 106.15, 282.23
90.39
115.13, 194.99, 149.57
90.19
122.73 199.67, 162.70
90.09
Total number of observed reflections229567380856298551235576
Unique reflections80099 {68039}142673 {121943}104265 {88337}91368
Average mosaicity0.570.380.640.50
Multiplicity2.9 (2.9)*2.7 (2.7)*2.9 (2.9)*2.6 (2.4)*
Completeness (%)95.4 (94.8) {80.6}95.0 (96.7) {79.0}96.2 (97.9) {82.0}92.9 (90.2)*
Wilson B-factor (Å2)82.781.446.6-
<I/σ (I)>4.9 (1.9)*4.8 (1.2)*5.8 (1.5)*4.8 (1.1)*
Rmergec0.14 (0.48)*0.13(0.65)*0.13(0.65)*0.14 (0.63)*
Structure refinement
Rwork0.23 (0.26)*0.20 (0.24)*0.18 (0.23)*-
Rfree0.26 (0.33)*0.24 (0.26)*0.24 (0.26)*-
Complexes per asymmetric unit222-
Number of atoms780307787836865-
Protein residues998999814661-
Ligand atoms496496248-
RMS bond lengths (Å)0.0040.0040.004-
RMS bond angles (°)0.940.980.93-
Ramachandran favored (%)94.094.095.0-
Ramachandran allowed (%)5.45.54.5
Ramachandran outliers (%)0.30.30.2-
Clashscore4.54.85.6-
Mean B values (Å2)
Overall108.498.348.6-
Macromolecules108.698.448.6-
Ligands78.591.536.4-
  1. *Numbers represent the highest-resolution shell.

    †Numbers represent the truncated data after treated with ellipsoidal truncation and anisotropic scaling.

  2. Rmerge = ΣhklΣi|Ii(hkl)-Iav(hkl)|/ΣhklΣiIi(hkl).

Table 4
Buried Surface Area between αβ-tubulin dimer and TOG domains or DRP.
https://doi.org/10.7554/eLife.38922.017
Interface1:2:2: sk-Alp14-monomer:
αβ-Tubulin: DRP(Å2)*
1:2:2: sk-Alp14-monomer-SL:
αβ-Tubulin: DRP (Å2)
1:2:1: sk-Alp14-monomer:
αβ-Tubulin: DRP-ΔN (Å2)
TOG1 and TOG2-interface 2273 ± 35257 ± 42-
TOG1-TOG2 dimer-interface 1661 ± 27702 ± 18-
αβ-tubulin and TOG1752 ± 12786 ± 42804 ± 24
αβ-tubulin and TOG2916 ± 23890 ± 22863 ± 12
β-tubulin and DRP or DRP-ΔN842 ± 68873 ± 21846 ± 20
α-tubulin and DRP
(inter-αβ-tubulin subunit)
108 ± 4781 ± 31-
  1. *Interface areas were determined by a single buried surface, and averaged among each non-crystallographic unit in the structure.

Table 5
Intra and inter dimer curvature angles (°) of αβ-Tubulins observed in structures.
https://doi.org/10.7554/eLife.38922.021
PDB IDIntra dimer (α1β1) angle (°)Inter dimer (β1α2) angle (°)
Stathmin:RB3 complex with GTP3RYH9.210.3
Stathmin:RB3 complex with GDP1SA013.013.5
αβ-tubulin:TOG1 complex with GTP4FFB13-
αβ-Tubulin:TOG2 complex with GTP4U3J13-
αβ-Tubulin:DRP complex with GDP4DRX11.0-
Sk-Alp14-monomer:αβ-Tubulin:DRP wheel with GDPCurrent11.3-
Sk-Alp14-monomer:αβ-Tubulin:DRP-ΔN with GDPCurrent11.216.4
  1. *Curvature of αβ-tubulin interface were determined as previously described by Rice and Brouhard 2015.

Key resources table
Reagent type
(species)
or resource
DesignationSourceIdentifierAdditional
information
Chemical
compound,
drug
Darpin D1
(Synthetic DNA)
InvitrogenN/A
Chemical
compound,
drug
GTPSigmaG-8877
Chemical
compound,
drug
GDPSigmaG7127
Chemical
compound,
drug
Crystallization
plates
TTP
Labtech
4150–05600
Chemical
compound,
drug
Crystallization
sparse matrix
screens
QiagenN/A
Chemical
compound,
drug
PEG-8000Sigma1546605
Chemical
compound,
drug
PEG-2000Sigma8.21037
Chemical
compound,
drug
Copper(II)
sulfate
SigmaC1297
Chemical
compound,
drug
1, 10-
phenanthroline
Sigma131377
Chemical
compound,
drug
TrypsinSigmaT6567
Chemical
compound,
drug
ChymotrypsinSigmaC6423
Chemical
compound,
drug
IodoacetamideSigmaI6125
Chemical
compound,
drug
4-VinylpyridineSigmaV3204
Other2:4:4 sk-
Alp14-550:αβ-
Tubulin:DRP
Protein
Data Bank
PDB: #6MZFDeposited Data
(Atomic
coordinates)
Other2:4:4
sk-Alp14-550-
SL:αβ-Tubulin:
DRP
Protein
Data Bank
PDB: #6MZEDeposited
Data (Atomic
coordinates)
Other1:2:1
sk-Alp14-550:
αβ-Tubulin:DRPΔN
Protein
Data Bank
PDB: #6MZGDeposited
Data (Atomic
coordinates)
OtherSaccharomyces
cerevisiae Stu2p
UniprotKB/
Swiss-Prot
P46675Protein
sequence
OtherSaccharomyces kluyveri
Stu2p or Alp14p
Lachancea kluyveri NRRL
Y-12651 chromosome
SKLU-Cont10078Protein sequence
OtherSchizosaccharomyces
pombe Alp14p

UniprotKB/
Swiss-Prot
O94534Protein sequence
OtherChaetomium
thermophilum
Stu2
UniprotKB/
Swiss-Prot
G0S3A7Protein sequence
OtherSoluBL21
bacterial
expression
system
AmsBioC700200Model
system
(expression
system)
Recombinant
DNA reagent
pLIC_V2-Sc
Stu2p-H6
Current
study
N/ARecombinant
DNA constructs
Expressed
in bacterial
strains
Recombinant
DNA reagent
pLIC_V2-Sk
Stu2p-H6
Current studyN/A
Recombinant
DNA reagent
pLIC_V2-Sc
Stu2-550-H6
(TOG1-TOG2
monomer)
Al-Bassam et al., 2006
Recombinant
DNA reagent
pLIC_V2-KL-Stu2
-monomer-H6
(residues 1–560)
Current studyNA
Recombinant
DNA reagent
pLIC_V2-CT
Stu2-mon
omer-H6
(residues 1–550)
Current
study
NA
Recombinant
DNA reagent
pLIC_V2-SK
Alp14-monomer-
H6 (residues 1-550)
Current studyN/A
Recombinant
DNA reagent
pLIC_V2-Sk
Alp14-monomer
-SL-H6
(residues 1–550;
linker
residues replaced
KL sequence;
see Materials and methods)
Current studyN/A
Recombinant
DNA reagent
pLIC_V2-
sk-wt-Alp14-
dimer-H6
(residues 1–724)
Current
study
N/A
Recombinant
DNA reagent
pLIC_V2
-sk-Alp14-
dimer- H6
Current
study
N/A
Recombinant
DNA reagent
S180C
and L304C
(residues
1–724)
Recombinant
DNA reagent
pLIC_V2-
sk-Alp14-
dimer- H6
Current studyN/A
Recombinant
DNA reagent
S41C and
E518C
(residues
1–724)
Recombinant
DNA reagent
pLIC_V2-wt
-Alp14-dimer-
H6 (residues
1–690)
Current studyN/A
Recombinant
DNA reagent
pLIC_V2-TOG1M
- H6 (residues
1–690: Y23A
and R23A)
Current studyN/A
Recombinant
DNA reagent
pLIC_V2-TOG2M
- H6 (residues
1–690: Y300A
and K381A)
Current studyN/A
Recombinant
DNA reagent
pLIC_V2-INT1-H6
(residues 1–690:
L206A, L208A,
F275R D276A,
L277A, V278A,
K320L, R359A)
Current studyN/A
Recombinant
DNA reagent
pLIC_V2-INT2-H6
(residues 1–690:
L39D, S40A,
D42A, L437D,
S440A, E478A
and R479A)
Current studyN/A
Recombinant
DNA reagent
pLIC_V2
-INT1 + 2 H6
(L206A, L208A,
F275R D276A,
L277A, V278A,
K320L, R359A
L39D, S40A,
D42A, L437D,
S440A, E478A
and R479A)
Current studyN/A
Recombinant
DNA reagent
pET303-H6-DRPCurrent
study
N/A
Recombinant
DNA reagent
pLIC_V2-H6-DRPΔNCurrent
study
N/A
Otherαβ-tubulin
purified
from porcine
brains
Current
study
N/ANative protein
purification
Otherαβ-tubulin
purified from
porcine brains
Castoldi and Popov, 2003
Software,
algorithm
ASTRA V6.0Wyatt
Technology
http://www.wyatt.
com/products/
software/astra.html
Software,
algorithm
NanoAnalyzeTA
Instruments
http://www.tainstruments.com/
Software,
algorithm
EMAN2http://blake.bcm.edu/emanwiki/EMAN2
Software,
algorithm
iMOSFLMBattye et al., 2011http://www.mrc-lmb.cam.ac.uk/harry/imosflm/ver721/quickguide.html
Software,
algorithm
PHASERTerwilliger, 2000http://www.phaser.cimr.cam.ac.uk/index.php/
Software,
algorithm
PHASERMcCoy, 2007Phaser_Crystallographic_
Software
Software,
algorithm
PyMolSchrodinger, LLChttp://www.pymol.org/
Software,
algorithm
UCSF-ChimeraPettersen et al., 2004https://www.cgl.ucsf.edu/chimera/
Software,
algorithm
DM from CCP4 suiteCowtan and Main, 1996http://www.ccp4.ac.uk/html/dmmulti.html
Software,
algorithm
PHENIXAdams et al., 2010https://www.phenix-online.org
Software,
algorithm
anisotropy serverStrong et al., 2006https://services.mbi.ucla.edu/anisoscale/
Software,
algorithm
Phyre protein
homology model
Kelley et al., 2015www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index
Software,
algorithm
Cr-yoloWagner et al., 2018http://sphire.
mpg.de/wiki/
Software,
algorithm
Relion 2.2Kimanius et al., 2016https://www2.mrc-lmb.cam.ac.uk/relion/index.php
Software,
algorithm
CryosparcPunjani et al., 2017https://cryosparc.com/
Software,
algorithm
MolProbityChen et al., 2012http://molprobity.biochem.duke.edu
Software,
algorithm
CootEmsley et al., 2010http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
Software,
algorithm
BLENDER
3D-animation
Blender
foundation
https://www.blender.org/

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