Structurally derived universal mechanism
for the catalytic cycle of the tail-anchored
targeting factor Get3
In the format provided by the
authors and unedited
Supplementary information
https://doi.org/10.1038/s41594-022-00798-4
Structurally derived universal mechanism for
the catalytic cycle of the tail-anchored
targeting factor Get3
Michelle Y. Fry
1
, Vladim
́
ıra Najdrov
́
a
2
, Ailiena O.
Maggiolo
1
, Shyam M. Saladi
1
, Pavel Dole
ˇ
zal
2
and William M.
Clemons Jr.
1*
1*
Division of Chemistry & Chemical Engineering, California Institute
of Technology, 1200 E. California Blvd, Pasadena, 91125, CA, USA.
2
Department of Parasitology, Faculty of Science, BIOCEV, Charles
University, Pr
̊
umyslov
́
a 595, Vestec, 25250, Czech Republic.
*Corresponding author(s). E-mail(s): clemons@caltech.com;
Contributing authors: myfry@caltech.edu;
najdrova.vladimira@seznam.cz; mag@caltech.edu;
saladi@caltech.edu; pavel.dolezal@natur.cuni.cz;
This PDF file includes:
Supplemental Figures S1-S7
References
1
2
Fry, et al. Supplemental
Supplemental Figures
Fig. S1
:
Conservation across clades of of eukaryotic Get3s
Sequence alignments for each clade are visualized using HMM logo plots where residues size
correlate to prevalence in the alignment (Clade I,
top
and Clade II,
bottom
) [1]. There are no
distinct motif differences between the clades.
Fry, et al. Supplemental
3
Fig. S2
:
Comparison of apo
Gi
Get3 with various structures of opisthokont Get3
homologs in the ‘open’ state.
A)
Two views of
Gi
Get3 apo1 (
magenta
) and apo2 (
purple
) aligned with chain A of
Af
Get3
(
grey
, PDBID:3IBG) [2].
B)
A comparison of the active sites of
Gi
Get3 apo2 (
purple
) and
Af
Get3 (
grey
). The P-loop, A-loop, and Switch I & II are colored as in Fig. 2.
C
) Cartoon
representations of fungal Get3 homologs in the open state (PDBIDs:3IBG, 2WOO, 3A36, &
3H84). Chain A is colored in grey and chain B is colored from N- to C-terminus using the
viridis color map (
purple to yellow
). Species and ligands are specified below the PDBID num-
bers.
D
) Surface and cartoon representations of the CBD of 2WOO, 3A36, and Apo1 where
residues are colored by hydrophobicity using the TM Tendency scale [3] and helices are num-
bered as in Fig. 2
.
4
Fry, et al. Supplemental
Fig. S3
:
Comparison of ATP-bound
Gi
Get3 to structures of opisthokont Get3s in
the ‘closed’ state.
Cartoon representation of
A
) fungal Get3s bound to nucleotide in the ‘closed’ state
(PDBIDs:2WOJ, 3IQW, and 3IQX) [4, 5] and
B
) opisthokont Get3s in complex with other
GET components (PDBIDS:4PWX, 4XTR, and 7RU9) [6–8]. For
A
&
B
, chain A is colored
in grey and chain B is colored from N- to C-terminus using the viridis color map (
purple to
yellow
). Species and ligands are specified below the PDBID numbers.
C
) Two views of sur-
face and cartoon representations of the CBD of 2WOJ, 7RU9, and
Gi
Get3
D
53
N
·
ATP where
residues are colored by hydrophobicity using the TM Tendency scale [3] and helices are num-
bered as in Extended Data Fig. 3.
D)
Two views of
Gi
Get3
D
53
N
·
ATP (
yellow
) and
Ct
Get3
·
AMPPNP (
grey
) aligned by chain A.
E
) A comparison of the active sites of
Gi
Get3 (
yellow
)
and
Ct
Get3 (
grey
). Nucleotide and important catalytic loops are shown as sticks and colored as
in Extended Data Fig. 3.
Fry, et al. Supplemental
5
Fig. S4
:
SPA map and model quality
The Gold-standard Fourier Shell Correlation curves for ‘masked’ (
orange
) and ‘unmasked’
(
blue
) half maps used to build the (
A
)
Gi
Get3 and (
B
)
Gi
Get3/TA complex maps from RELION
3.1.2. While a complete model could be built into the overall map, a focused refined map of the
NBD improved the local resolution and provided more detail. Resolution was determined using
an FSC=0.143 criterion.
C
) The FSC curves of the model vs. map for the ‘unmasked’ (
blue
)
and ‘masked’ (
orange
)
Gi
Get3/TA complex model. Overall refers to the overall map and NBD
refers to the focused refined map. Resolution was determined using an FSC=0.5 criterion.