Finch, AJ et al., Supplemental Material Includes 7 Supplemental Figures, 7 Supplemental Tables, (Tables 1 and 2 are provided as Excel files), Supplemental Materials and Methods, Supplemental References. Supplemental Figures Figure S1. Targeted disruption of the mouse Sbds gene, related to Fig. 1 a. Genomic organization of the mouse Sbds locus (Sbds+); b. targeting vector; c. the product of homologous recombination (Sbdsfl); d. structure of the Sbds locus following Cre recombination (Sbds-). Red boxes represent Sbds exons I-V, yellow triangles represent loxP sites. The 5' external probe used for genotyping by filter hybridization is shown as a gray box. B, BamH1. Figure S2. Sbds deletion does not alter the ratio of 60S to 40S ribosomal subunits, related to Fig. 2. The ratios of 60S to 40S subunits are compared between Sbdsfl/- mice treated with 6 x 250 µg poly(I:C) over 2 weeks in the absence (-Cre) or presence (+Cre) of the pMx1-cre transgene. Figure S3. A negative genetic interaction network for EFL1 and SDO1, related to Fig. 3. Genes are represented as nodes, and negative genetic interactions (e.g. synthetic lethal/sick interactions) are represented as edges that connect the nodes. Synthetic lethal/sick interactions confirmed by random spore and/or tetrad analysis are shown as black edges. Network edges representing unconfirmed quantitative negative interactions that satisfied a stringent confidence threshold (e <-0.12, p <0.05) 
 1 are shown in grey. Nodes are colored based on a general biological process annotation described previously (Costanzo et al. 2010). Figure S4. EFL1 catalytic residues are required for genetic complementation of efl1Δ cells in vivo, related to Fig. 3. a. Schematic of the domain structure of yeast EFL1 showing the position of critical loss of function missense mutations. b. Sequence alignment of the consensus GTP-binding domain (G domain) for H. sapiens EFL1 (HsEFL1, NP_078856), S. cerevisiae EFL1 (ScEfl1p, NP_014236) and S. cerevisiae EF-2 (ScEF-2). The amino sequences were aligned using MacVector 11.1 (http://www.macvector.com). The conserved motifs G1, G2, G3, G4 and G5 of the G domain are boxed in red. Residues mutated in (c) are shown in red. c. EFL1 catalytic residues are required for function in vivo. The ability of a yeast EFL1-expressing plasmid to complement the growth defect of efl1Δ cells is impaired by mutations in the G1 P-loop that spans the α and β phosphates of the bound GTP cofactor (T33A), the G3 switch II region involved in Mg2+ coordination and γ- phosphate binding (D102A, H106A, H106I), the G4 motif involved in binding the guanine nucleobase of GTP (D159A) or mutation of a residue predicted to contact the SRL (W240A). Figure S5. The SBDS protein is highly conserved from archaea to human, related to Fig. 4. a. Structure-based sequence alignment of representative SBDS protein orthologs. Numbering corresponds to the amino acid sequence for human SBDS. Shading intensity indicates the degree of amino acid identity. GenBankTM accession numbers: 
 2 H. sapiens (NP_057122), M. musculus (P70122), S. cerevisiae (NP_013122), D. melanogaster (NP_648057), D. discoideum (AAO50830), A. fulgidus (NP_069327), M. thermautotrophicus (NP_275828). Secondary structure elements for the A. fulgidus and H. sapiens SBDS orthologs are shown above the alignment with domain I colored in red, domain II in yellow, domain III in green. The alignment was generated with MacVector 11.1 (http://www.macvector.com). b-d. The fold of the human and archaeal SBDS proteins is conserved. Superposition of the structures of human SBDS (pdb code 2L9N) domain I (red) (b), domain II (yellow) (c) and domain III (green) (d) with the respective domains of A. fulgidus SBDS (pdb code 1T95) (gray). Rms deviation for domain I (residues A16-T89) is 1.27 Å; domain II (residues D97-K164) is 1.48 Å and domain III (residues H171- L237) is 1.13 Å. Figures were generated using PyMOL (http://www.pymol.org). Figure S6. Chemical shift perturbations caused by SDS-associated disease mutants, related to Fig. 4. Overlays of the 1H,15N HSQC spectra for SBDS WT (blue) and 25 SDS-associated mutants (red). a. (I) P6L, (II) N8K, (III) R19Q, (IV) F27L, (V) A30S, (VI) Y32C, (VII) K33E, (VIII) N34I. b. (IX) E44G, (X) F57L, (XI) K67E, (XII) K118N, (XIII) C119R, (XIV) C119Y, (XV) N121T, (XVI) T129A. c. (XVII) S143L, (XVIII) S143W, (XIX) K148R, (XX) K148T, (XXI) Q153R, (XXII) R169C, (XXIII) R169L, (XXIV) R175W. d. (XXV) R218Q. Figure S7. The SDS-associated SBDS variants R126T and K151N bind to 60S subunits in vitro, related to Fig. 6. Recombinant WT SBDS and variants (R126T, K151N) were bound together with EFL1 to RRL 60S subunits over the indicated 
 3 range of KCl concentrations and pelleted through 30% (w/v) sucrose cushions. Bound SBDS, EFL1 and Rpl28 were visualized by immunoblotting. Supplemental Tables. Table S1. Quantitative scoring of EFL1 positive and negative genetic interactions (Excel file). Table S2. MS/MS analysis of murine eIF6 tryptic peptides. Observed phosphopeptides are shaded in yellow. The peptide containing S235 (LNEAKPSTIATS235MR) is shaded in blue. Lower case amino acids indicate modifications (Excel file). 
 4 Table S3. Summary of experimental restraints and structural statistics for the 20 accepted lowest energy structures of the human SBDS protein Structural
constraints
 
 Intra‐residue
 1490
 Sequential
 1119
 Medium‐range
(2
≤
|i‐j|
≤
4)
 

596
 Long‐range
(|i‐j|
>
4)
 

822
 Dihedral
TALOS
constraints
 

296
 Hydrogen
bond
constraints
 

116
 Total
 4439
 
 Statistics
for
accepted
structures
 Statistics
parameter

 
 R.m.s
deviation
for
distance
constraints
(űSD)
 










0.0052
±
0.0010
 R.m.s
deviation
for
dihedral
constraints
(o±SD)
 









0.0727
±
0.0486
 
 Mean
CNS
energy
term
(kcal.mol‐1±SD)
 E
(overall)
 157.7
±
26.9
 E
(van
der
Waals)
 



40.5
±
13.1
 E
(NOE
and
hydrogen
bond
constraints)
 

8.8
±
3.7
 E
(chi‐1
dihedral
and
TALOS
constraints)
 

0.5
±
1.0
 
 
 R.m.s
deviations
from
the
ideal
geometry
 Bond
lengths
(űSD)
 






















0.0012
±
0.0002
 Bond
angles
(o±SD)
 




0.29
±
0.01
 Improper
angles
(o±SD)
 






















0.15
±
0.02
 
 Average
atomic
r.m.s
deviation
from
the
mean
structure
(űSD)
 Domain
I
 

 Residues
A16‐T89
(N,
Cα,
C
atoms)
 



0.44
±
0.12
 aSecondary
structure
(N,
Cα,
C
atoms)
 



0.37
±
0.09
 Residues
A16‐T89
(all
heavy
atoms)
 



0.99
±
0.12
 Domain
II
 
 Residues
D97‐K164
(N,
Cα,
C
atoms)
 



















0.78
±
0.25
 bSecondary
structure
(N,
Cα,
C
atoms)
 



0.55
±
0.14
 Residues
D97‐K164
(all
heavy
atoms)
 



















1.42
±
0.27
 Domain
III
 














 Residues
H171‐L237
(N,
Cα,
C
atoms)
 



















0.66
±
0.11
 cSecondary
structure
(N,
Cα,
C
atoms)
 



















0.63
±
0.10
 Residues
H171‐L237
(all
heavy
atoms)
 



















1.30
±
0.10
 
 Ramachandran Quality parameters (%) Molprobity 
 Residues in favored region of Ramachandran Plot 

















92.8
 Residues
in
allowed
region
of
Ramachandran
Plot
 

















99.1
 
 
 PROCHECK
 
 Most
favored
regions
 



















90.4
 Additionally
allowed
regions
 



















9.5
 Generously
allowed
regions
 





















0.0
 Disallowed
regions
 





















0.1
 
 5 aresidues A16-R22, K25-C31, K33-S41, L47-V50, F57-N59, Q64-V65, K68-F75, Q80-T89. bresidues D97-D117, V130-D139, T150-K164. cresidues H171-L178, E182- L193, K195-D201, E207-P214, R218-K229, L234-L237. 
 6 Table S4. Summary of SBDS missense mutations associated with Shwachman- Diamond syndrome 
 Mutation Reference N8K (Boocock et al. 2003) E44G K67E I87S R126T R169C I167M (Nakashima et al. 2004) R19Q (Shammas et al. 2005) C31W K33E N34I L71P K118N S143L + K148R Q153R R169L I87T (Makitie et al. 2004) Y32C (Nicolis et al. 2005) C84R (Kuijpers et al. 2005) R175W (Erdos et al. 2006) N121T P6L C. Bellanné-Chantelot and J. Donadieu, personal communication F57L C119Y, C119R T129A K151N R169L R218Q A154V A30S M. Schwarz, personal communication 
 7 Table S5. Impact of SDS-associated mutations 
 Chemical shift SBDS mutants perturbationa Classb I II III Domain I P6L + – – B N8K + – – B R19Q + – – B F27L * – – A A30S + – – B Y32C * – – A K33E + – – B N34I + – – B E44G + – – A F57L * – – A K67E + – – B C84R * – – A Domain II K118N – + + B C119R – * * A C119Y – * * A N121T – * * A R126T – + – B T129A – + – B S143W – + – B S143L – + – B K148R – + – B K148T – + – B K151N – + – B Q153R – + – A R169C – + + A R169L – + + A Domain III R175W – – + B R218Q – – + B 
 8 a. (–) indicates no chemical shift perturbation compared to WT SBDS; (+), local or global chemical shift perturbation; (*), stability of the domain was compromised leading to unfolding and/or aggregation. b. (A) stability mutants, (B) surface mutants. 
 9 Table S6. Yeast strains
 
 Name
 Genotype
 Source
 SE1
 S288c;
MATa/α,
lyp1Δ/+,
mfα1Δ::MFα1pr­LEU2/+;
 AJW
 can1Δ::MFA1pr­HIS3/+,
his3Δ1/his3Δ1,
leu2Δ0/leu2Δ0,
 ura3Δ0/ura3Δ0,
met15Δ0/met15Δ0,
 sdo1Δ::NatMX4/SDO1,
efl1Δ::KanMX4/EFL1
 C375
 S288c,
MATα,
can1Δ::MFA1pr­HIS3,
mfα1Δ::MFα1pr­ AJW
 
 LEU2,
lyp1Δ,
his3Δ1,
leu2Δ0,
ura3Δ0,
met15Δ0,
 
 sdo1Δ::NatMX4,
TIF6­I58T
 NS0
 S288c,
MATα,
can1Δ::MFA1pr­HIS3,
lyp1Δ,
his3Δ1,
 This
study
 leu2Δ0,
ura3Δ0,
met15Δ0,
efl1Δ::NatMX4,
TIF6­R61G
 Y5538
 S288c,
MATa/α,
lyp1Δ/+,
mfα1Δ::MFα1pr­LEU2/+;
 C.
Boone
 can1Δ::MFA1pr­HIS3/+,
his3Δ1/his3Δ1,
leu2Δ0/leu2Δ0,
 ura3Δ0/ura3Δ0,
met15Δ0/met15Δ0
 MATa, CAN1, ade2, trp1, ura3-52, BCY123
 A.
 Newman,
 his3, leu2-3, 112, pep4::his+, prb1::leu2+, bar1::HisG+, LMB
 lys2::pGAL1/10-GAL4+ 
 
 
 10